A method for detecting microorganisms by adding fluorescent dyes directly to solid growth media

Abstract

The present invention relates to a method for detecting and / or enumerating a microorganism or group of microorganisms of interest, comprising the steps of: a) contacting in a container a sample suspected of containing said microorganism or group of microorganisms with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms, and with at least one fluorescent dye; b) incubating said container under conditions and for a time sufficient to form microcolonies of said microorganism or group of microorganisms; and c) detecting and / or enumerating microcolonies that emit a fluorescent signal of said at least one fluorescent dye, thereby detecting, identifying and / or enumerating said microorganism or group of microorganisms contained in a sample.

Description

[Technical field]

[0001] The present invention relates to the field of microbiology, and more particularly to a method for the rapid detection, and optionally enumeration, of a microorganism or group of microorganisms of interest in a sample. [Background technology]

[0002] Microorganisms can contaminate any product designed for consumption or use in any environment, human and animal, such as food, beverage, medical device, pharmaceutical or cosmetic. The presence of these microorganisms can not only deteriorate or spoil the product, but also cause disease when consumed or administered by humans or animals. To ensure the safety of these products, microbial testing is necessary to check the risk of contamination under normal use conditions, thereby preventing the proliferation of poisoning or infection. Especially for the medical device, pharmaceutical or cosmetic industries, the need for sterility is crucial, and potential defects of the product can occur at all stages of production, so it is important to test the microbial quality of products and raw materials along the entire supply chain.

[0003] Of course, microorganisms can infect humans or animals, and clinical diagnosis requires the detection and / or quantification of the microorganisms responsible for the infection. For example, the diagnosis of urinary tract infection (UTI) can be performed by quantitative urine culture. Traditionally, the presence of more than 1,000 bacteria / ml in urine represents significant bacteriuria and is considered to be indicative of a UTI.

[0004] For all these industrial and clinical applications, the contamination thresholds and the methods used may vary, but it is always important to reveal the presence of the relevant microorganisms at the earliest possible stage.

[0005] Louis Pasteur's method, cultivation on Petri dishes, has been the most effective way to identify and count microorganisms since the 19th century. To this day, this technique remains the reference method in numerous industries to detect and count microorganisms. The method is simple: extract the microorganisms from a sample, place them on dedicated Petri dishes containing a suitable medium and count the colonies when they become visible. This method is reliable but time-consuming (up to several days depending on the target microorganism).

[0006] To shorten the length of the assay, it has been proposed to use indicators such as chromogenic substrates, fluorogenic substrates or fluorescent or radiolabeled antibodies to detect the smaller sized microcolonies (e.g., International Patent Applications WO96 / 14431, WO2013 / 050598, WO2008 / 118400). However, for most of these methods, for optimal results the growth and indicator steps must be separated: a growth step for fast cell growth without harmful indicators that slow growth, and an indicator step for dedicated coloring and identification that effectively allows the smaller sized microcolonies to be detected.

[0007] Over the years, numerous other attempts have been made to reduce the time required for the assay using, in particular, immunological methods using labeling with fluorescent antibodies or fluorogenic substrates, nucleic acid amplification methods or culture-independent approaches based on flow cytometry methods. However, these methods remain expensive, have a non-trivial workflow, require highly skilled experts and / or concentrated samples, and for most of them it is extremely difficult to distinguish between live and dead microorganisms. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO96 / 14431 [Patent Document 2] WO2013 / 050598

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[0009] [Non-licensed document 1] Ismail et al., Int J Environ Res Public Health. 2013 Nov 14;10(11):6169-83

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[0010] Thus, there remains a need for rapid, sensitive, user-friendly, and cost-effective methods for detecting and enumerating microorganisms in a sample. [Means for solving the problem]

[0011] Here we provide a method that meets this need.

[0012] In a first aspect, the present invention provides a method for detecting or enumerating a microorganism or group of microorganisms of interest, comprising the steps of: a) contacting in a container a sample suspected of containing said microorganism or group of microorganisms with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms, and with at least one fluorescent dye; b) incubating the container under conditions and for a time sufficient to form microcolonies of the microorganism or group of microorganisms; and c) detecting or enumerating microcolonies that emit a fluorescent signal of said at least one fluorescent dye; thereby to detect or enumerate said microorganism or group of microorganisms contained in a sample.

[0013] Optionally, prior to step a), the microorganisms of the sample are concentrated on a membrane filter, and in step a) said membrane and the microorganisms are contacted with a solid growth medium and said at least one fluorescent dye.

[0014] Step a) is a1) contacting a sample suspected of containing said microorganism or group of microorganisms with at least one fluorescent dye, and a2) contacting said sample in a container with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms. May contain Steps a1) and a2) may be performed simultaneously or sequentially in any order.

[0015] Preferably, step a1) is carried out before step a2).

[0016] Optionally, before step a2), the microorganisms of the sample are concentrated on a membrane filter. In particular, the sample may be concentrated on a membrane filter before or after step a1), preferably after step a1). In particular, the at least one fluorescent dye may be filtered on the same membrane as the sample before or after filtration of the sample. The fluorescent dye may be added to the sample before filtration / concentration on the membrane, or may be filtered on the same membrane before or after filtration of the sample, preferably after filtration of the sample. A membrane filter may then be placed on the surface of the growth medium.

[0017] Preferably, the membrane filter is made of mixed cellulose esters (MCE), polyvinylidene difluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate or nylon, more preferably made of MCE or PVDF.

[0018] The solid growth medium may be selective for said microorganism or group of microorganisms.

[0019] The at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, acridine dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes, and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof. Preferably, the at least one fluorescent dye may be selected from the group consisting of MB660R, Acridine Orange, DRAQ5, Cytrak Orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably Sulfo-Cy5, Cy5.5, preferably Sulfo-Cy5.5, BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta 650, Seta 375, Setau 647, SeTau 488, Alexa Fluor 488, ATTO 647 and DAPI, derivatives thereof and combinations thereof. More particularly, the at least one fluorescent dye may be selected from the group consisting of MB660R-DBCO, MB660R-acid, Acridine Orange, DRAQ5, Cytrak Orange, Alexa Fluor 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, ATTO 647-acid and DAPI, derivatives thereof and combinations thereof.

[0020] In some embodiments, the at least one fluorescent dye may be selected from the group consisting of anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, xanthene-based dyes, and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof.Preferably, the at least one fluorescent dye is selected from the group consisting of DRAQ5™, Alexa Fluor™ 350, Cy5™, preferably sulfo-Cy5, BODIPY™ 500 / 510, prodan, Alexa Fluor™ 405, Seta™ 650, Setau™ 647, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647, and DAPI, derivatives thereof, and combinations thereof. More preferably, the at least one fluorescent dye is DRAQ5™, Alexa Fluor™ 350-NHS ester, Sulfo-Cy5 acid, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Seta™ 650-DBCO, SeTau™ 647-maleimide, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI and combinations thereof. Alternatively, the at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade blue™, Seta™ 650, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647 and DAPI, derivatives thereof and combinations thereof.More preferably, the at least one fluorescent dye is DRAQ5™, Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, Sulfo-Cy5 acid, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta™ 650-DBCO, SeTau 488-NHS, SeTau™ 647-maleimide, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI and combinations thereof.

[0021] Alternatively, the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and combinations thereof. In particular, the at least one fluorescent dye may be selected from the group consisting of MB660R, DRAQ5, Cytrak Orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta 650, Seta 375, Setau 647, SeTau 488, Alexa Fluor 488 and ATTO 647, derivatives thereof and combinations thereof. More particularly, the at least one fluorescent dye may be selected from the group consisting of MB660R-DBCO, MB660R-acid, DRAQ5, Cytrak Orange, Alexa Fluor 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO and ATTO 647-acid, derivatives thereof and combinations thereof.

[0022] In particular, the microorganism or group of microorganisms of interest are - may belong to the thermoacidophilic bacteria, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius and Alicyclobacillus contaminans and combinations thereof; and / or may belong to the acetic acid bacteria, preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter and Asaia, and / or combinations thereof; and / or may belong to the lactic acid bacteria, preferably selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof; and / or may belong to the anaerobic bacteria, preferably selected from the bacteria of the genera Clostridium and Cutibacterium and combinations thereof; and / or - may belong to the aerobic mesophilic bacteria, preferably selected from the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria and Burkholderia and combinations thereof; and / or may belong to the gram-negative bacteria, preferably selected from the genera Escherichia and Pseudomonas and combinations thereof; and / or - may belong to the group of yeasts and molds, preferably selected from the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof; and / or - preferably from the genera Acinetobacter, Aeromonas, Brevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobactrum, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus teus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Vibrio, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus, and combinations thereof.

[0023] Preferably, in the method of the present invention, - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of thermoacidophilic bacteria, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius and Alicyclobacillus contaminans and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof. Preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably from the group consisting of MB660R and derivatives, more preferably from the group consisting of MB660R-acid and MB660R-DBCO, even more preferably MB660R-DBCO; and / or - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Acetobacter bacteria (AAB), preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter and Asaia and combinations thereof, more preferably selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans and Asaia siamensis and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488-acid, Alexa Fluor Preferably, the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof. More preferably, said at least one fluorescent dye is selected from the group consisting of MB660R and ATTO647 and derivatives and combinations thereof, preferably selected from the group consisting of MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof, more preferably selected from the group consisting of MB660R-acid and ATTO647-acid and combinations thereof; and / or - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of lactic acid bacteria (LAB), preferably selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof, more preferably selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum and Weissella confusa and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof. Preferably, said at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R and DAPI and their derivatives and combinations, in particular selected from the group consisting of Alexa Fluor 488-acid, MB660R-acid and DAPI and combinations thereof, preferably selected from the group consisting of xanthene-based dyes, more preferably selected from the group consisting of Alexa Fluor 488-acid and MB660R-acid and combinations thereof, even more preferably MB660R-acid; and / or - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of anaerobic bacteria, preferably selected from bacteria of the genera Clostridium and Cutibacterium and combinations thereof, more preferably selected from the group consisting of Clostridium sporogenes and Cutibacterium acnes and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of MB660R, Cy5™, preferably Sulfo-Cy5 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of MB660R-DBCO, Sulfo-Cy5 acid and DAPI and combinations thereof. Preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and cyanine-based dyes, preferably from the group consisting of MB660R and Cy5, preferably Sulfo-Cy5 and derivatives and combinations thereof, more preferably from the group consisting of MB660R-DBCO and Sulfo-Cy5 acid and combinations thereof. More preferably, said at least one fluorescent dye comprises MB660R-DBCO and Sulfo-Cy5 acid, and / or - the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably selected from Methylobacterium extorquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterococcus faecalis, Listeria grayi, Shigella sonnei, Kocuria rhizophila, and Burkholderia cepacia, and combinations thereof; and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes, and DAPI, and combinations thereof, preferably DRAQ5™, Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 and SeTau 488, Alexa Fluorand / or belongs to or comprises a microorganism belonging to the group of aerobic mesophilic bacteria (AMB), selected from the group consisting of DRAQ5™, Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, Sulfo-Cy5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI and combinations thereof, more preferably selected from the group consisting of DRAQ5™, Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, Sulfo-Cy5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI and combinations thereof; - the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium , Lactobacillus faecalis, Lactobacillus sonnei, Kocuria rhizophila and Borrelia cepacia, and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, acridine dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI, and combinations thereof, preferably acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, BODIPY dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-Maleimide, SeTau 488-NHS, Alexa Fluor 488-Acid, Alexa FluorThe present invention relates to or comprises a microorganism belonging to the group of aerobic mesophilic bacteria (AMB), selected from the group consisting of 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI, and combinations thereof. Preferably, the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, BODIPY dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488-acid, Alexa Fluor488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof. More preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of MB660R and derivatives and combinations thereof, optionally in combination with a fluorescent dye selected from the group consisting of cyanine-based dyes, preferably Cy5, in particular Sulfo-Cy5, more preferably MB660R-DBCO, optionally in combination with Sulfo-Cy5-acid, and / or - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-negative bacteria, preferably selected from bacteria belonging to the genera Escherichia, Proteus and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5-acid, MB660R-DBCO and DAPI and combinations thereof. Preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably Sulfo-Cy5, MB660R, DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Sulfo-Cy5-Acid, MB660R-DBCO and DAPI and combinations thereof; and / or the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-negative bacteria, preferably selected from bacteria belonging to the genera Escherichia and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli and Pseudomonas aeruginosa and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine-based dyes, pyrene-based dyes, coumarin-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, Alexa Fluor 350, Alexa Fluor 405, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5-acid, Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO, MB660R-DBCO and DAPI and combinations thereof; Preferably, the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, pyrene-based dyes, coumarin-based dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, Alexa Fluor 350, Alexa Fluor 405 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5-acid, Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO and MB660R-DBCO and combinations thereof. More preferably, the at least one fluorescent dye is selected from the group consisting of pyrene-based dyes, coumarin-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor 350 and Alexa Fluor 405 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO and combinations thereof. Even more preferably, said at least one fluorescent dye is a combination of Alexa Fluor 350-NHS ester and Alexa Fluor 405-DBCO; and / or - the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-positive bacteria, preferably selected from bacteria belonging to the genera Staphylococcus and Enterococcus and combinations thereof, more preferably selected from Staphylococcus aureus and Enterococcus faecalis and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably Sulfo-Cy5, MB660R, DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Sulfo-Cy5-Acid, MB660R-DBCO and DAPI and combinations thereof. Preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5, preferably Sulfo-Cy5 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Sulfo-Cy5-acid and MB660R-DBCO and combinations thereof; and / or - the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Acinetobacter, Aeromonas, Blevendimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Vibrio, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus and combinations thereof, more preferably selected from Acinetobacter baumanii, Aeromonas hydrophila, hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobactrum anthropic, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia pickettii pickettii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Bacillus subtilis, Salmonella faecalis, Micrococcus luteusand combinations thereof, and said at least one fluorescent dye is selected from the group consisting of coumarin-based dyes, cyanine-based dyes, pyrene-based dyes and xanthene-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 405 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Sulfo-Cy5-acid, Alexa Fluor™ 405-DBCO and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of (i) a mixture of MB660R and Sulfo-Cy5, preferably MB660R-DBCO and Sulfo-Cy5-acid, and (ii) Alexa Fluor™ 350 and Alexa Fluor™ 405, preferably Alexa and / or comprising a microorganism belonging to the group of heterotrophic bacteria, selected from the group consisting of a mixture of Alexa Fluor™ 350-NHS ester and Alexa Fluor™ 405-DBCO; The microorganism or group of microorganisms of interest is preferably one of the following genera: Acinetobacter, Aeromonas, Blevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, The bacteria are selected from the group consisting of bacteria belonging to the genera Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus, and combinations thereof, and more preferably selected from Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium purpura, and the like. and the at least one fluorescent dye is selected from the group consisting of acridine dyes, xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine-rotaxane-based dyes, and DAPI, and combinations thereof, and preferably selected from the group consisting of acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5-acid, sulfo-Cy5.5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa The microorganism belongs to or comprises a microorganism belonging to the group of heterotrophic bacteria, selected from the group consisting of Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI and combinations thereof.Preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5-acid, Sulfo-Cy5.5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647-acid and combinations thereof, and / or. - the microorganism or group of microorganisms of interest is preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably Candida albicans, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum, the at least one fluorescent dye is selected from the group consisting of acridine dyes, xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of acridine orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, prodan, Alexa Fluor 405, Cascade Blue, BODIPY 500 / 510, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and DAPI and their derivatives and combinations, more preferably selected from the group consisting of Acridine Orange, Alexa Fluor 350-NHS Ester, Pacific Blue-NHS Ester, Sulfo-Cy5-Acid, Prodan, Alexa Fluor 405-DBCO, Cascade Blue, BODIPY 500 / 510, Seta 650-DBCO, SeTau647-Maleimide, SeTau 488-NHS, Alexa Fluor 488-Acid, MB660R-Acid, MB660R-DBCO and DAPI and combinations thereof. Preferably, the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, coumarin-based dyes, cyanine-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Prodan, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 and SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5-acid, Prodan, Alexa Fluor™ 405-DBCO .... blue™, Seta 650-DBCO, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488-acid, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI and combinations thereof. More preferably, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably MB660R and derivatives and combinations thereof, preferably MB660R-DBCO; and / or the microorganism or group of microorganisms of interest preferably comprises one or several bacteria selected from the group of Thermoacidophilic Bacteria (TAB), Acetic Acid Bacteria (AAB), Lactic Acid Bacteria (LAB), Anaerobes, Aerobic Mesophilic Bacteria, Gram-negative and Gram-positive bacteria and combinations thereof, and preferably one or several microbacteria selected from yeasts and molds, said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 and SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, Sulfo-Cy5-acid, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488-acid, MB660R-acid and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of Sulfo-Cy5-acid and MB660R-DBCO and combinations thereof.

[0024] In some embodiments, the microorganism or group of microorganisms of interest preferably comprises one or several bacteria selected from the group of Thermoacidophilic Bacteria (TAB), Acetic Acid Bacteria (AAB), Lactic Acid Bacteria (LAB), Anaerobes, Aerobic Mesophilic Bacteria, Gram-negative and Gram-positive Bacteria, Heterotrophic Bacteria and combinations thereof, and preferably one or several microbacteria selected from yeasts and molds, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably Alexa Fluor 350, Pacific blue, Cy5, preferably Sulfo-Cy5, Alexa Fluor 405, Cascade blue™, Seta dye, preferably Seta 650 or Seta 375, SeTau dye, preferably SeTau 647 and SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, Pacific blue™-NHS ester, Sulfo-Cy5-acid, Alexa Fluor 405-DBCO, Cascade blue, Seta 650-DBCO, Seta 375-NHS, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488-acid, MB660R-acid and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of Sulfo-Cy5-acid and MB660R-DBCO and combinations thereof.

[0025] In particular, the microorganism or group of microorganisms of interest may belong to or include microorganisms belonging to the group of aerobic mesophilic bacteria (AMB), preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably selected from Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Chlamydia trachomatis ... and the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes, and DAPI, and combinations thereof, and is preferably selected from the group consisting of DRAQ5™, Alexa Fluor 50 ... Fluor™ 350, Cy5™, preferably Sulfo-Cy5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably DRAQ5™, Alexa Fluor™ 350-NHS ester, Sulfo-Cy5-acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI, and combinations thereof.

[0026] In particular, the microorganism or group of microorganisms of interest is preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, coumarin-based dyes, cyanine-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably Alexa Fluor™ 350, Cy5™, preferably sulfo-Cy5, prodan, Alexa The dye may belong to or comprise a microorganism belonging to the group of yeasts and molds, selected from the group consisting of Alexa Fluor™ 405, Seta dye, SeTau dye, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Sulfo-Cy5-acid, Prodan, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488-acid, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI and combinations thereof.

[0027] In particular, the microorganism or group of microorganisms of interest may belong to or include a microorganism belonging to the group of Gram-negative bacteria, preferably selected from bacteria belonging to the genera Escherichia and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli and Pseudomonas aeruginosa and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5 (trademark), preferably Sulfo-Cy5, MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Sulfo-Cy5-acid and MB660R-DBCO and combinations thereof.

[0028] In particular, the microorganism or group of microorganisms of interest may preferably comprise one or several bacteria selected from the group of Thermoacidophilic Bacteria (TAB), Acetic Acid Bacteria (AAB), Lactic Acid Bacteria (LAB), Anaerobes, Aerobic Mesophilic Bacteria, Gram-negative and Gram-positive bacteria and combinations thereof, and preferably one or several microbacteria selected from yeasts and molds, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Sulfo-Cy5-acid, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488-acid, MB660R-acid and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of Sulfo-Cy5-acid and MB660R-DBCO and combinations thereof.

[0029] In particular, the microorganism or group of microorganisms of interest may belong to the thermoacidophilic bacteria and said at least one fluorescent dye may comprise a dye selected from the group of xanthene-based dyes, preferably selected from MB™ 660R and derivatives thereof, more preferably MB™ 660R DBCO.

[0030] In particular, the microorganism or group of microorganisms of interest may belong to or comprise a microorganism selected from the group consisting of Aspergillus braziliensis, Candida albicans and Escherichia coli and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of Bodipy 500 / 510, Alexa Fluor 350 or derivatives thereof, preferably Alexa Fluor 350-NHS ester, Pacific blue or derivatives thereof, preferably Pacific blue-NHS ester, Prodan, Alexa Fluor 405 or derivatives thereof, preferably Alexa Fluor 405-DBCO, Cascade Blue, Seta 650 or derivatives thereof, preferably Seta 650-DBCO, Setau 488 or derivatives thereof, preferably Setau 488-NHS, Setau 647 or derivatives thereof, preferably Setau 647-maleimide and any combination thereof.

[0031] In embodiments where the microorganisms of the sample are concentrated on a membrane filter prior to step a) or step a2), the membrane filter may be removed from the solid growth medium after step b) and prior to step c) and the microcolonies thereon may be detected and / or enumerated in step c), or the membrane filter may be maintained on the solid growth medium to perform step c).

[0032] Preferably, in step c) a fluorescence microscope or a solid phase cytometer is used to detect and / or enumerate microcolonies emitting a fluorescent signal corresponding to said at least one fluorescent dye.

[0033] In another aspect, the present invention also relates to the use of the method of the present invention in determining whether a sample contains at least one microorganism of interest, in determining whether a sample is sterile or not, or in determining the degree of biological contamination of a sample.

[0034] In another aspect, the present invention also relates to the use of a kit for detecting and / or enumerating a microorganism or group of microorganisms of interest according to the method of the present invention, said kit comprising: (i) - at least one fluorescent dye, preferably as defined above, and optionally, - at least one membrane filter, preferably as defined above, and / or - at least one container containing a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest or materials required to obtain said at least one container, and / or - a leaflet providing instructions for using said kit, or (ii) - at least one vessel or materials necessary to obtain said at least one vessel, preferably containing at least one fluorescent dye as defined above and a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest, and Optionally, - preferably at least one membrane filter as defined above, and / or - a leaflet providing instructions for using said kit Includes. [Brief description of the drawings]

[0035] [Figure 1] A. acidoterrestris 24 hours, PVDF membrane, MB660R-DBCO 100μM, light power 100%, gain 1, exposure time 100ms [Diagram 2] E. coli 17 hours, MCE black membrane, Alexa Fluor™ 350-NHS ester 500 μM, light power 50%, gain 1, exposure time 200 ms [Diagram 3] E. coli 17 hours, PVDF membrane, Alexa Fluor™ 350-NHS ester 150 μM, light power 20%, gain 1, exposure time 200 ms [Figure 4] E. coli 17 hours, MCE black membrane, Alexa Fluor™ 405-DBCO 500μM, light power 50%, gain 1, exposure time 200ms [Diagram 5] E. coli 15 hours, PVDF membrane, MB660R-DBCO 250μM, light power 100%, gain 1, exposure time 100ms [Figure 6] E. coli 18 hours, MCE black membrane, sulfo-Cy5-acid 150 μM, light power 100%, gain 1, exposure time 400 ms [Figure 7] E. coli 18 h, PVDF membrane, sulfo-Cy5-acid 50 μM, light power 100%, gain 1, exposure time 400 ms [Figure 8] C. albicans 21 hours, PVDF membrane, MB660R-DBCO 250 μM, light power 100%, gain 1, exposure time 100 ms [Figure 9] A. brasiliensis 21 hours, PVDF membrane, MB660R-DBCO 250μM, light power 100%, gain 1, exposure time 200ms [Figure 10] Mixture C. albicans + A. braziliensis 20 h, PVDF membrane, sulfo-Cy5-acid 50 μM, light power 100%, gain 1, exposure time 400 ms [Figure 11] Mixture C. albicans + A. braziliensis 21 h, MCE black membrane, Alexa Fluor™ 405-DBCO 500 μM, light power 50%, gain 1, exposure time 200 ms [Figure 12] Mixture C. albicans + A. braziliensis 21 h, MCE black membrane, Alexa Fluor™ 350-NHS ester 500 μM, light power 50%, gain 1, exposure time 200 ms [Figure 13] Mixture C. albicans + A. braziliensis 21 h, PVDF membrane, Alexa Fluor™ 350-NHS ester 500 μM, light power 20%, gain 1, exposure time 300 ms [Figure 14] E. faecalis + C. albicans + A. braziliensis 24 hours, TSA agar plate 25°C, PVDF membrane, MB660R 100μM + Sulfo-Cy5-acid 100μM, light power 100%, gain 1, exposure time 200ms [Figure 15] Structure of fluorescent dyes. [Figure 16] Using different methods for contacting the fluorescent dye with the bacteria: Bacillus subtilis, black or white membrane, MB660R-DBCO (50 μM) and Sulfo-Cy5-acid (50 μM). [Figure 17] Detection of Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus and Alicyclobacillus hervarius, MB660R-DBCO, on a membrane placed on an agar medium or directly on the agar medium (without a membrane). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The inventors have demonstrated herein that microorganisms can be cultured on solid media in the presence of fluorescent dyes to allow early detection of microcolonies. Indeed, it has been surprisingly found that the fluorescent dyes do not show significant toxicity and therefore do not affect the growth of the microorganisms to be detected. The presence of these dyes during the growth phase of the microorganisms allows very early detection and enumeration of the microorganisms at the microcolony stage. In particular, the inventors have shown that this method can be used to rapidly detect and enumerate thermoacidophile bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophiles, yeasts, molds and bacteria of relevant importance in UTI diagnosis, as well as to assess the sterility or bioburden of a sample. The method developed by the inventors is a cost-effective method using standard equipment and reagents, does not require highly skilled specialists to implement, and provides faster and more accurate results than other methods, while having an easy workflow that can distinguish between live and dead microorganisms. The present method therefore meets the need for a rapid, sensitive, user-friendly, and cost-effective method for detecting and enumerating microorganisms in a sample of interest.

[0037] Thus, in a first aspect, the present invention provides a method for detecting or enumerating a microorganism or group of microorganisms of interest, comprising the steps of: a) contacting in a container a sample suspected of containing said microorganism or group of microorganisms with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms, and with at least one fluorescent dye; b) incubating the container under conditions and for a time sufficient to form microcolonies of the microorganism or group of microorganisms; and c) detecting or enumerating microcolonies that emit a fluorescent signal of said at least one fluorescent dye; thereby to detect or enumerate said microorganism or group of microorganisms contained in a sample.

[0038] The method of the present invention can be used to detect a microorganism or a group of microorganisms in a sample, i.e. to reveal the presence of a microorganism present in a sample, by the detection of microcolonies in step c) of the method. It can also be used to enumerate a microorganism or a group of microorganisms in a sample. In this case, it is assumed that each microcolony detected in step c) arises from an individual cell that has undergone cell division. The number of said microorganisms in a sample can therefore be determined by counting the number of microcolonies, optionally taking into account a dilution or concentration factor. The method of the present invention can also be used to identify a microorganism or a group of microorganisms. As used herein, the term "identification" does not necessarily require the determination of the genus and species of a given microcolony. The term can also refer to the classification of a microorganism in a taxonomic group (of any rank) or in a specific group (e.g. thermoacidophilic bacteria, acetic bacteria, lactic acid bacteria, yeasts or molds, aerobic mesophilic bacteria, anaerobic bacteria).

[0039] Step a) of the method of the invention comprises contacting in a container a sample suspected of containing a microorganism or a group of microorganisms with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms, and with at least one fluorescent dye.

[0040] Step a) is a1) contacting a sample suspected of containing said microorganism or group of microorganisms with at least one fluorescent dye, and a2) contacting said sample in a container with a solid growth medium containing nutrients to support the growth of said microorganism or group of microorganisms. May contain Steps a1) and a2) may be performed simultaneously or sequentially in any order.

[0041] In some embodiments, step a1) is performed before step a2). Optionally, before step a2), the microorganisms of the sample are concentrated on a membrane filter. In particular, the sample may be concentrated on a membrane filter before or after step a1), preferably after step a1). In particular, the at least one fluorescent dye may be filtered on the same membrane as the sample before or after filtration of the sample.

[0042] In certain embodiments, steps a1) and a2) are carried out simultaneously.

[0043] The method may further comprise, prior to step a), providing a sample suspected of containing the microorganism or group of microorganisms of interest.

[0044] sample The sample can be obtained from a liquid (e.g. water, fruit juice, beer, wine, biological fluids such as urine), a solid (e.g. food, medicines or cosmetics, medical devices or any solid surface) or a gas (e.g. air). Depending on the form of the product / environment to be tested, the sample can be used directly in the method of the invention or can be subjected to preliminary steps.

[0045] In particular, the sample can be a liquid sample or a liquefied sample. As used herein, the term "liquefied sample" refers to a liquid sample obtained from a solid sample. In some cases, the solid sample may be dissolved or suspended in a liquid medium by physical and / or chemical treatment. Microorganisms can also be extracted from various surfaces or devices using any method known to those skilled in the art, such as wiping, rubbing, for example, using a wipe, printing, for example, by agar contact, rinsing or immersion, or ultrasonic treatment, especially for removing biofilms (see, for example, Ismail et al., Int J Environ Res Public Health. 2013 Nov 14;10(11):6169-83, which is incorporated herein by reference).

[0046] The liquid sample may contain suspended solids. However, if necessary, the remaining suspended solids can be removed from the liquid medium using a suitable method, preferably one that minimizes loss of microorganisms, for example by low speed centrifugation or filtration using a suitable pore size. The liquid medium used to suspend the solid sample can be any suitable solvent, for example, sterile water, a buffer solution or a liquid culture medium.

[0047] Optionally, the liquid or liquefied sample may be diluted (eg, serially diluted) or concentrated prior to step a) using any suitable method, for example, centrifugation or filtration.

[0048] The sample used / analyzed in the method of the present invention can be any sample in which it is desired to determine whether it is contaminated with a microorganism.

[0049] Examples of samples include, but are not limited to, biological samples (e.g., saliva, nasopharyngeal, urinary, fecal, blood, plasma, cerebrospinal fluid or mucus samples), environmental samples (e.g., residential, commercial or industrial water, wastewater, cooling water, boiler water, ground water, recreational water, process water, water treatment unit effluent, soil or other environmental materials), medical devices or any parts thereof, food or beverages for human or animal consumption (e.g., dairy products, raw materials, drinking water, fruit juice, beer, wine, water used in the composition of a product), pharmaceuticals or cosmetics as well as ingredients of such foods, beverages, pharmaceuticals or cosmetics.

[0050] Membrane Filters In some preferred embodiments, the method may further comprise concentrating the microorganisms of the sample on a membrane filter prior to step a).

[0051] Then, in step a), this membrane is contacted with a solid growth medium and at least one fluorescent dye as described below. Alternatively, the sample may be contacted with said at least one fluorescent dye before filtration or after filtration but before contact with the solid growth medium. In particular, the sample may be mixed with said at least one fluorescent dye and then filtered on the membrane, or said at least one fluorescent dye may be filtered on the same membrane as the sample before or after filtration of the sample. In particular, the fluorescent dye may be added to the sample before filtration / concentration on the membrane, or may be filtered on the same membrane before or after filtration of the sample, preferably after filtration of the sample. A membrane filter may then be placed on the surface of said growth medium.

[0052] The passage of nutrients through the filter during incubation allows the growth of organisms in the form of microcolonies on the upper surface of the membrane.

[0053] Liquid, liquefied or gas samples may be filtered / concentrated using a sterile membrane filter suitable for retaining the microorganisms contained in the sample, usually a microfiltration membrane filter with a pore size smaller than the target microorganism. The sample may be passed through the membrane using a filter funnel and a vacuum system. Preferably, the membrane filter has a nominal pore size not larger than 0.45 μm, with a pore size between 0.22 μm and 0.45 μm. The diameter of the filter may vary depending on the device used to filter the sample and the size of the vessel containing the growth medium. For example, the vessel may be a Petri dish with a diameter of 90 mm or 55 mm, and the membrane may have a diameter of 47 mm.

[0054] The membrane filter can be made of any suitable material, for example, mixed cellulose ester (MCE), polyvinylidene fluoride (PVDF), polyester sulfone (PES), nitrocellulose, polytetrafluoroethylene, polycarbonate or nylon. Preferably, the membrane filter is made of mixed cellulose ester (MCE), polyvinylidene fluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate or nylon. More preferably, the membrane filter is made of mixed cellulose ester (MCE) or polyvinylidene fluoride (PVDF). The membrane filter can be white or colored, for example, black, and / or can be a grid membrane.

[0055] The volume of the sample to be filtered may vary depending on the nature of the sample and the expected microbial content, this volume is usually between 1 mL and 1 L and can be easily adjusted by one skilled in the art.

[0056] microorganisms The method of the invention makes it possible to detect or enumerate different microorganisms or groups of microorganisms that can grow in a culture device, in particular on a suitable solid growth medium, ie culturable microorganisms.

[0057] As used herein, the term "microorganism" refers to bacteria, archaea or microfungi. Preferably, the term refers to bacteria, yeasts (i.e. unicellular microfungi) or molds (i.e. multicellular and filamentous microfungi). In particular, the method of the present invention makes it possible to detect or enumerate viable microorganisms. As used herein, the term "viable microorganism" refers to a microorganism that is capable of growing when incubated in a suitable liquid culture medium or on a suitable solid culture medium. Indeed, in contrast to some other methods of the prior art, the method of the present invention does not detect dead microorganisms or fragments thereof, such as DNA or cell fragments, which allows a clear distinction between viable microorganisms.

[0058] As used herein, the term "group of microorganisms" refers to a group comprising at least two distinct microorganisms. The microorganisms may be selected from the group consisting of bacteria, archaea and microbes and combinations thereof, preferably selected from the group consisting of bacteria, yeasts and molds and combinations thereof. In particular, the group of microorganisms may comprise a mixture of two or more bacteria, two or more fungi or one or more bacteria and one or more fungi.

[0059] The microorganism of interest can be a pathogenic or non-pathogenic microorganism. Preferably, the microorganism is a pathogenic or non-pathogenic microorganism capable of deteriorating or spoiling a product (e.g., food, beverage, medical device, pharmaceutical or cosmetic product) or the environment (e.g., swimming pool, groundwater).

[0060] In some embodiments, the microorganism or group of microorganisms of interest are bacteria. The bacteria can be aerobic, anaerobic or facultative anaerobes and gram-negative or gram-positive.

[0061] In particular, the microorganism or group of microorganisms of interest may belong to the group of thermoacidophilic bacteria (TAB), the group of acetic acid bacteria (AAB), the group of lactic acid bacteria (LAB), the group of anaerobes, the group of aerobic mesophilic bacteria, the group of gram-negative bacteria, the group of gram-positive bacteria, the group of heterotrophic bacteria, or a combination thereof. More particularly, the microorganism or group of microorganisms of interest may belong to the group of thermoacidophilic bacteria (TAB), the group of acetic acid bacteria (AAB), the group of lactic acid bacteria (LAB), the group of anaerobes, the group of aerobic mesophilic bacteria, the group of gram-negative bacteria, or the group of gram-positive bacteria, or a combination thereof.

[0062] In one embodiment, the microorganism or group of microorganisms of interest belongs to the group of Alicyclobacillus species, also known as Thermoacidophilic Bacteria (TAB).

[0063] Alicyclobacillus species are spore-forming bacteria that can grow at high temperatures (up to 70°C) and in acidic conditions. The spores can survive normal pasteurization procedures, and as a result, these bacteria are potential spoilage organisms of fruit juice, fruit juice-related products (juice from concentrate, concentrated fruit juice, water-extracted fruit juice, dehydrated / powdered fruit juice, non-alcoholic or alcoholic beverages including fruit products such as fruit nectars and flavored waters) or syrups. Normally, TAB are not harmful, but spoilage can occur if the strain produces guaiacol, a natural phenolic chemical that has a smoky flavor and odor and causes fruit juice to spoil, or other unpleasant-smelling substances. The most common guaiacol-producing TAB is Alicyclobacillus acidoterrestris.

[0064] Preferably, the target microorganism or group of microorganisms belonging to the group of Alicyclobacillus species is selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius and Alicyclobacillus contaminans and combinations thereof.

[0065] In another embodiment, the microorganism or group of microorganisms of interest belongs to the group of acetic acid bacteria (AAB).

[0066] AAB are strictly aerobic microorganisms with catalase positive and oxidase negative, ellipsoid to rod morphology cells that can exist singly, in pairs or chains. They are also mesophilic microorganisms and their optimum growth temperature is between 25 and 30°C. The optimum pH for their growth is 5.0 to 6.5, but they can also grow at lower pH values.

[0067] These bacteria are widespread in nature and play an important role in the production of foods and beverages such as vinegar. However, the unwanted growth of AAB in other fermented beverages, such as wine, cider, beer and other soft drinks, results in an undesirable sour taste. By way of illustration, strains of the genus Gluconobacter belonging to this group are considered to be typical spoilers of soft drinks. Indeed, in these substrates, sugars and alcohols are incompletely oxidized, leading to the accumulation of organic acids, such as the production of acetic acid from ethanol or gluconic acid from glucose.

[0068] Preferably, the target microorganism or group of microorganisms belonging to the group of acetic acid bacteria (AAB) is selected from the group consisting of Acetobacter, Acidomonas, Asaea, Gluconacetobacter, Gluconobacter, Ameyamaea, Bombella, Commensalibacter, Endobacter, Granulibacter, Komagataebacter, K Omagataeibacter, Kozakia, Neoasaia, Neokomagataea, Nguyenibacter, Saccharibacter, Swaminathania, Swingsia and Tanticharoenia, and combinations thereof.

[0069] More preferably, the target microorganism or group of microorganisms belonging to the group of acetic acid bacteria (AAB) is a bacterium belonging to the genus Acetobacter, such as Acetobacter pasteurianus, Acetobacter malorum, Acetobacter cerevisiae, Acetobacter oeni, Acetobacter pomorum and Acetobacter aceti, a bacterium belonging to the genus Asaia, such as Asaia bogorensis, Asaia siamensis, Asaia krungthepensis and Asaia ranensis, lannaensis, Gluconacetobacter spp., such as Gluconacetobacter liquefaciens, Gluconacetobacter entanii, and Gluconacetobacter intermedius, Gluconobacter spp., such as Gluconobacter oxydans, and combinations thereof.

[0070] Even more preferably, the target microorganism or group of microorganisms belonging to the group of acetic acid bacteria (AAB) is selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans and Asaea thiamensis and combinations thereof.

[0071] In another embodiment, the microorganism or group of microorganisms of interest belongs to the group of lactic acid bacteria (LAB).

[0072] This group of bacteria often plays a positive role in the food industry, but they can also cause serious spoilage, especially in wine and foods with a low pH or that are vacuum-packed. Furthermore, these bacteria are sometimes considered indicators, since the conditions favorable for their growth are also favorable for Clostridium botulinum, a highly pathogenic bacterium. These bacteria form a heterogeneous group, but have in common that they are catalase-negative (some strains can produce heme-dependent catalase), non-spore-forming, strictly fermentative, facultatively aerobic and produce lactic acid as the main product of glucose fermentation.

[0073] The microorganism or group of microorganisms of interest belonging to the group of lactic acid bacteria (LAB) are preferably selected from the genus Lactobacillus (e.g. Lactobacillus casei, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus lindneri, Lactobacillus fructivorans, Lactobacillus acidophilus), the genus Leuconostoc (e.g. Leuconostoc gelidum, Leuconostoc gasicomitatum and Leuconostoc mesenteroides). mesenteroides), Pediococcus (e.g., Pediococcus damnosus, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus parvulus, Pediococcus inopinatus, Pediococcus halophilus, Pediococcus dextrinicus and Pediococcus urinaeequii), urinaeequi), Lactococcus (e.g., Lactococcus piscium), Enterococcus (e.g., Enterococcus faecalis and Enterococcus faecium), Carnobacterium (e.g., Carnobacterium divergens),divergens, Carnobacterium maltaromaticum, Streptococcus (e.g., Streptococcus lactis) and Weissella (e.g., Weissella confuse, Weissella viridescens), and combinations thereof.

[0074] More preferably, the microorganism or group of microorganisms of interest belonging to the group of lactic acid bacteria (LAB) is selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof.

[0075] Even more preferably, the target microorganism or group of microorganisms belonging to the group of lactic acid bacteria (LAB) is selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Weissella confusa and combinations thereof.

[0076] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to the group of anaerobes.

[0077] Anaerobes may be divided into three categories: obligate anaerobes, which are harmed by the presence of oxygen; aerotolerant organisms, which cannot use oxygen for growth but tolerate its presence; and facultative anaerobes, which can grow without oxygen but will use it when it is present. Most pathogenic food-borne microorganisms are facultative anaerobes. Anaerobes, especially those of the Clostridium genus, can spoil vacuum-packed or canned foods. Anaerobes, especially Cutibacterium acnes, may also be involved in post-operative infections due to their ability to survive on internal implants and surgical devices.

[0078] The microorganism or group of microorganisms of interest belonging to the group of anaerobic bacteria is preferably a bacterium belonging to the genus Clostridium, such as, for example, Clostridium sporogenes, Clostridium algidicarnis, Clostridium frigoris, Clostridium bowmanii, Clostridium frigidicarmis, Clostridium ruminantium, Clostridium estertheticum, Clostridium gasigenes, Clostridium perfringens, Clostridium perfringens or Clostridium botulinum, Cutibacterium, for example, Cutibacterium acnes, and Brochothrix, for example, Brochothrix thermosphacta, and combinations thereof.

[0079] More preferably, the target microorganism or group of microorganisms belonging to the group of anaerobic bacteria is selected from bacteria belonging to the genera Clostridium or Cutibacterium and combinations thereof, even more preferably from the group consisting of Clostridium sporogenes and Cutibacterium acnes and combinations thereof.

[0080] In another embodiment, the microorganism or group of microorganisms of interest belongs to the group of aerobic mesophilic bacteria (AMB). Aerobic mesophilic bacteria are bacteria that grow aerobically at mesophilic temperatures, i.e., between 25° C. and 40° C. The detection and enumeration of aerobic mesophilic bacteria is an important indicator for the quality control of food, cosmetics, medical devices and non-sterile pharmaceutical preparations.

[0081] The microorganism or group of microorganisms of interest belonging to the group of aerobic mesophilic bacteria is preferably selected from the genus Methylobacterium (e.g. Methylobacterium extroquens), Pseudomonas (e.g. Pseudomonas aeruginosa), Bacillus (e.g. Bacillus subtilis, Bacillus cereus), Escherichia (e.g. Escherichia coli), Staphylococcus (e.g. Staphylococcus aureus), Acinetobacter (e.g. Acinetobacter baumannii), Cronobacter (e.g. Cronobacter sakazakii), Klebsiella (e.g. Klebsiella pneumoniae), Salmonella (e.g. Salmonella typhimurium), Enterococcus (e.g. Enterococcus faecalis), Listeria (e.g. Listeria grey, Listeria monocytogenes), monocytogenes), Shigella (e.g., Clostridium sonnei), Vibrio (e.g., Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus), Campylobacter (e.g., Campylobacter jejuni, Campylobacter coli), Yersinia (e.g., Yersinia enterocolitica), Kocuria (e.g., Kocuria rhizophila) and Burkholderia (e.g., Burkholderia cepacia), and combinations thereof.

[0082] More preferably, the target microorganism or group of microorganisms belonging to the group of aerobic mesophilic bacteria is selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria, Burkholderia, and combinations thereof. The target microorganism or group of microorganisms belonging to the group of aerobic mesophilic bacteria may more preferably be selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria, Burkholderia, and combinations thereof.

[0083] Even more preferably, the target microorganism or group of microorganisms belonging to the group of aerobic mesophilic bacteria is selected from the group consisting of Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Listeria greyi, Salmonella sonnei, Kocuria rhizophila and Burkholderia cepacia, and combinations thereof. Even more preferably, the target microorganism or group of microorganisms belonging to the group of aerobic mesophilic bacteria is selected from the group consisting of Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila and Burkholderia cepacia, and combinations thereof.

[0084] In another embodiment, the microorganism or group of microorganisms of interest belongs to the group of Gram-negative or Gram-positive bacteria.

[0085] Gram-negative bacteria are characterized by a cell envelope composed of a thin peptidoglycan cell wall sandwiched between the cytoplasmic inner membrane and the bacterial outer membrane. They represent a significant medical challenge because their outer membrane protects them from numerous antibiotics (including penicillin), detergents that would normally damage the peptidoglycan of the (inner) cell membrane, and lysozyme, an antibacterial enzyme produced by animals that forms part of the innate immune system. Furthermore, the outer leaflet of this membrane contains complex lipopolysaccharides (LPS), whose lipid A component can cause a toxic reaction when these bacteria are lysed by immune cells. They are involved, for example, in most cases of urinary tract infections. Although Escherichia coli is the most common Gram-negative pathogen, other Gram-negative bacteria such as Klebsiella, Chlamydia, Proteus, Pseudomonas, Enterobacter, Acinetobacter, Serratia, Haemophilus, Yersinia, and Salmonella are of major importance.

[0086] The microorganism or group of microorganisms of interest belonging to the group of Gram-negative bacteria is preferably selected from the group consisting of Escherichia (e.g. Escherichia coli), Klebsiella (e.g. Klebsiella pneumoniae), Proteus (e.g. Proteus mirabilis), Chlamydia (e.g. Chlamydia trachomatis), Pseudomonas (e.g. Pseudomonas aeruginosa), Enterobacter (e.g. Enterobacter faecalis), faecalis) and Enterobacter aerogenes), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Serratia (e.g., Serratia marcescens), Haemophilus (e.g., Haemophilus influenzae) and Salmonella (e.g., Salmonella typhimurium), and combinations thereof. More preferably, the microorganism or group of microorganisms of interest belonging to the group of Gram-negative bacteria are selected from the group consisting of Escherichia (e.g. Escherichia coli), Klebsiella (e.g. Klebsiella pneumoniae), Proteus (e.g. Proteus mirabilis), Pseudomonas (e.g. Pseudomonas aeruginosa), Enterobacter (e.g. Enterobacter faecalis and Enterobacter aerogenes), Acinetobacter (e.g. Acinetobacter baumannii), Cronobacter (e.g. Cronobacter sakazakii), Shigella (e.g. Clostridium sonnei), , Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Serratia (e.g., Serratia marcescens) and Salmonella (e.g., Salmonella typhimurium) and combinations thereof, in particular selected from Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter baumannii, Cronobacter sakazakii, Salmonella sonnei, Kocuria rhizophila, Burkholderia cepacia, Serratia marcescens and Salmonella typhimurium.

[0087] More particularly, the target microorganism or group of microorganisms belonging to the group of Gram-negative bacteria may be selected from bacteria of the genera Escherichia, Proteus and Pseudomonas and combinations thereof, more preferably from Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and combinations thereof. Even more particularly, the target microorganism or group of microorganisms belonging to the group of Gram-negative bacteria may be selected from bacteria of the genera Escherichia and Pseudomonas and combinations thereof, more preferably from Escherichia coli, Pseudomonas aeruginosa and combinations thereof, even more preferably from Escherichia coli bacteria.

[0088] In particular, the microorganism or group of microorganisms of interest belonging to the group of Gram-negative bacteria may belong to the genera Escherichia, Klebsiella, Enterobacter and Salmonella and combinations thereof, more particularly to the family Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes and Salmonella typhimurium and combinations thereof.

[0089] Gram-positive bacteria are also clinically relevant. Among the Gram-positive bacteria isolated in bacterial UTIs, Staphylococcus saprophyticus, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus agalactiae are the most common Gram-positive pathogens.

[0090] The target microorganism or group of microorganisms belonging to the group of Gram-positive bacteria is preferably selected from bacteria belonging to the genera Staphylococcus, Enterococcus, Streptococcus and combinations thereof. More particularly, the target microorganism or group of microorganisms belonging to the group of Gram-positive bacteria may be selected from Staphylococcus aureus and Enterococcus faecalis and combinations thereof.

[0091] In another embodiment, the microorganism or group of microorganisms of interest belongs to the group of heterotrophic bacteria, in particular heterotrophic bacteria that are usually recoverable from pharmaceutical water systems, i.e. tap water (or drinking water), purified water, WFI (water for injection) (see, for example, Tim Sandle, September 2015. SOJ Microbiology & Infectious Diseases 3(2):1-8, incorporated herein by reference). Indeed, water is an important part of the pharmaceutical industry. It is used for cleaning, as a component of aqueous sterile and non-sterile formulations, for hand washing, as steam supply to autoclaves, among other uses. Due to its importance in pharmaceutical production, the microbial control of water is very important. As water is always present, each grade of pharmaceutical water is a potential source of microbial contamination, especially if not properly managed. In pharmaceutical products, there are three types of water: a) tap water (or drinking water), b) purified water, and c) WFI (water for injection). Each of these is a different grade, with increasing likelihood of microbial control going down the list (i.e., stricter restrictions apply to water for injection than to tap water). Tap water is supplied by a public utility company and is of "potable water" (drinking) quality. Results from monitoring the water system are assessed in terms of heterotrophic microbial counts against predefined alert and action levels.

[0092] The target microorganism or group of microorganisms belonging to the group of heterotrophic bacteria are preferably selected from the group consisting of Acinetobacter (e.g. Acinetobacter baumannii), Aeromonas (e.g. Aeromonas hydrophila), Brevundimonas (e.g. Brevundimonas diminuta), Burkholderia (e.g. Burkholderia cepacia), Citrobacter (e.g. Citrobacter freundii), Edwardsiella (e.g. Edwardsiella cepacia), (e.g., Enterobacter aerogenes), Escherichia (e.g., Escherichia coli), Ochrobacterium (e.g., Ochrobacterium anthropicus), Klebsiella (e.g., Klebsiella pneumoniae), Methylobacterium (e.g., Methylobacterium extroquens), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea aggrecanase), romerans), Proteus (e.g., Proteus mirabilis), Pseudomonas (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens), Ralstonia (e.g., Ralstonia picketii), Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), Shigella (e.g., Salmonella sonnei), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., The bacteria are selected from bacteria belonging to the genus Monas (e.g., Stenotrophomonas maltophilia), Vibrio (e.g., Vibrio parahaemolyticus), Yersinia (e.g., Yersinia enterocolitica), Bacillus (e.g., Bacillus subtilis), Enterococcus (e.g., Enterococcus faecalis), Micrococcus (e.g., Staphylococcus aureus), Staphylococcus aureus, and combinations thereof.More preferably, the target microorganism or group of microorganisms belonging to the group of heterotrophic bacteria is selected from the group consisting of Acinetobacter (e.g. Acinetobacter baumannii), Aeromonas (e.g. Aeromonas hydrophila), Brevundimonas (e.g. Brevundimonas diminuta), Burkholderia (e.g. Burkholderia cepacia), Citrobacter (e.g. Citrobacter freundii), Edwardsiella (e.g. (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Escherichia (e.g., Escherichia coli), Ochrobacterium (e.g., Ochrobacterium anthropicus), Klebsiella (e.g., Klebsiella pneumoniae), Methylobacterium (e.g., Methylobacterium extroquens), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., For example, the bacteria may be selected from the group consisting of bacteria belonging to the genus Pantoea agglomerans, Proteus (e.g., Proteus mirabilis), Pseudomonas (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens), Ralstonia (e.g., Ralstonia picketii), Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), Shigella (e.g., Salmonella sonnei), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Yersinia (e.g., Yersinia enterocolitica), Bacillus (e.g., Bacillus subtilis), Enterococcus (e.g., Enterococcus faecalis), Micrococcus (e.g., Staphylococcus aureus), and combinations thereof.

[0093] More particularly, the target microorganism or group of microorganisms belonging to the group of heterotrophic bacteria are preferably selected from the group consisting of Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, The bacterium is selected from Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Pseudomonas sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus, and combinations thereof. More particularly, the target microorganism or group of microorganisms belonging to the group of heterotrophic bacteria are Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloense, The bacterium may be selected from the group consisting of Bacillus subtilis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus, and combinations thereof.

[0094] In particular, the microorganism or group of microorganisms of interest are selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), the genus Gluconobacter (e.g., Gluconobacter lig ... oxidans), Asaia spp. (e.g., Asaia thiamensis), Lactobacillus spp. (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella spp. (e.g., Weissella confusa), Clostridium spp. (e.g., Clostridium sporogenes), Cutibacterium spp. (e.g., Cutibacterium acnes), Methylobacterium spp. (e.g., Methylobacterium extroquens), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Bacillus spp. (e.g., Bacillus subtilis), Escherichia spp. (e.g., Escherichia coli ), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Micrococcus (also named Kocuria) (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), E. coli, and Escherichia coli. Monas (e.g., Aeromonas hydrophila), Brevundimonas (e.g., Brevundimonas diminuta), Citrobacter (e.g., Citrobacter freundii), Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Ochrobacterium (e.g., Ochrobacterium anthropicus), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea agglomerans), Proteus (e.g., Proteus mirabilis),The microorganism or group of microorganisms of interest may be selected from the group consisting of bacteria belonging to the genus Ralstonia (e.g., Ralstonia picketii), Serratia (e.g., Serratia marcescens), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Vibrio (e.g., Vibrio parahaemolyticus), Yersinia (e.g., Yersinia enterocolitica), and combinations thereof. More particularly, the microorganism or group of microorganisms of interest may be selected from the group consisting of bacteria belonging to the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), Acetobacter (e.g., Acetobacter aceti), Gluconoa, and the like. Setobacter (e.g., Gluconoacetobacter liquefaciens), Gluconobacter (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., , Cutibacterium acnes), Methylobacterium spp. (e.g., Methylobacterium extroquens), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Bacillus spp. (e.g., Bacillus subtilis), Escherichia spp. (e.g., Escherichia coli), Staphylococcus spp. (e.g., Staphylococcus aureus), Acinetobacter spp. (e.g., Acinetobacter baumannii), Cronobacter spp. (e.g., Cronobacter sakazakii), Klebsiella spp. (e.g., Bacillus), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Shigella (e.g., Salmonella sonnei), Micrococcus (also named Kocuria) (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Aeromonas (e.g., Aeromonas hydrophila), Brevundimonas (e.g., Brevundimonas diminuta), Citrobacter (e.g.,The bacteria may be selected from the group consisting of bacteria belonging to the genus Citrobacter freundii, Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Ochrobacterium (e.g., Ochrobacterium anthropicus), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea agglomerans), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia picketii), Serratia (e.g., Serratia marcescens), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Yersinia (e.g., Yersinia enterocolitica), and combinations thereof.

[0095] More particularly, the microorganism or group of microorganisms of interest may be Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confuse, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Bacillus subtilis ... The bacterium may be selected from the group consisting of Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Listeria greyi, Salmonella sonnei, Kocuria rhizophila and Bordetella cepacia, Aeromonas hydrophila, Brevundimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropic, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, Serratia marcescens, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, and combinations thereof.More particularly, the microorganism or group of microorganisms of interest are Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, The bacterial strain may be selected from the group consisting of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila and Bordetella cepacia, Aeromonas hydrophila, Brevundimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropic, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, Serratia marcescens, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, and combinations thereof.

[0096] Alternatively, the microorganism or group of microorganisms of interest may be a member of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), Acetobacter (e.g., Acetobacter aceti), Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), Gluconobacter (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporum), rogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter The bacterium may be selected from the group consisting of bacteria belonging to the genus Chlamydia trachomatis (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), and combinations thereof.The microorganism or group of microorganisms of interest may also be selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus conminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), the genus Gluconobacter (e.g., Gluconobacter oxydans), the genus Asaia (e.g., Asaia thiamensis), the genus Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), the genus Weissella (e.g., Weissella confusa), the genus Clostridium (e.g., Clostridium taurate), the genus Clostridium spp. (e.g., Clostridium niger ... (e.g., Rostridium sporogenes), Cutibacterium spp. (e.g., Cutibacterium acnes), Methylobacterium spp. (e.g., Methylobacterium extroquens), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Bacillus spp. (e.g., Bacillus subtilis), Escherichia spp. (e.g., Escherichia coli), Staphylococcus spp. (e.g., Staphylococcus aureus), Acinetobacter spp. (e.g., baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), and combinations thereof.More particularly, the microorganism or group of microorganisms of interest are Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaea thiamensis, The bacterium may be selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Listeria greyi, Enterobacter sonnei, Kocuria rhizophila, and Burkholderia cepacia, and combinations thereof. The microorganism or group of microorganisms of interest may also be Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaea siamensis ... The bacterium may be selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Enterobacter sonnei, Kocuria rhizophila, and Bordetella cepacia, and combinations thereof.

[0097] In some other embodiments, the microorganism or group of microorganisms of interest is a microfungus, in particular a yeast or mold. Yeasts and molds can cause spoilage of various products such as food, wine, beverages, cosmetics or pharmaceutical preparations. In particular, they can invade and grow in any type of food at virtually any time, they invade crops such as grains, nuts, beans and fruits in the field before harvest and during storage. They also grow on processed foods, food mixtures and are important spoilage organisms in dairy products, particularly in fermented dairy foods such as cheese and low moisture products. It is estimated that 5-10% of food is wasted and must be disposed of due to fungal contamination. Preferably, the fungi to be detected or enumerated by the method of the present invention belong to the phylum Ascomycota, more preferably to the classes Saccharomycetes or Deuteromycetes.

[0098] In one embodiment, the microorganism or group of microorganisms of interest is selected from the group consisting of fungi belonging to the genera Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces bailii), Aspergillus (e.g., Aspergillus brasiliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium varioti, Penicillium chrysogenum), and combinations thereof. Preferably, the microorganism or group of microorganisms of interest is selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Diotrichum candidum, Penicillium variotii and Penicillium chrysogenum and combinations thereof.

[0099] In particular, the microorganism or group of microorganisms of interest are selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), the genus Gluconobacter (e.g., Gluconobacter bufense ... ter oxydans), Asaia spp. (e.g., Asaia thiamensis), Lactobacillus spp. (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella spp. (e.g., Weissella confusa), Clostridium spp. (e.g., Clostridium sporogenes), Cutibacterium spp. (e.g., Cutibacterium acnes), Methylobacterium spp. (e.g., Methylobacterium extroquens), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Bacillus spp. (e.g., Bacillus subtilis), Escherichia spp. (e.g., Escherichia coli) , Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Aeromonas (e.g., Aeromonas hydrophila), Brevibacterium cepacia, Bundimonas (e.g., Brevundimonas diminuta), Citrobacter (e.g., Citrobacter freundii), Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Ochrobacterium (e.g., Ochrobacterium anthropicus), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea agglomerans), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia picketii),The fungus may be selected from the group consisting of bacteria belonging to the genus Serratia (e.g., Serratia marcescens), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Vibrio (e.g., Vibrio parahaemolyticus), and Yersinia (e.g., Yersinia enterocolitica), and fungi belonging to the genus Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces bailii), Aspergillus (e.g., Aspergillus braziliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium varioti, Penicillium chrysogenum), and combinations thereof. The microorganism or group of microorganisms of interest may also be selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), the genus Gluconobacter (e.g., Gluconobacter oxydans), the genus Asaia (e.g., Asaia thiamensis), the genus Lactobacillus (e.g., Lactobacillus casei, Lactobacillus planiculosus ... thalum), Weissella (e.g., Weissella confuse), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis),Shigella (e.g., Shigella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Aeromonas (e.g., Aeromonas hydrophila), Brevundimonas (e.g., Brevundimonas diminuta), Citrobacter (e.g., Citrobacter freundii), Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Ochrobacterium (e.g., Ochrobacterium anthropicus), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea agglomerans), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia picketii) ), Serratia (e.g., Serratia marcescens), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Yersinia (e.g., Yersinia enterocolitica), and fungi of the genus Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces bailii), Aspergillus (e.g., Aspergillus braziliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium variotii, Penicillium chrysogenum), and combinations thereof.

[0100] More particularly, the microorganism or group of microorganisms of interest are Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaea thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Faecalis Listeria grayi, Lactobacillus sonnei, Kochlia rhizophila, Burkholderia cepacia, Aeromonas hydrophila, Brevundimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropicum, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, Serratia marcescens sensu, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii and Penicillium chrysogenum, and combinations thereof.The microorganism or group of microorganisms of interest may also be Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confuse, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium ... The bacterial strain may be selected from the group consisting of Enterobacter pneumoniae, Enterobacter faecalis, Enterobacter sonnei, Kocuria rhizophila, Bordetella cepacia, Aeromonas hydrophila, Blevendimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropic, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, Serratia marcescens, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Candida albicans, Zygosaccharomyces bailly, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, and Penicillium chrysogenum, and combinations thereof.

[0101] Alternatively, the microorganism or group of microorganisms of interest may be a member of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), Acetobacter (e.g., Acetobacter aceti), Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), Gluconoacetobacter niger, ... Bacter (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Bacillus subtilis), The fungus may be selected from the group consisting of bacteria belonging to the genus Holderia (e.g., Burkholderia cepacia), and fungi belonging to the genus Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces bailii), Aspergillus (e.g., Aspergillus brasiliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium varioti, Penicillium chrysogenum), and combinations thereof.The microorganism or group of microorganisms of interest may also be a member of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), Acetobacter (e.g., Acetobacter aceti), Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), Gluconobacter (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), and fungi belonging to the genera Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces baylii), Aspergillus (e.g., Aspergillus brasiliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium varioti, Penicillium chrysogenum), and combinations thereof.More particularly, the microorganism or group of microorganisms of interest may be Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporum ... rogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterococcus faecalis, Listeria greyi, Propionibacterium sonnei, Kocuria rhizophila, Burkholderia cepacia, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii and Penicillium chrysogenum, and combinations thereof.The microorganism or group of microorganisms of interest may also be Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium truncatula ... sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Enterobacter sonnei, Kocuria rhizophila, Burkholderia cepacia, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii and Penicillium chrysogenum, and combinations thereof.

[0102] In a preferred embodiment, the methods of the invention are used to detect or enumerate populations of microorganisms of interest. In particular, the group of microorganisms is - two or more microorganisms selected from the group of thermoacidophilic bacteria (TAB) as defined above, i.e. belonging to the genus Alicyclobacillus, preferably two or more microorganisms selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius and Alicyclobacillus contaminans and combinations thereof, or - two or more microorganisms selected from the group of acetic acid bacteria (AAB) as defined above, preferably selected from bacteria belonging to the genera Acetobacter, Acidomonas, Asaia, Gluconacetobacter, Gluconobacter, Ameyamaea, Bombella, Commensalibacter, Endobacter, Granulibacter, Komagataebacter, Kozakia, Neoasiaia, Neokomagataea, Nguenibacter, Saccharibacter, Swaminatania, Swingsia and Tanticaloenia and combinations thereof, more preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter and Asaia and combinations thereof, even more preferably selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans and Asaia thiamensis and combinations thereof, or - two or more microorganisms selected from the group of lactic acid bacteria (LAB) as defined above, preferably selected from bacteria belonging to the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Carnobacterium, Streptococcus and Weissella and combinations thereof, more preferably selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof, even more preferably selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum and Weissella confusa and combinations thereof, or - two or more microorganisms selected from the group of anaerobic bacteria, preferably selected from bacteria of the genera Clostridium and Cutibacterium and combinations thereof, more preferably selected from the group consisting of Clostridium sporogenes and Cutibacterium acnes and combinations thereof, or - selected from the group of aerobic mesophilic bacteria (AMB) as defined above, preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably selected from Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Clostridium perfringens ... selected from the group consisting of Novabacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Listeria greyi, Listeria sonnei, Kocuria rhizophila and Burkholderia cepacia and combinations thereof, even more preferably two or more microorganisms selected from the group consisting of Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Listeria sonnei, Kocuria rhizophila and Burkholderia cepacia and combinations thereof, or - two or more microorganisms selected from the group of gram-negative bacteria as defined above, preferably selected from bacteria belonging to the genera Escherichia, Proteus and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa and combinations thereof, even more preferably selected from bacteria belonging to the genera Escherichia and Pseudomonas and combinations thereof, in particular selected from Escherichia coli and Pseudomonas aeruginosa and combinations thereof, or - two or more microorganisms selected from the group of Gram-positive bacteria as defined above, preferably selected from bacteria belonging to the genera Staphylococcus, Enterococcus, Streptococcus and combinations thereof, more preferably selected from Staphylococcus aureus and Enterococcus faecalis and combinations thereof, or - selected from the group of bacteria of heterotrophic bacteria as defined above, preferably from the genera Acinetobacter, Aeromonas, Blevendimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Vibrio, Yersinia, Bacillus, Enterococcus The bacteria are selected from the group consisting of bacteria belonging to the genera Acinetobacter baumannii, Aeromonas hydrophila, Blevendimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Bacillus sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus and combinations thereof, and even more preferably, Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Bacillus cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropicus, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Mycobacterium sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Mycobacterium faecalis, Mycobacterium luteus (Kocuria rhizophila),two or more microorganisms selected from Staphylococcus aureus and combinations thereof; or - two or more microorganisms selected from the group as defined above of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria, heterotrophic bacteria and combinations thereof, preferably selected from the group as defined above of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria and combinations thereof, or - two or more microorganisms selected from yeasts and molds as defined above, preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum and combinations thereof, or - two or more microorganisms selected from the group as defined above of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria, heterotrophic bacteria, yeasts and molds and combinations thereof, preferably selected from the group as defined above of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria, yeasts and molds and combinations thereof may include.

[0103] In particular, the group of microorganisms to be detected or enumerated by the method of the invention is - one or several bacteria, preferably selected from the group as defined above: thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria, heterotrophic bacteria and combinations thereof, - preferably one or several microfungi selected from yeasts and molds as defined above may include.

[0104] Preferably, the group of microorganisms to be detected or enumerated by the method of the invention is - one or several bacteria, preferably selected from the group as defined above: Thermoacidophilic Bacteria (TAB), Acetic Acid Bacteria (AAB), Lactic Acid Bacteria (LAB), Anaerobes, Aerobic Mesophilic Bacteria, Gram-negative Bacteria and Gram-positive Bacteria and combinations thereof, - preferably one or several microfungi selected from yeasts and molds as defined above may include.

[0105] The microorganism or group of microorganisms to be detected or enumerated by the method of the invention may vary depending on the nature of the sample.

[0106] for example: - samples obtained from fruit juice, fruit juice-related products or syrups may be suspected of containing thermoacidophilic bacteria; - samples obtained from vacuum-packed or canned foods may be suspected of containing anaerobic bacteria; - samples obtained from medicinal raw materials may be suspected of containing mesophilic aerobic bacteria, especially from the genus Bacillus, anaerobic bacteria, especially from the genus Clostridium, or fungi, such as fungi of the genera Penicillium and Aspergillus, - samples obtained from cosmetic products may be suspected of containing aerobic mesophilic bacteria, in particular Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Bacillus species; and - the sample obtained from the medical device or urine (i.e. for the diagnosis of UTI) may be suspected of containing Gram-negative bacteria, in particular Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis or Klebsiella pneumoniae, or Gram-positive bacteria, in particular Staphylococcus aureus or Enterococcus faecalis, and - Samples obtained from pharmaceutical water systems were tested for the following pathogens: Acinetobacter, Aeromonas, Blevendimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Vibrio, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus and combinations thereof, preferably selected from bacteria belonging to the genus Acinetobacter, Aeromonas, Brevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus ... More preferably, the bacteria selected from the group consisting of Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia pithivum, ketii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Bacillus subtilis, Salmonella faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus and combinations thereof, and even more preferably, Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli,may be suspected of containing heterotrophic bacteria as defined above, selected from Ochrobacterium anthropicum, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus, and combinations thereof;

[0107] For each field in which the method of the invention can be used (e.g. medicine, cosmetics, food and beverages, environmental analysis, diagnostics, etc.), the skilled artisan will know which microorganisms may be suspected to be contained in the sample and will therefore be able to adapt the method of the invention to these microorganisms.

[0108] Growth medium In step a) of the method of the invention, the sample is contacted in a vessel with a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest.

[0109] As used herein, the term "growth medium" or "culture medium" refers within the context of the present invention to a nutrient medium used for the growth of a microorganism or a group of microorganisms. A growth medium can be a defined medium, synthesized from individual chemicals and therefore of known exact molecular composition, or an undefined medium, containing several complex components consisting of a mixture of many chemical species in unknown proportions, e.g., yeast extract or casein hydrolysate.

[0110] The growth medium used in the method of the present invention can be a non-selective growth medium or a selective growth medium. A non-selective growth medium is a common medium for bacterial growth, such as tryptone soy agar (TSA) for fungal growth, such as Sabouraud dextrose agar (SDA) or for both bacterial and fungal growth, such as Reasoner's 2A agar (R2A). A non-selective medium usually contains the nutrients necessary to support the growth of a wide variety of microorganisms. A selective growth medium is a medium used for the growth of only selected microorganisms. In fact, the composition of a selective medium ensures the growth of cells that have certain characteristics, such as antibiotic resistance or the ability to synthesize certain metabolic products, such as amino acids. This medium thus provides an environment that favors the growth of target microorganisms over non-target microorganisms that may be present in the sample.

[0111] The expressions "solid growth medium" or "solid culture medium", as used herein, refer to a growth medium that allows microorganisms to form microcolonies on its surface, e.g., a medium that has a gel-like appearance or is in the form of a gel, a gel being a colloidal system in which a porous network of interconnected particles spans the volume of a liquid medium, allowing nutrients to diffuse through the medium and become available to the microorganisms. Preferably, a solid growth medium, as used herein, is prepared by adding to a liquid growth medium a sufficient amount of a gelling agent, e.g., agar, agarose, alginate, carrageenan, cellulose, gelatin, pectin and combinations thereof. Usually, a solid growth medium contains a gelling agent, preferably agar, at a concentration of 0.5% to 3%, preferably 1% to 2.5%. Preferably, the solid growth medium used in the method of the present invention is an agar growth medium.

[0112] The solid growth medium is poured into a container, which can be any sterile container adapted for microbial culture, in particular a conventional Petri plate.

[0113] The choice of growth medium essentially depends on the microorganism or group of microorganisms of interest. However, this choice may also depend on the field of application (e.g. pharmaceuticals, cosmetics, food and beverages, environmental analysis, diagnostics, etc.) and on the standard methods used in this field. By way of example, IFU Microbiological Method 2 recommends Orange Serum Agar (OSA) medium for the simultaneous detection of yeasts, molds, lactic acid bacteria and acetic bacteria from samples obtained from non-alcoholic beverages. On the other hand, R2A or TSA medium is recommended for bioburden testing in the pharmaceutical industry. Similarly, some applications require selective media to detect only certain microorganisms or groups of certain microorganisms (e.g. MacConkey agar for the analysis of urine samples to diagnose UTI caused by Gram-negative bacteria), while other applications, e.g. sterility tests or bioburden tests, usually require non-selective media to detect or enumerate as many microorganisms as possible. Of course, as explained below, the conditions and incubation times also play a role in the selectivity or non-selectivity of the method. Those skilled in the art can readily consider these parameters to select an appropriate growth medium.

[0114] Examples of growth media suitable for supporting the growth of thermoacidophilic bacteria include, but are not limited to, YSG (yeast-starch-glucose) agar (Matsubara et al. 2002, Int J Syst Evol Microbiol 52, 1681-1685), PDA (potato dextrose agar; Pettipher et al. 1997, Lett Appl Microbiol 24, 185-189), OSA (orange serum agar; Pettipher et al. 2000, Food Austr 57, 293-295), K agar (Walls et al. 1998, Dairy Food Environ Sanit 18, 499-503), and BAT (Bacillus acidoterrestris) agar. Preferably, the growth medium used to support the growth of thermoacidophilic bacteria is a growth medium recommended for the detection of thermoacidophilic bacteria according to IFU Standard Method No. 12.

[0115] Examples of growth media suitable for supporting the growth of lactic acid bacteria include, but are not limited to, Rogosa agar (Rogosa et al. 1951, J Bacteriol 62, 132-133), MRS (De Man, Rogosa and Sharpe) agar (De Man et al. 1960, J Appl Bacteriol 23, 30-35), Lactobacilli selective agar (Mitsuoka, 1978, Intestinal Bacteria and Health. Tokyo, Harcourt Brace Jovanovich Japan), LAMVAB (Hartemink et al. 1997, J Microbiol Methods 29, 77-84), TSA-BCP and PCA-BCP (Trypticase Soy Agar and Plate Count Agar with BromoCresol Purple; Lin et al. 2006, Food Microbiol 23, 30-35), and lactic acid bacteria selective agar (Mitsuoka, 1978, Intestinal Bacteria and Health. Tokyo, Harcourt Brace Jovanovich Japan). 74-81) or Raka Ray agar. Preferably, the growth medium used to support the growth of lactic acid bacteria is a growth medium recommended for the detection of lactic acid bacteria according to IFU standard method no. 5.

[0116] Examples of growth media suitable for supporting the growth of yeast or mold include, but are not limited to, YM (yeast extract malt extract) and YMG (yeast extract malt extract glucose) agar (Wickerham, 1939 J. Tropical Med. Hyg., 42, 176), OSA (orange serum agar), PDA (potato dextrose agar), tomato juice agar (MacFaddin JF, 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams & Wilkins, Baltimore, Md), MEA (malt extract agar) medium, glucose peptone agar (Glathe et al., Zentralbl Bakteriol Parasitenkd Infektionskr Hyg. 1968;122(1):3-21), Wallerstein nutrient agar (Green and Gray. 1950. Wallerstein Lab. Commun. 12:43), Wort agar (Parfitt EH (1933) J. Dairy Sci. 16. 141-147) and Sabouraud dextrose agar (Sabouraud, R. (1892) Ann. Dermatol. Syphilol. 3:1061). In particular, YM and YMG agar media are selective growth media with a low pH useful for culturing yeasts, molds or other acid-tolerant or acidophilic organisms (i.e. capable of growing in an acidic natural environment having a pH in the range of 3-4) while preventing the growth of most bacteria and other acid-intolerant organisms. Preferably, the growth medium used to support the growth of yeasts or molds is a growth medium recommended for the detection of yeasts or molds according to the USP61 standard, the BS EN ISO 16212-2017 standard or the IFU standard method numbers 3 and 4.

[0117] Examples of growth media suitable for supporting the growth of acetic acid bacteria include, but are not limited to, YM (yeast extract malt extract) agar, YMG (yeast extract malt extract glucose) agar, GYC (glucose yeast extract calcium carbonate, 10% glucose, 1.0% yeast extract, 2.0% calcium carbonate, 1.5% agar, pH 6.8) agar, YPE (yeast extract-peptone-ethanol) agar, YPG (yeast extract polypeptone glycerol), YPM / YPS (yeast extract polypeptone mannitol or sorbitol) agar, and YPGD (yeast extract polypeptone glucose) agar.

[0118] Examples of growth media suitable for supporting the growth of aerobic mesophilic bacteria include, but are not limited to, TSA (tryptone soy agar), PCA (plate count agar), R2A (Reasoner's 2A agar), SCDA (soybean casein digest agar), and FTM (fluid thioglycollate medium) agar. In particular, TSA and PCA media are non-selective growth media often used to evaluate the viable bacterial growth of a sample, and R2A medium is a culture medium adapted for the growth of bacteria normally present in drinking water. Preferably, the growth medium used to support the growth of aerobic mesophilic bacteria is a growth medium recommended for the detection of aerobic mesophilic bacteria according to the USP61 standard or the BS EN ISO 21149-2017 standard.

[0119] Examples of growth media suitable for supporting the growth of anaerobic bacteria include, but are not limited to, TSA (tryptone soy agar) medium, enriched clostridial agar (Barnes et al. (1963) J. Appl. Bact. 26. 415-427), anaerobic agar (Brewer JH, 1942, Science, 95:587), and Columbia agar (Ellner et al. Amer. J. Clin. Path., 29; 181-183 (1958)), optionally supplemented with defibrinated sheep blood. In some embodiments, the growth medium used to support the growth of anaerobic bacteria may be a growth medium recommended for the detection of anaerobic bacteria according to ISO 7937:2004 or ISO 14189:2013 standards, particularly for the detection of Clostridium perfringens.

[0120] Examples of growth media suitable for supporting the growth of gram-positive bacteria include, but are not limited to, MSA (mannitol salt agar) medium or phenylethyl alcohol agar (Brewer and Lilley. 1949. December meeting of the Maryland Association of Medical and Public Health Laboratories).

[0121] Examples of growth media suitable for supporting the growth of gram-negative bacteria include, but are not limited to, MacConkey agar, Hekton Enteric Agar (King and Metzger. 1968. Appl. Microbiol. 16:577-578) or XLD (Xylose-Lysine-Desoxycholate) Agar (Taylor, 1965. Am. J. Clin. Pathol., 44:471-475). In particular, MacConkey agar contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, especially Enterobacteriaceae. In some embodiments, the growth medium used to support the growth of gram-negative bacteria may be a growth medium recommended for the detection of gram-negative bacteria belonging to the Enterobacteriaceae family according to the ISO 21528:2017 standard.

[0122] Examples of growth media suitable for supporting bacterial and fungal growth include, but are not limited to, Reasoner's 2A agar (R2A), tryptone yeast extract agar, or orange serum agar. Preferably, the growth medium used to support the growth of all microorganisms (bacteria, yeasts, and molds) is a growth medium recommended for enumeration of culturable microorganisms (total count) according to IFU Standard Method No. 2 or ISO 6222:1999 standard.

[0123] Optionally, one or several additional selective components such as antibiotics, pH indicators, selective growth inhibitors, etc. can be added to the medium to increase the selectivity of the medium.

[0124] All of these growth media are well known to those of skill in the art and are commercially available or readily prepared.

[0125] Fluorescent dyes In step a) of the method of the invention, the sample is contacted not only with a solid growth medium as described above but also with at least one fluorescent dye.

[0126] As used herein, the terms "fluorochrome", "fluorophore" or "fluorochrome" are used synonymously and refer to a compound capable of re-emission upon light excitation. Usually, a fluorophore absorbs light in a certain wavelength range (excitation spectrum) and re-emits it in a longer wavelength range (emission spectrum), with respective excitation and emission maxima. The term refers to a compound that does not require any chemical modification to exhibit fluorescent properties. In particular, said at least one fluorescent dye is not a fluorogenic substrate, i.e. a substrate that generates a fluorescent substance after reaction with a microbial enzyme or group of enzymes. Furthermore, said at least one fluorescent dye is not linked to a functional moiety, e.g. an antibody, that is capable of specifically recognizing a microorganism or group of microorganisms. Preferably, the fluorescent dye used in the method of the invention does not limit the growth of the microorganism of interest, i.e. the growth of the microorganism of interest is not significantly affected by the presence of the fluorescent dye. Preferably, said at least one fluorescent dye is not encapsulated, e.g. in liposomes or lipid vesicles. Preferably, said at least one fluorescent dye is not a DNA and / or RNA intercalating agent. Preferably, the at least one fluorescent dye is not Sulforhodamine B, Hoechst dye, Rhodamine B, Neutral Red, Bromothymol Blue, Eosin, Methylene Blue, Amphotericin B or Nile Red.

[0127] In the method of the present invention, the sample may be contacted with one or several fluorescent dyes. Preferably, each fluorescent dye used in the method can stain at least one target microorganism. In some embodiments, several fluorescent dyes are used, each fluorescent dye can stain one or several of the target microorganisms, and all target microorganisms are stained by at least one of these fluorescent dyes. The fluorescent dyes used in the method of the present invention may be membrane-permeable fluorescent dyes and stain the inside of the microorganism, or may only be adsorbed on the outer surface of the microorganism.

[0128] The skilled person can choose one or several fluorescent dyes depending on the microorganism or group of microorganisms to be detected.

[0129] In particular, one skilled in the art can evaluate the ability of a fluorescent dye to stain a microorganism of interest as described in the experimental section. Briefly, a culture of the microorganism is filtered on a 0.45 μm membrane filter, e.g., an MCE or PVDF membrane, and the filtration membrane is deposited on a drop of the fluorescent dye to be tested on a solid growth medium suitable for supporting the growth of the microorganism. The medium is incubated under conditions and for a time sufficient to form microcolonies of the microorganism. Detection of microcolonies that emit a fluorescent signal indicates that the fluorescent dye can be used in the method of the present invention to detect the microorganism. If a group of microorganisms must be detected, one skilled in the art may repeat this assay for each microorganism of the group, using the same or different fluorescent dyes, until a suitable combination is determined that allows the detection of all the microorganisms of interest.

[0130] In one embodiment, the sample is contacted with at least one fluorescent dye selected from the group consisting of acridine dyes, e.g., acridine orange, anthracene-based dyes, e.g., anthraquinone dyes, coumarin-based dyes, cyanine-based dyes, e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, xanthene-based dyes, e.g., fluorescein dyes, eosin dyes, rhodamine dyes and carbopyronine dyes, squaraine-based dyes, squarainerotaxane-based dyes and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof. Preferably, the sample is contacted with at least one fluorescent dye selected from the group consisting of anthracene-based dyes, such as anthraquinone dyes, coumarin-based dyes, cyanine-based dyes, such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, xanthene-based dyes, such as fluorescein dyes, eosin dyes, rhodamine dyes and carbopyronine dyes, squaraine-based dyes, squarainerotaxane-based dyes and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof.

[0131] In particular, the at least one fluorescent dye is acridine dyes, in particular acridine orange, anthracene-based dyes, in particular DRAQ5™ (for example 1,5-bis(2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracene-9,10-dione), CyTRAK Orange™ (Edward, R. (2009) Mol. Cells 27(4): 391-396; CAS Registry Number 1195771-25-5) and their derivatives, coumarin-based dyes, in particular Alexa Fluor™ 350 (for example 7-amino-3-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxoethyl]-4-methyl-2-oxochromene-6-sulfonic acid), Pacific blue™ (for example 6,8-difluoro-7-hydroxy-2-oxochromene-3-carboxylic acid) and their derivatives, - cyanine-based dyes, in particular Cy5™ or Cy5.5™, including the sulfonated and non-sulfonated forms, preferably the sulfonated forms, such as sulfonated Cy5™ (e.g. sulfo-Cy5 acid, FIG. 15) or sulfonated Cy5.5™ (e.g. sulfo-Cy5.5 acid, FIG. 15), iodocyanine green (e.g. sodium; 4-[(2Z)-2-[(2E,4E,6E)-7-[1,1-dimethyl-3-(4-sulfonatobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate) and derivatives thereof, - dipyrromethene-based dyes, in particular BODIPY™ dyes (based on the core structure 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), such as BODIPY™ 500 / 510 (for example 12-(2,2-difluoro-12-methyl-1-aza-3-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-3,5,7,9,11-pentaen-4-yl)dodecanoic acid) and derivatives thereof, - naphthalene-based dyes, in particular Prodan (6-propionyl-2-dimethylaminonaphthalene) and their derivatives; - pyrene-based dyes, in particular Alexa Fluor™ 405 (for example 8-[2-[4-(2,5-dioxopyrrolidin-1-yl)oxycarbonylpiperidin-1-yl]-2-oxoethoxy]pyrene-1,3,6-trisulfonate; triethylazanium), Cascade blue™ (for example [4-[[4-(diethylamino)phenyl]-[4-(ethylamino)naphthalen-2-yl]methylidene]cyclohexa-2,5-dien-1-ylidene]-diethylazanium) and their derivatives, - squaraine-based dyes, in particular Seta dyes, such as Seta 650 (CAS Registry Number 2043938-22-1), Seta 375 (CAS Registry Number 2043938-18-5) and their derivatives; - squaraine rotaxane-based dyes, in particular SeTau dyes, such as SeTau 647 (e.g. SeTau 647 Di-NHS, CAS Registry Number 1442086-02-3), SeTau 488 (e.g. SeTau-488-NHS, Seta Biomedicals, Product Number K9-3152, Molecular Weight: 1642.73, λ Ab Max[nm]:486;ε[M -1 cm -1 ]:59000;λ Em Maximum [nm]: 532; QY [%]: 27) and their derivatives, - xanthene-based dyes, in particular rhodamine dyes, such as MB™ 660R (e.g. MB™ 660R acid, see FIG. 15), ROX (carboxy-X-rhodamine), TAMRA (carboxytetramethylrhodamine), TEX™ 615 (CAS Registry Number 1209486-62-3) and their derivatives, fluorescein dyes, such as Alexa Fluor™ 488 (e.g. Alexa Fluor™ 488 acid, see FIG. 15), 6-FAM (6-carboxyfluorescein), HEX (hexachlorofluorescein), JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), MAX (CAS Registry Number 1610888-25-9), TET (tetrachlorofluorescein) and SUN (see, e.g., FIG. 15) and their derivatives and carbopyronine-based dyes, such as ATTO™ 647 (e.g., ATTO™ 647 acid, see FIG. 15) and their derivatives, and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof may be selected from the group consisting of:

[0132] More particularly, the at least one fluorescent dye is - anthracene-based dyes, in particular DRAQ5™, CyTRAK Orange™ and their derivatives; - coumarin-based dyes, in particular Alexa Fluor™ 350, Pacific blue™ and their derivatives; - cyanine-based dyes, in particular Cy5™ or Cy5.5™, including the sulfonated and non-sulfonated forms, preferably the sulfonated forms, such as sulfonated Cy5™ or sulfonated Cy5.5™, iodocyanine green and derivatives thereof; - dipyrromethene-based dyes, in particular the BODIPY™ dyes, such as BODIPY™ 500 / 510 and their derivatives; - naphthalene-based dyes, in particular prodan and their derivatives; - pyrene-based dyes, in particular Alexa Fluor™ 405, Cascade blue™ and their derivatives, - squaraine-based dyes, in particular the Seta dyes, such as Seta 650, Seta 375 and their derivatives; - squaraine rotaxane-based dyes, in particular the SeTau dyes, such as SeTau 647, SeTau 488 and derivatives thereof; - xanthene-based dyes, in particular rhodamine dyes, such as MB™ 660R, ROX, TAMRA, TEX™ 615 and their derivatives, fluorescein dyes, such as Alexa Fluor™ 488, 6-FAM, HEX, JOE, MAX, TET and SUN and their derivatives, and carbopyronine-based dyes, such as ATTO™ 647 and their derivatives, and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof may be selected from the group consisting of:

[0133] More particularly, the at least one fluorescent dye may be selected from the group consisting of anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes, xanthene-based dyes, and 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof. Even more particularly, the at least one fluorescent dye may be selected from the group consisting of anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes, and xanthene-based dyes, and combinations thereof.

[0134] In certain embodiments, the at least one fluorescent dye may be selected from the group consisting of acridine orange, DRAQ5™, Cytrak Orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably sulfo-Cy5™, CY5.5, preferably sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta™ 650, Seta™ 375, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647 and DAPI, derivatives thereof and combinations thereof.

[0135] In another particular embodiment, the at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5™, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta™ 650, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647 and DAPI, derivatives thereof and combinations thereof.

[0136] In another particular embodiment, the at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5™, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Seta™ 650, Setau™ 647, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647 and DAPI, derivatives thereof and combinations thereof.

[0137] In another particular embodiment, the at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak Orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5™, CY5.5, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta™ 650, Seta™ 375, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488, ATTO™ 647 and DAPI, derivatives thereof and combinations thereof.

[0138] In another particular embodiment, the at least one fluorescent dye may be selected from the group consisting of Acridine Orange, DRAQ5™, Cytrak Orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5™, CY5.5, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta™ 650, Seta™ 375, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488 and ATTO™ 647, derivatives thereof and combinations thereof.

[0139] In another particular embodiment, the at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak Orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5™, CY5.5, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta™ 650, Seta™ 375, Setau™ 647, SeTau 488, MB™ 660R, Alexa Fluor™ 488 and ATTO™ 647, derivatives thereof and combinations thereof.

[0140] As used herein, the term "derivative" refers to a fluorescent compound that includes the core structure of the parent compound (i.e., an aromatic ring skeleton that defines the color or absorption / emission wavelength of the dye, e.g., the core structure of an anthracene, coumarin, cyanine, dipyrromethene, naphthalene, pyrene, squaraine, squaraine rotaxane, xanthene, rhodamine, fluorescein, or carbopyronine dye) and differs from the parent compound due to one or several chemical modifications, e.g., due to modifications in the substituents. For example, a derivative may differ from a parent compound by replacement of at least one substituent with another substituent, by deletion of at least one substituent, by addition of at least one substituent, and combinations thereof. For example, hydrogen may be replaced by a halogen, e.g., fluorine or chlorine, a hydroxyl group (-OH) may be replaced by a carboxylic acid moiety (-COOH), or a carboxylic acid moiety may be replaced with hydrogen. Any kind of substituent modification may be contemplated, provided that the resulting derivative is a fluorophore and is suitable for carrying out the method of the present invention.

[0141] Preferably, the derivative of the fluorescent dye is selected from the group consisting of ester derivatives, in particular NHS ester (succinimidyl ester) derivatives, TFP ester (tetrafluorophenyl ester) derivatives, STP (4-sulfo-2,3,5,6-tetrafluorophenol, sodium salt) ester derivatives or sulfo-NHS ester derivatives, carboxylic acid derivatives, dibenzocyclooctyne (DBCO) derivatives, tetrazine derivatives, hydrazide derivatives, azide derivatives, trans-cyclooctene (TCO) derivatives, alkyne derivatives, maleimide derivatives. More preferably, the derivative of the fluorescent dye is selected from the group consisting of carboxylic acid derivatives, dibenzocyclooctyne (DBCO) derivatives, NHS ester derivatives and maleimide derivatives.

[0142] In a more particular embodiment, said at least one fluorescent dye is selected from the group consisting of DRAQ5™, Alexa Fluor™ 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta™ 650-DBCO, SeTau 488-NHS, SeTau™ 647-maleimide, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI and combinations thereof.

[0143] In another more specific embodiment, said at least one fluorescent dye is selected from the group consisting of DRAQ5™, Alexa Fluor™ 350 NHS ester, Sulfo-Cy5 acid, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Seta™ 650-DBCO, SeTau™ 647-maleimide, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI, and combinations thereof.

[0144] In another more particular embodiment, said at least one fluorescent dye may be selected from the group consisting of Acridine Orange, DRAQ5™, Cytrak Orange, Alexa Fluor™ 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta™ 650-DBCO, Seta 375-NHS, SeTau™ 647-maleimide, SeTau 488-NHS, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI, derivatives thereof and combinations thereof.

[0145] In another more particular embodiment, said at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak Orange, Alexa Fluor™ 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta™ 650-DBCO, Seta 375-NHS, SeTau™ 647-maleimide, SeTau 488-NHS, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO, ATTO™ 647-acid and DAPI, derivatives thereof and combinations thereof.

[0146] In another more particular embodiment, said at least one fluorescent dye may be selected from the group consisting of Acridine Orange, DRAQ5™, Cytrak Orange, Alexa Fluor™ 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta™ 650-DBCO, Seta 375-NHS, SeTau™ 647-maleimide, SeTau 488-NHS, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO and ATTO™ 647-acid, derivatives thereof and combinations thereof.

[0147] In another more particular embodiment, said at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak Orange, Alexa Fluor™ 350 NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, preferably Sulfo-Cy5.5, BODIPY™ 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta™ 650-DBCO, Seta 375-NHS, SeTau™ 647-maleimide, SeTau 488-NHS, MB™ 660R-acid, MB™ 660R-DBCO, Alexa Fluor™ 488-acid, Alexa Fluor™ 488-DBCO and ATTO™ 647-acid, derivatives thereof and combinations thereof.

[0148] Preferably, in the methods of the invention the sample is contacted with 1, 2, 3 or 4 fluorescent dyes, more preferably with 1 or 2 fluorescent dyes.

[0149] In some embodiments, the at least one fluorescent dye is selected from the group consisting of acridine orange-DAPI, acridine orange-Seta 375-NHS, acridine orange-MB660R acid, acridine orange-Alexa Fluor 350-NHS ester, acridine orange-Pacific Blue-NHS ester, acridine orange-sulfoCy5-acid, acridine orange-BODIPY, acridine orange-prodan, acridine orange-Alexa Fluor 405-DBCO, acridine orange-Cascade blue, acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfoCy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647-acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-PacificBlue-NHS ester, Alexa Fluor 488 acid-sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor 488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-Maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 acid-Sulfo Cy5 acid, ATTO647 acid-Seta 650-DBCO, ATTO647 acid-SeTau 647-Maleimide, ATTO647 acid-MB660R-Acid, ATTO647 acid- Preferably selected from the group consisting of MB660R-DBCO, Sulfo Cy5.5 acid-Sulfo Cy5 acid, Sulfo Cy5 acid-Seta 650-DBCO, Sulfo Cy5 acid-SeTau 647-maleimide and Seta 650-DBCO-SeTau 647-maleimide, preferably Acridine Orange-Seta 375-NHS, Acridine Orange-MB660R acid, Acridine Orange-Alexa Fluor 350-NHS ester, Acridine Orange-PacificBlue-NHS ester, Acridine orange-sulfo Cy5 acid, Acridine orange-BODIPY, Acridine orange-Prodan, Acridine orange-Alexa Fluor 405-DBCO, Acridine orange-Cascade blue, Acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfo Cy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-Prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647 acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-Pacific Blue-NHS ester, Alexa Fluor 488 acid-Sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-Prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-Sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-Prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 Acid-Sulfo Cy5 Acid, ATTO647 Acid-Seta 650-DBCO, ATTO647 Acid- SeTau 647-Maleimide, ATTO647 Acid- MB660R-Acid, ATTO647 Acid- MB660R-DBCO, Sulfo Cy5.5 Acid-Sulfo Cy5 Acid, Sulfo Cy5 Acid-Seta 650-DBCO, Sulfo Cy5 Acid-SeTau 647-Maleimide and Seta 650-DBCO-SeTau 647-maleimide.

[0150] In a particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of thermoacidophilic bacteria (TAB) as defined above, i.e. belonging to the genus Alicyclobacillus, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius and Alicyclobacillus contaminans and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, even more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor In a particular preferred embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of thermoacidophilic bacteria (TAB) as defined above, i.e. belonging to the genus Alicyclobacillus, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius and Alicyclobacillus contaminans and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of MB660R and derivatives thereof, preferably selected from the group consisting of MB660R-acid and MB660R-DBCO and combinations thereof, more preferably MB660R-DBCO.

[0151] In another particular embodiment, the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Acetobacter, Acidomonas, Asaia, Gluconacetobacter, Gluconobacter, Ameyamaea, Bombella, Commensalibacter, Endobacter, Granulibacter, Komagataebacter, Kozakia, Neoasiaia, Neokomagataea, Nguenibacter, Saccharibacter, Swaminatania, Swingsia and Tanticarroenia and combinations thereof, more preferably from the genera Acetobacter, Gluconobacter, Gluconacetobacter and Asaia. and combinations thereof, even more preferably selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans and Asaea thiamensis and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes, carbopyronine-based dyes and DAPI and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, even more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and combinations thereof. Preferably, the at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647 acid and combinations thereof.

[0152] In a particular preferred embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of acetic acid bacteria (AAB) as defined above, preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter and Asaia and combinations thereof, even more preferably selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans and Asaia thiamensis and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488 acid, MB660R-acid and ATTO647 acid and combinations thereof, more preferably selected from the group consisting of MB660R and ATTO647 and derivatives and combinations thereof, in particular selected from the group consisting of MB660R-acid and ATTO647 acid and combinations thereof.

[0153] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of lactic acid bacteria (LAB) as defined above, preferably selected from bacteria belonging to the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Carnobacterium, Streptococcus and Weissella and combinations thereof, more preferably selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof, even more preferably selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum and Weissella confuse and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes, carbopyronine-based dyes and DAPI and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, even more preferably selected from the group consisting of Alexa Fluor In a particular preferred embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of lactic acid bacteria (LAB) as defined above, preferably selected from bacteria belonging to the genera Lactobacillus and Weissella and combinations thereof, even more preferably selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum and Weissella confuse and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and combinations thereof.In a preferred embodiment, the at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R and DAPI and their derivatives and combinations, preferably selected from the group consisting of Alexa Fluor 488 acid, MB660R-acid and DAPI and their combinations, more preferably selected from the group consisting of Alexa Fluor 488 acid and MB660R-acid and their combinations, even more preferably MB660R-acid. In another preferred embodiment, the at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488 and MB660R and their derivatives and combinations, preferably selected from the group consisting of Alexa Fluor 488 acid and MB660R-acid and their combinations.

[0154] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of anaerobic bacteria, preferably selected from bacteria of the genera Clostridium and Cutibacterium and combinations thereof, more preferably selected from the group consisting of Clostridium sporogenes and Cutibacterium acnes and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of MB660R, Cy5™, preferably sulfo-Cy5 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of MB660R-DBCO, sulfo-Cy5 acid and DAPI and combinations thereof. In particular, in this embodiment, said at least one fluorescent dye may be a combination of MB660R and Cy5™, preferably sulfo-Cy5, in particular a combination of MB660R-DBCO and sulfo-Cy5 acid.

[0155] In another particular embodiment, the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Listeria, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Listeria greyi, Kocuria sonnei, Kocuria rhizophila and cepa. The microorganism belongs to or comprises a microorganism belonging to the group of aerobic mesophilic bacteria (AMB) as defined above, selected from the group consisting of bacteria, fungi, fungi, and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of DRAQ5™, Alexa Fluor 50 ... Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably DRAQ5™, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta 650-DBCO, SeTau647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and combinations thereof. In particular, said at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably DRAQ5™, Alexa Fluor™ 350-NHS ester, Sulfo-Cy5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647-acid and DAPI, and combinations thereof.

[0156] More particularly, the target microorganism or group of microorganisms is preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria and Burkholderia, and combinations thereof, more preferably selected from the group consisting of Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila and Burkholderia cepacia, and combinations thereof. The at least one fluorescent dye may belong to or comprise a microorganism belonging to the group of aerobic mesophilic bacteria (AMB) as defined above, wherein the at least one fluorescent dye is selected from the group consisting of acridine dyes, xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of acridine orange, DRAQ5™, Cytrak orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5, Cy5.5™, preferably Sulfo-Cy5.5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably Acridine orange, DRAQ5™, Cytrak orange, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and combinations thereof. In particular, said at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably sulfo-Cy5, Cy5.5™, preferably sulfo-Cy5.5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably DRAQ5™, Cytrak orange, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647 acid and combinations thereof.

[0157] In a preferred embodiment, the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria and Burkholderia and combinations thereof, more preferably Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, The microorganism may belong to or comprise a microorganism belonging to the group of aerobic mesophilic bacteria (AMB) as defined above, selected from the group consisting of Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Escherichia faecalis, Escherichia sonnei, Kocuria rhizophila and Burkholderia cepacia and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of MB660R and derivatives and combinations thereof, optionally in combination with a fluorescent dye selected from the group consisting of Cy5™, preferably Sulfo-Cy5. Preferably, the at least one fluorescent dye is MB660R-DBCO, optionally in combination with Sulfo-Cy5 acid.

[0158] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-negative bacteria as defined above, preferably selected from bacteria belonging to the genera Escherichia and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli and Pseudomonas aeruginosa and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, pyrene-based dyes, coumarin-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 350, Alexa Fluor™ 405, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, Alexa Fluor™ 350-NHS ester, Alexa Fluor™ 405-DBCO, MB660R-DBCO and DAPI and combinations thereof. Even more preferably, the at least one fluorescent dye is selected from the group consisting of Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 350, Alexa Fluor™ 405 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Sulfo-Cy5 acid, Alexa Fluor™ 350-NHS ester, Alexa Fluor™ 405-DBCO and MB660R-DBCO and combinations thereof.

[0159] More particularly, the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and their derivatives and combinations, more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof. Alternatively, the at least one fluorescent dye may be selected from the group consisting of pyrene-based dyes, coumarin-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350 and Alexa Fluor™ 405 and their derivatives and combinations, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Alexa Fluor™ 405-DBCO and combinations thereof. In particular, the at least one fluorescent dye may be a combination of Alexa Fluor™ 350-NHS ester, Alexa Fluor™ 405-DBCO.

[0160] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-negative bacteria as defined above, preferably selected from bacteria belonging to the genera Escherichia, Proteus and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO and DAPI and combinations thereof. More particularly, the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof.

[0161] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram-positive bacteria as defined above, preferably selected from bacteria belonging to the genera Staphylococcus and Enterococcus and combinations thereof, more preferably selected from Staphylococcus aureus and Enterococcus faecalis and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO and DAPI and combinations thereof. More particularly, the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof.

[0162] In another particular embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of Gram positive or negative bacteria as defined above, preferably selected from bacteria belonging to the genera Escherichia, Proteus, Pseudomonas, Staphylococcus and Enterococcus and combinations thereof, more preferably selected from Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis and combinations thereof, and said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO and DAPI and combinations thereof. More particularly, the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5™, preferably sulfo-Cy5, MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof.

[0163] In another particular embodiment, the microorganism or group of microorganisms of interest is preferably selected from the genus Acinetobacter, Aeromonas, Blevendimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Yersinia, Bacillus, Enterobacter ... The bacterium is selected from the group consisting of bacteria belonging to the genera Staphylococcus, Micrococcus and Staphylococcus and combinations thereof, more preferably Acinetobacter baumannii, Aeromonas hydrophila, Blevendimonas diminuta, Burkholderia cepacia, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobacterium anthropiformis, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus cerevisiae, and combinations thereof. or comprises a microorganism belonging to the group of heterotrophic bacteria as defined above, selected from Bacillus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus and combinations thereof, and said at least one fluorescent dye is selected from the group of heterotrophic bacteria as defined above, lysine dyes, xanthene based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine based dyes, anthracene based dyes, coumarin based dyes, cyanine based dyes, dipyrromethene based dyes, naphthalene based dyes, pyrene based dyes, squaraine based dyes, squaraine rotaxane based dyes and DAPI and combinations thereof, preferably acridine orange, DRAQ5™, Cytrak orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably sulfo-Cy5, Cy5.5™, preferably sulfo-Cy5.5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably acridine orange, DRAQ5™, Cytrak orange, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and combinations thereof.

[0164] In particular, said at least one fluorescent dye may be selected from the group consisting of DRAQ5™, Cytrak orange, Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably sulfo-Cy5, Cy5.5™, preferably sulfo-Cy5.5, BODIPY™ dyes, preferably BODIPY 500 / 510, Prodan, Alexa Fluor™ 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and ATTO647 and derivatives and combinations thereof, more preferably DRAQ5™, Cytrak orange, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647 acid and combinations thereof.

[0165] In a preferred embodiment, the microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Acinetobacter, Aeromonas, Brevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobacterium, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Yersinia, Bacillus, Enterococcus, Micrococcus and Staphylococcus and combinations thereof, more preferably Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Burkholderia cepacia, Citrobacter fumigatus, Bacillus subtilis ... aerobic bacteria, such as Escherichia coli, Ochrobacterium anthropicus, Klebsiella pneumoniae, Methylobacterium extroquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia picketii, Salmonella typhimurium, Serratia marcescens, Salmonella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Bacillus faecalis, Bacillus luteus (Kocuria rhizophila), Staphylococcus aureus and combinations thereof, and wherein said at least one fluorescent dye is selected from i) Alexa Fluor 300 (Alexander) Fluorescence Microscopy (Fluorescence Microscopy) a combination of Fluor 350 or a derivative thereof and Alexa Fluor 405 or a derivative thereof, and (ii) a combination of MB660R or a derivative thereof and Sulfo-Cy5 or a derivative thereof, preferably selected from a combination of AF350-NHS ester and AF405-DBCO, and a combination of MB660R-DBCO and Sulfo-Cy5 acid.

[0166] In another particular embodiment, the microorganism or group of microorganisms of interest is an enzyme as defined above, preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum and combinations thereof. The at least one fluorescent dye is selected from the group consisting of acridine dyes, xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squarainerotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of acridine orange, Alexa Fluor, Fluorescein ... Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5, Prodan, Alexa Fluor™ 405, Cascade Blue, BODIPY 500 / 510, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Acridine Orange, Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, BODIPY 500 / 510, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid, MB660R-DBCO and DAPI, and combinations thereof.More particularly, said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably sulfo-Cy5, prodan, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488, MB660R, ATTO647 and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, sulfo-Cy5 acid, prodan, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI, and combinations thereof.Even more particularly, said at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably sulfo-Cy5, Prodan, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, sulfo-Cy5 acid, Prodan, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid, MB660R-DBCO and DAPI, and combinations thereof.

[0167] In another particular embodiment, the microorganism or group of microorganisms of interest is preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum and combinations thereof. The microorganism belongs to or comprises a microorganism belonging to the group of yeasts and molds as defined above, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Pacific Blue, Cy5™, preferably Sulfo-Cy5, Prodan, Alexa Fluor™ 405, Cascade Blue, BODIPY 500 / 510, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably Alexa Fluor™ 350-NHS ester, Pacific Blue-NHS ester, Sulfo-Cy5 acid, Prodan, Alexa Fluor™ 405-DBCO, Cascade Blue, BODIPY 500 / 510, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof.

[0168] In a preferred embodiment, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism belonging to the group of yeasts and molds as defined above, preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of MB660R and derivatives and combinations thereof, more preferably MB660R-DBCO.

[0169] In another particular embodiment, the microorganism or group of microorganisms of interest is: - one or several bacteria, preferably selected from the group as defined above: Thermoacidophilic Bacteria (TAB), Acetic Acid Bacteria (AAB), Lactic Acid Bacteria (LAB), Anaerobes, Aerobic Mesophilic Bacteria, Gram-negative Bacteria and Gram-positive Bacteria and combinations thereof, - preferably one or several microfungi selected from yeasts and molds as defined above Including, The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof, and even more preferably selected from the group consisting of Sulfo-Cy5 acid and MB660R-DBCO and combinations thereof. In particular, the at least one fluorescent dye may be a combination of Sulfo-Cy5 acid and MB660R-DBCO.

[0170] Preferably, in this embodiment, the microorganism or group of microorganisms of interest is selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens ... ence), Gluconobacter (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., , Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila) , bacteria belonging to the genus Burkholderia (e.g., Burkholderia cepacia), and fungi belonging to the genus Candida (e.g., Candida albicans), Zygosaccharomyces (e.g., Zygosaccharomyces bailii), Aspergillus (e.g., Aspergillus brasiliensis), Geotrichum (Geotrichum candidum), Saccharomyces (e.g., Saccharomyces cerevisiae), and Penicillium (e.g., Penicillium varioti, Penicillium chrysogenum), and combinations thereof.

[0171] More preferably, in this embodiment, the microorganism or group of microorganisms of interest is selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium truncatula ... The bacterial strain selected from the group consisting of U.S. sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterococcus faecalis, Listeria greyi, Salmonella sonnei, Kocuria rhizophila, Burkholderia cepacia, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii and Penicillium chrysogenum, and combinations thereof.

[0172] More preferably, in this embodiment, the microorganism or group of microorganisms of interest is selected from the group consisting of bacteria belonging to the genera Weissella (e.g., Weissella confusa), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Enterococcus (e.g., Enterococcus faecalis), and fungi belonging to the genera Candida (e.g., Candida albicans) and Aspergillus (e.g., Aspergillus braziliensis), and combinations thereof, in particular selected from the group consisting of Weissella confusa, Cutibacterium acnes, Methylobacterium extroquens, Enterococcus faecalis, Candida albicans, and Aspergillus braziliensis, and combinations thereof.

[0173] In another particular embodiment, the microorganism or group of microorganisms of interest is: - one or several bacteria, preferably selected from the group as defined above: thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophilic bacteria, gram-negative and gram-positive bacteria, heterotrophic bacteria and combinations thereof, - preferably one or several microfungi selected from yeasts and molds as defined above Including, The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta Preferably, the at least one fluorescent dye is selected from the group consisting of Sulfo-Cy5 acid and MB660R-DBCO, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof, and more preferably, selected from the group consisting of Sulfo-Cy5 acid and MB660R-DBCO and combinations thereof. In particular, the at least one fluorescent dye may be a combination of Sulfo-Cy5 acid and MB660R-DBCO.

[0174] Preferably, in this embodiment, the microorganism or group of microorganisms of interest is selected from the group consisting of the genus Alicyclobacillus (e.g., Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans), the genus Acetobacter (e.g., Acetobacter aceti), the genus Gluconoacetobacter (e.g., Gluconoacetobacter liquefaciens), Gluconobacter niger, and the genus Gluconobacter niger. (e.g., Gluconobacter oxydans), Asaia (e.g., Asaia thiamensis), Lactobacillus (e.g., Lactobacillus casei, Lactobacillus plantarum), Weissella (e.g., Weissella confusa), Clostridium (e.g., Clostridium sporogenes), Cutibacterium (e.g., Cutibacterium acnes), Methylobacterium (e.g., Methylobacterium extroquens), Pseudomonas (e.g., Pseudomonas aeruginosa), Bacillus (e.g., Bacillus subtilis), Escherichia coli, Lysobacter (e.g., Escherichia coli), Staphylococcus (e.g., Staphylococcus aureus), Acinetobacter (e.g., Acinetobacter baumannii), Cronobacter (e.g., Cronobacter sakazakii), Klebsiella (e.g., Klebsiella pneumoniae), Salmonella (e.g., Salmonella typhimurium), Enterococcus (e.g., Enterococcus faecalis), Listeria (e.g., Listeria grayi), Shigella (e.g., Salmonella sonnei), Kocuria (e.g., Kocuria rhizophila), Burkholderia (e.g., Burkholderia cepacia), Aeromonas (e.g., Aeromonas cepacia), hydrophila), Brevundimonas (e.g., Brevundimonas diminuta), Citrobacter (e.g., Citrobacter freundii), Edwardsiella (e.g., Edwardsiella tarda), Enterobacter (e.g., Enterobacter aerogenes), Ochrobacterium (e.g., Ochrobacterium anthropicus), Moraxella (e.g., Moraxella osloensis), Pantoea (e.g., Pantoea agglomerans), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g.,Ralstonia picketii), Serratia (e.g., Serratia marcescens), Sphingomonas (e.g., Sphingomonas paucimobilis), Stenotrophomonas (e.g., Stenotrophomonas maltophilia), Vibrio (e.g., Vibrio parahaemolyticus), Yersinia (e.g., Yersinia enterocolitica), and combinations thereof.

[0175] More preferably, in this embodiment, the microorganism or group of microorganisms of interest is Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter lignin, Alicyclobacillus ligninus ... cufaciens, Gluconobacter oxydans, Asaea thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Murine typhimurium Bacillus faecalis, Listeria greyi, Bacillus sonnei, Cocuria rhizophila, Burkholderia cepacia, Aeromonas hydrophila, Brevundimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropicum, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, Serratia marcescens, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Vibrio parahaemolyticus, Yersinia enterocolitica, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium varioti, and Penicillium chrysogenum, and combinations thereof.

[0176] Even more preferably, in this embodiment, the microorganism or group of microorganisms of interest is Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confuse, Clostridium sporogenes, Cutibacterium acnes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Lactobacillus subtilis, Lactobacillus casei ... Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila, Borrelia cepacia, Aeromonas hydrophila, Brevundimonas diminuta, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Ochrobacterium anthropicum, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Ralstonia picketii, The bacterium is selected from the group consisting of Serratia marcescens, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Candida albicans, Zygosaccharomyces bailii, Aspergillus braziliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, and Penicillium chrysogenum, and combinations thereof.

[0177] - the microorganism is a microorganism A as defined in Table 1 or the group of microorganisms of interest comprises a microorganism A as defined in Table 1, - said at least one fluorescent dye is fluorescent dye B as defined in Table 1 Any embodiment of the methods of the present invention are disclosed herein.

[0178] In embodiments in which the sample is filtered on a filtration membrane, said filtration membrane is preferably Membrane C as defined in Table 1.

[0179] Specifically disclosed are each combination of microorganisms, fluorescent dyes, and optionally membranes in Table 1. Thus, by way of example, specifically disclosed herein is the use of Alexa Fluor 488 acid to detect or enumerate Alicyclobacillus acidoterrestris in a sample, optionally after filtration onto a PVDF membrane, or the use of DAPI to detect or enumerate Acetobacter acetate in a sample, optionally after filtration onto an MCE membrane.

[0180] [Table 1A]

[0181] [Table 1B]

[0182] [Table 1C]

[0183] [Table 1D]

[0184] [Table 1E]

[0185] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidiphilus, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus aureus, and combinations thereof, and said at least one fluorescent dye is Alexa Fluor 488 or a derivative thereof, preferably Alexa Fluor 488 acid.

[0186] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidiphilus, Acetobacter aceti, Gluconoacetobacter liquefaciens, Asaea thiamensis, Lactobacillus casei, Lactobacillus plantarum and Escherichia coli and combinations thereof, and the at least one fluorescent dye is Alexa Fluor 488 or a derivative thereof, preferably Alexa Fluor 488-DBCO.

[0187] In some embodiments, the microorganism or group of microorganisms of interest is Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Cannabinoid vesicles ... Ia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confuse, Zygosaccharomyces bailii, Aspergillus braziliensis, Candida albicans, Saccharomyces cerevisiae, Penicillium variotii, Penicillium chrysogenum, Geotrichum candidum, Cutibacterium acnes, Clostridium sporogenes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta, Aeromonas hydrophila, Pseudomonas fluorescens, Citrobacter freundii, Enterobacter aerogenes, Pantoea agglomerans, Stenotrophomonas maltophilia, The microorganisms in the sample include or belong to a microorganism selected from the group consisting of Serratia marcescens, Yersinia enterocolitica, Edwardsiella tarda, Moraxella osloensis, Ochrobacterium anthropi, Proteus mirabilis, Sphingomonas paucimobilis and Salmonella typhimurium and combinations thereof, and the at least one fluorescent dye is MB660R or a derivative thereof, preferably MB660R-acid and / or MB660R-DBCO.

[0188] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaia thiamensis, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus aureus, and combinations thereof, and said at least one fluorescent dye is MB660R or a derivative thereof, preferably MB660R-acid.

[0189] In some embodiments, the microorganism or group of microorganisms of interest is Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter difficile ... oxydans, Lactobacillus plantarum, Weissella confusa, Zygosaccharomyces baylii, Aspergillus braziliensis, Candida albicans, Saccharomyces cerevisiae, Penicillium variotii, Penicillium chrysogenum, Geotrichum candidum, Cutibacterium acnes, Clostridium sporogenes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Colon Bacillus Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella faecalis, Salmonella sonnei, Kocuria rhizophila, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta, Aeromonas hydrophila, Pseudomonas fluorescens, Citrobacter freundii, Enterobacter aerogenes, Pantoea agglomerans, Stenotrophomonas maltovii, The at least one fluorescent dye may be selected from the group consisting of: Bacillus subtilis, Serratia marcescens, Yersinia enterocolitica, Edwardsiella tarda, Moraxella osloensis, Ochrobacterium anthropi, Proteus mirabilis, Sphingomonas paucimobilis, and Salmonella typhimurium, and combinations thereof, and the at least one fluorescent dye is MB660R or a derivative thereof, preferably MB660R-DBCO. Optionally, the at least one fluorescent dye may further comprise Cy5, preferably sulfo-Cy-5 or a derivative thereof, preferably sulfo-Cy5 acid.

[0190] In some embodiments, the microorganism or group of microorganisms of interest is Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus hervarius, Alicyclobacillus contaminans, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Lactobacillus plantarum, Weissella confusa, Zygosaccharomyces baylii, Aspergillus braziliensis, Caenorhabditis cerevisiae ... The at least one fluorescent dye may be selected from the group consisting of: Candida albicans, Saccharomyces cerevisiae, Penicillium variotii, Penicillium chrysogenum, Geotrichum candidum, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter faecalis, Enterobacter sonnei, Kocuria rhizophila and Burkholderia cepacia, and combinations thereof, and the at least one fluorescent dye is MB660R or a derivative thereof, preferably MB660R-DBCO.

[0191] In some embodiments, the microorganism or group of microorganisms of interest is Cutibacterium acnes, Clostridium sporogenes, Acinetobacter baumannii, Klebsiella pneumoniae, Clostridium sonnei, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta, Aeromonas hydrophila, Pseudomonas fluorescens, Citrobacter freundii, Enterobacter aerogenes, Pantoea agglomerans, Stenotrophomonas maltophilia, Serratia marcescens, Entomobasidium erythropoei, Entomobasidium cepacia ... The microorganism comprises or belongs to a microorganism selected from the group consisting of Lucinia enterocolitica, Edwardsiella tarda, Moraxella osloensis, Ochrobacterium anthropi, Proteus mirabilis, Sphingomonas paucimobilis and Salmonella typhimurium and combinations thereof, and the at least one fluorescent dye is a combination of MB660R or a derivative thereof and Cy5, preferably Sulfo-Cy-5 or a derivative thereof, preferably a combination of MB660R-DBCO and Sulfo-Cy5 acid.

[0192] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidiphilus, Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Asaea thiamensis, Lactobacillus plantarum, Weissella confusa, Escherichia coli, and Staphylococcus aureus, and combinations thereof, and the at least one fluorescent dye is ATTO647 or a derivative thereof, preferably ATTO647 acid.

[0193] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Acetobacter aceti, Gluconoacetobacter liquefaciens, Gluconobacter oxydans, Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, Zygosaccharomyces bailii, Saccharomyces cerevisiae, Clostridium sporogenes, Methylobacterium extroquens, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter faecalis, Burkholderia cepacia, and Proteus mirabilis, and combinations thereof, and said at least one fluorescent dye is DAPI.

[0194] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Aspergillus braziliensis, Candida albicans, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta and Aeromonas hydrophila and combinations thereof, and the at least one fluorescent dye is Alexa Fluor 350 or a derivative thereof, preferably Alexa Fluor 350-NHS ester. Optionally, the at least one fluorescent dye further comprises Alexa Fluor 405 or a derivative thereof, preferably Alexa Fluor 405-DBCO.

[0195] In some embodiments, the microorganism or group of microorganisms of interest is selected from the group consisting of Aspergillus braziliensis, Candida albicans, Cutibacterium acnes, Clostridium sporogenes, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter faecalis, Enterobacter sonnei, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta, Aeromonas hydrophila, Pseudomonas fluorescens, Citrobacter freundii, Enterobacter pneumoniae ... The at least one fluorescent dye may be selected from the group consisting of: Lactobacter aerogenes, Pantoea agglomerans, Stenotrophomonas maltophilia, Serratia marcescens, Yersinia enterocolitica, Edwardsiella tarda, Moraxella osloensis, Ochrobacterium anthropi, Proteus mirabilis, Sphingomonas paucimobilis, and Salmonella typhimurium, and combinations thereof, and the at least one fluorescent dye is Cy5, preferably Sulfo-Cy5 or a derivative thereof, preferably Sulfo-Cy5 acid. Optionally, the at least one fluorescent dye may further comprise MB660R or a derivative thereof, preferably MB660R-DBCO.

[0196] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Aspergillus brasiliensis, Candida albicans, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, and combinations thereof, and said at least one fluorescent dye is Cy5, preferably sulfo-Cy5 or a derivative thereof, preferably sulfo-Cy5 acid.

[0197] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises Escherichia coli and said at least one fluorescent dye is Cy5.5, preferably Sulfo-Cy5.5 or a derivative thereof, preferably Sulfo-Cy5.5 acid.

[0198] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Aspergillus braziliensis, Candida albicans, Methylobacterium extroquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepacia, Ralstonia picketii, Brevundimonas diminuta and Aeromonas hydrophila and combinations thereof, and the at least one fluorescent dye is Alexa Fluor 405 or a derivative thereof, preferably Alexa Fluor 405-DBCO. Optionally, the at least one fluorescent dye further comprises Alexa Fluor 350 or a derivative thereof, preferably Alexa Fluor 350-NHS ester.

[0199] In some embodiments, the microorganism or group of microorganisms of interest belongs to or includes a microorganism selected from the group consisting of Aspergillus braziliensis and Escherichia coli, and combinations thereof, and the at least one fluorescent dye is acridine orange.

[0200] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Aspergillus braziliensis, Candida albicans and Escherichia coli and combinations thereof, and said at least one fluorescent dye is selected from the group consisting of Bodipy 500 / 510, Alexa Fluor 350 or a derivative thereof, preferably Alexa Fluor 350-NHS ester, Pacific blue or a derivative thereof, preferably Pacific blue-NHS ester, Prodan, Alexa Fluor 405 or a derivative thereof, preferably Alexa Fluor 405-DBCO, Cascade Blue, Seta 650 or a derivative thereof, preferably Seta 650-DBCO, Setau 488 or a derivative thereof, preferably Setau 488-NHS, Setau 647 or a derivative thereof, preferably Setau 647-maleimide, and any combination thereof.

[0201] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Candida albicans and Escherichia coli and combinations thereof, and the at least one fluorescent dye is Seta 375 or a derivative thereof, preferably Seta 375-NHS.

[0202] In some embodiments, the microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Escherichia coli and Escherichia faecalis and combinations thereof, and said at least one fluorescent dye is DRAQ5.

[0203] In some embodiments, the microorganism or group of microorganisms of interest belongs to or includes E. coli and said at least one fluorescent dye is Cytrak Orange.

[0204] The solution containing the fluorescent dye can be incorporated into the growth medium before pouring the medium into a contained container, or can be poured or streaked onto the surface of a solid growth medium. Preferably, the fluorescent dye is poured or streaked onto the surface of a solid growth medium before or after contacting the growth medium with the sample. In embodiments where the sample is concentrated on a membrane filter before step a), the fluorescent dye can be poured (either uniformly or as droplets) onto the surface of the solid growth medium, and then a membrane filter can be placed on the surface of the growth medium, thereby contacting both the growth medium and the fluorescent dye. Alternatively, the fluorescent dye can be added to the sample before filtration / concentration on the membrane, or filtered before or after filtration of the sample, preferably on the same membrane after filtration of the sample. A membrane filter can then be placed on the surface of the growth medium.

[0205] The concentration and volume of each fluorescent dye can be easily adjusted by one skilled in the art. Typically, fluorescent dyes are used at concentrations ranging from 1 μM to 1 mM, preferably ranging from 1 μM to 500 μM, preferably ranging from 5 μM to 250 μM. In an embodiment in which the fluorescent dye is poured onto the surface of a solid growth medium and then a membrane filter (47 mm) is placed on the surface of said growth medium, the volume of the droplet containing the fluorescent dye is typically between 100 μL and 300 μL, preferably between 150 μL and 200 μL.

[0206] Incubation conditions and times In step b) of the method of the invention, a vessel containing a sample as defined above, a growth medium and at least one fluorescent dye is incubated under conditions and for a time sufficient to form microcolonies of the microorganism or group of microorganisms of interest, thus growth of the microorganisms being carried out in the presence of the fluorescent dye.

[0207] As used herein, the term "microcolony" refers to colonies that are invisible or barely visible to the naked eye, grown for hours or days depending on the microorganism. Typically, the size of a microcolony is less than 500 μm, preferably between 10 μm and 200 μm.

[0208] The incubation time required to obtain microcolonies varies depending on the microorganism, in particular on the generation time of the microorganism. This incubation time can be easily adjusted by a person skilled in the art depending on the desired microorganism or group of microorganisms and parameters that affect the generation time, such as temperature and type of growth medium. Usually, the incubation time ranges from 3 hours to 96 hours. The growth medium or the surface of the membrane filter can be periodically monitored microscopically for growth using a fluorescence microscope or another suitable system at one or more specific wavelengths to adjust the incubation time.

[0209] Likewise, the incubation conditions, in particular the atmospheric and temperature conditions, also vary depending on the microorganism. These conditions can be adapted to favor the microorganism or group of microorganisms of interest and / or to limit contamination with other microorganisms.

[0210] In particular, incubation can be carried out under aerobic, semi-aerobic (usually between 2 and 10% oxygen) or anaerobic conditions. In semi-aerobic or anaerobic conditions, the concentration of carbon dioxide can be increased to favor capnophilic microorganisms, usually reaching values ​​between 1 and 20% carbon dioxide.

[0211] The temperature usually ranges between 15°C and 50°C, preferably between 20°C and 45°C. However, extremophilic microorganisms can be cultured at temperatures below 15°C or above 50°C. The incubation temperature can be easily adjusted by the skilled artisan depending on the microorganism or group of microorganisms of interest. Thermophilic microorganisms, such as TAB, are usually cultured between 37°C and 48°C, preferably at about 45°C. Mesophilic microorganisms are usually cultured between 30°C and 40°C, preferably at about 35°C. Acid acetic bacteria and microorganisms are usually cultured between 20°C and 30°C, preferably at about 25°C. Lactic acid bacteria are usually cultured between 20°C and 40°C, preferably at about 30°C.

[0212] In embodiments where the microorganism or group of microorganisms of interest belongs to the thermoacidophilic bacteria, the incubation time is preferably at least 20 hours, more preferably between 20 and 30 hours, even more preferably about 24 hours, and the temperature is preferably at least 40°C, more preferably between 40°C and 50°C, even more preferably about 45°C.

[0213] In embodiments where the microorganism or group of microorganisms of interest belongs to the acetic acid bacteria, the incubation time is preferably at least 35 hours, more preferably between 35 and 72 hours, even more preferably about 48 hours, and the temperature is preferably at least 20°C, more preferably between 20°C and 30°C, even more preferably about 25°C.

[0214] In embodiments where the microorganism or group of microorganisms of interest belongs to lactic acid bacteria, the incubation time is preferably at least 20 hours, more preferably between 20 and 48 hours, even more preferably about 24 hours, and the temperature is preferably at least 20°C, more preferably between 20°C and 40°C, even more preferably about 30°C.

[0215] In embodiments where the microorganism or group of microorganisms of interest belongs to the aerobic mesophilic group, the incubation time is preferably at least 8 hours, more preferably between 10 and 36 hours, even more preferably between 10 and 24 hours, and the temperature is preferably at least 25° C., more preferably between 25° C. and 40° C., even more preferably about 35° C. However, some mesophilic bacteria, e.g., Methylobacterium extroquens, may require longer incubation times, e.g., up to 96 hours.

[0216] In embodiments where the microorganism or group of microorganisms of interest belongs to the anaerobes, the incubation time is preferably at least 10 hours, more preferably between 20 and 72 hours, even more preferably between 20 and 24 hours, and the temperature is preferably at least 20° C., more preferably between 20 and 45° C., even more preferably about 37° C. However, some anaerobes, such as Cutibacterium acnes, may require longer incubation times, for example up to 96 hours.

[0217] In embodiments where the microorganism or group of microorganisms of interest belongs to heterotrophic bacteria as defined above, the incubation time is preferably at least 8 hours, more preferably between 14 and 48 hours, even more preferably between 24 and 42 hours, and the temperature is preferably between 20° C. and 37° C., more preferably between 32° C. and 37° C., even more preferably about 32.5° C. However, some heterotrophic bacteria, such as Methylobacterium extroquens, may require longer incubation times, for example up to 96 hours at 22.5° C.

[0218] In embodiments where the microorganism or group of microorganisms of interest belongs to the microbes, the incubation time is preferably at least 20 hours, more preferably between 30 and 50 hours, even more preferably between 30 and 40 hours, and the temperature is preferably at least 20°C, more preferably between 20°C and 35°C, even more preferably about 25°C.

[0219] Of course, if the population of microorganisms of interest includes microorganisms belonging to different populations, the incubation time as well as the temperature must be adjusted to allow the growth of each microorganism.

[0220] In some embodiments, particularly where the method is used to assess the sterility of a sample, the incubation time may be extended to detect the growth of any microorganisms present in the sample. Typically, in such cases, the container may be incubated for at least 48 hours, or even at least 72 hours.

[0221] detection In step c) of the method of the invention, microcolonies which form on the surface of the growth medium or on the surface of the membrane filter as defined above and which emit a fluorescent signal of at least one fluorescent dye are detected and / or enumerated.

[0222] The microcolonies that emit fluorescent signals can be detected and / or enumerated using any method known to those skilled in the art. In particular, the microcolonies can be detected and / or enumerated using a fluorescent solid-phase hemocytometer or microscope, preferably an automated solid-phase hemocytometer or microscope, or any other imaging system suitable for detecting fluorescent emission. Automated systems for counting microcolonies of microorganisms that emit fluorescent signals in culture devices are known in the art. Such automated systems generally include an imaging system, an image analysis algorithm for determining colony counts, and a data management system for displaying, and optionally storing and manipulating colony count data and images. As an example, the microcolony semi-automated counter MICA sold by the company DIAMIDEX can fluorometrically detect and count microorganisms at their early stage of microcolonies.

[0223] In some embodiments where step b) is performed on a membrane filter and thus microcolonies grow on the surface of the membrane filter, said filter may be removed from the solid growth medium after step b) and before step c) and the microcolonies thereon are detected and / or enumerated in step c). This step remains optional and the microcolonies on the surface of the membrane filter can be detected and / or enumerated directly in the incubation vessel.

[0224] Detection of at least one microcolony indicates the presence of the microorganism of interest in the sample.

[0225] It is further assumed that each microcolony detected in step c) arises from an individual cell that has undergone cell division, and therefore the number of microcolonies can be determined in the sample by counting the number of microcolonies, optionally taking into account a dilution or concentration factor.

[0226] Depending on the use of selective characteristics, e.g. selective growth media, selective incubation parameters, etc., the method of the invention may also allow the identification of microorganisms in a sample. In particular, the detection of microcolonies may reveal the presence of microorganisms belonging to specific taxonomic or functional groups (e.g. thermoacidophiles, acetic bacteria, lactic acid bacteria, gram-negative bacteria, yeasts or molds, aerobic mesophiles, anaerobes, etc.).

[0227] The method of the invention may comprise further steps after step c) to further characterise the microorganisms of the detected microcolonies, such as detection of enzymatic activity or metabolites, assays using immunochemical markers, specific staining, biochemical assays including tests or reagents specific for the identification and / or detection of particular microorganisms, including antibiotics.

[0228] Use of the method of the present invention The present invention also relates to the use of the method of the present invention for detecting or enumerating any microorganism or group of microorganisms of interest in a sample, in particular for detecting or enumerating thermoacidophile bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophile bacteria (AMB), yeasts and molds, gram-positive or gram-negative bacteria, heterotrophic bacteria or any combination thereof in a sample, preferably for detecting or enumerating thermoacidophile bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobes, aerobic mesophile bacteria (AMB), yeasts and molds, gram-positive or gram-negative bacteria or any combination thereof in a sample.

[0229] The present invention also relates to the use of the method of the present invention in determining whether a sample contains at least one microorganism of interest, in particular at least one microorganism belonging to the group Thermoacidophile bacteria (TAB), Acetic acid bacteria (AAB), Lactic acid bacteria (LAB), Anaerobes, Aerobic mesophile bacteria, Yeasts and molds, Gram-positive or Gram-negative bacteria, Heterotrophic bacteria or any combination thereof, preferably at least one microorganism belonging to the group Thermoacidophile bacteria (TAB), Acetic acid bacteria (AAB), Lactic acid bacteria (LAB), Anaerobes, Aerobic mesophile bacteria, Yeasts and molds, Gram-positive or Gram-negative bacteria or any combination thereof.

[0230] All method-related embodiments of the invention are also contemplated in this aspect.

[0231] The present invention also relates to the use of the method of the present invention in determining whether a sample is sterile.

[0232] As used herein, the term "sterile" refers to the absence of any microorganism of interest in a sample. Depending on the application field (e.g., pharmaceuticals, cosmetics, food and beverages, environmental analysis, diagnostics, etc.) and standard method, the microorganisms to be considered in sterility testing may vary. However, in general, the method of the present invention is designed to favor maximum detection of microorganisms that may be contained in a sample.

[0233] In this aspect, the method of the invention is used to detect a microorganism or group of microorganisms of interest, the absence of said microorganism or group of microorganisms of interest indicating that the sample is sterile, and detection of said microorganism or group of microorganisms of interest indicating that the sample is not sterile.

[0234] For such applications, the growth medium is usually a non-selective medium, such as R2A or TSA. Usually, the culture is incubated at a temperature between 20° C. and 40° C., preferably at about 35° C., for at least 72 hours. The growth medium and culture conditions may vary widely according to the field of application (e.g., pharmaceutical field, water analysis, etc.) and according to standard methods recommended in this field. Those skilled in the art can easily adjust the method of the present invention taking these parameters into account.

[0235] If desired, the methods of the invention may be repeated to detect different microorganisms or groups of microorganisms, for example using different growth media, incubation conditions, fluorescent dyes, etc.

[0236] Preferably, for such applications, the sample is provided with at least one fluorescent dye selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, and preferably selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, and even more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa The antibody is contacted with at least one fluorescent dye selected from the group consisting of Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof.In particular, the at least one fluorescent dye may be selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R, and derivatives and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO, and combinations thereof.

[0237] In particular, the at least one fluorescent dye may be selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 405, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof.

[0238] More particularly, the at least one fluorescent dye may be a combination of a xanthene-based dye and a cyanine-based dye, preferably Cy5™, preferably a combination of Sulfo-Cy5 or a derivative thereof and MB™ 660R or a derivative thereof, more preferably a combination of MB™ 660R DBCO and Sulfo-Cy5 acid.

[0239] In some embodiments, the at least one fluorescent dye is selected from the group consisting of acridine orange-DAPI, acridine orange-Seta 375-NHS, acridine orange-MB660R acid, acridine orange-Alexa Fluor 350-NHS ester, acridine orange-Pacific Blue-NHS ester, acridine orange-sulfoCy5 acid, acridine orange-BODIPY, acridine orange-prodan, acridine orange-Alexa Fluor 405-DBCO, acridine orange-Cascade blue, acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfoCy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647-acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-PacificBlue-NHS ester, Alexa Fluor 488 acid-sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor 488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-Maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 acid-Sulfo Cy5 acid, ATTO647 acid-Seta 650-DBCO, ATTO647 acid- SeTau 647-Maleimide, ATTO647 acid-MB660R-acid, ATTO647 acid- Preferably selected from the group consisting of MB660R-DBCO, Sulfo Cy5.5 acid-Sulfo Cy5 acid, Sulfo Cy5 acid-Seta 650-DBCO, Sulfo Cy5 acid-SeTau 647-maleimide and Seta 650-DBCO-SeTau 647-maleimide, preferably Acridine Orange-Seta 375-NHS, Acridine Orange-MB660R acid, Acridine Orange-Alexa Fluor 350-NHS ester, Acridine Orange-PacificBlue-NHS ester, Acridine orange-sulfo Cy5 acid, Acridine orange-BODIPY, Acridine orange-Prodan, Acridine orange-Alexa Fluor 405-DBCO, Acridine orange-Cascade blue, Acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfo Cy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-Prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647 acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-Pacific Blue-NHS ester, Alexa Fluor 488 acid-Sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-Prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-Sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-Prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 Acid-Sulfo Cy5 Acid, ATTO647 Acid-Seta 650-DBCO, ATTO647 Acid-SeTau 647-Maleimide, ATTO647 Acid-MB660R-Acid, ATTO647 Acid-MB660R-DBCO, Sulfo Cy5.5 Acid-Sulfo Cy5 Acid, Sulfo Cy5 Acid-Seta 650-DBCO, Sulfo Cy5 Acid-SeTau 647-Maleimide and Seta 650-DBCO-SeTau 647-maleimide.

[0240] In this aspect, all embodiments relating to the method of the invention are also contemplated.

[0241] The present invention also relates to the use of the method of the present invention in determining the biological contamination of a sample.

[0242] As used herein, the term "biomassage" refers to the number of viable microorganisms of interest present in a sample. Depending on the field of application (e.g., pharmaceuticals, cosmetics, food and beverages, environmental analysis, diagnostics, etc.) and standard method, the microorganisms to be considered in a biomassage test may vary. However, in general, the method of the present invention is designed to favor maximum detection of microorganisms that may be contained in a sample.

[0243] In this aspect, the method of the invention is used to detect and enumerate a microorganism or group of microorganisms of interest, and the enumeration of said microorganism or group of microorganisms of interest is used to assess the biological contamination of a sample.

[0244] For such applications, the growth medium is usually a non-selective medium, such as R2A, TSA or SDA agar. Usually, the culture is incubated at a temperature between 20° C. and 40° C., preferably at about 35° C., for at least 30 hours. The growth medium and the conditions of the culture may vary widely according to the field of application (e.g., pharmaceutical field, water analysis, etc.) and according to standard methods recommended in this field. The skilled person can easily adjust the method of the invention taking these parameters into account.

[0245] If desired, the methods of the invention may be repeated using, for example, different growth media, incubation conditions, fluorescent dyes, etc., to detect and enumerate different microorganisms or groups of microorganisms.

[0246] Preferably, for such applications, the sample is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squarainerotaxane-based dyes and combinations thereof, more preferably selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, and even more preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa The antibody is contacted with at least one fluorescent dye selected from the group consisting of Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof.More particularly, said at least one fluorescent dye may be selected from the group consisting of Alexa Fluor™ 350, Pacific blue™, Cy5™, preferably sulfo-Cy5, Alexa Fluor™ 405, Cascade blue™, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, and even more preferably, the at least one fluorescent dye is selected from the group consisting of Alexa Fluor™ 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Cascade blue™, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof.

[0247] In particular, the at least one fluorescent dye may be selected from the group consisting of Alexa Fluor™ 350, Cy5™, preferably Sulfo-Cy5, Alexa Fluor™ 405, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, preferably selected from the group consisting of Alexa Fluor™ 350-NHS ester, Sulfo-Cy5 acid, Alexa Fluor™ 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof. More particularly, said at least one fluorescent dye may be a combination of a xanthene-based dye and a cyanine-based dye, preferably Cy5™, preferably a combination of sulfo-Cy5 or a derivative thereof and MB™660R or a derivative thereof, more preferably a combination of MB™660R DBCO and sulfo-Cy5 acid. Optionally, one or several additional fluorescent dyes may be added to said at least one fluorescent dye as defined above, in particular one or several additional fluorescent dyes selected from the group consisting of anthracene-based dyes, dipyrromethene-based dyes and naphthalene-based dyes and combinations thereof, preferably DRAQ5™, BODIPY™ dyes, preferably selected from the group consisting of BODIPY 500 / 510 and prodan and derivatives and combinations thereof, more preferably selected from the group consisting of DRAQ5™, BODIPY 500 / 510 and prodan and combinations thereof.

[0248] In some embodiments, the at least one fluorescent dye is selected from the group consisting of acridine orange-DAPI, acridine orange-Seta 375-NHS, acridine orange-MB660R acid, acridine orange-Alexa Fluor 350-NHS ester, acridine orange-Pacific Blue-NHS ester, acridine orange-sulfoCy5 acid, acridine orange-BODIPY, acridine orange-prodan, acridine orange-Alexa Fluor 405-DBCO, acridine orange-Cascade blue, acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfoCy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647-acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-PacificBlue-NHS ester, Alexa Fluor 488 acid-sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor 488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-Maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 acid-Sulfo Cy5 acid, ATTO647 acid-Seta 650-DBCO, ATTO647 acid-SeTau 647-maleimide, ATTO647acid-MB660R-acid, ATTO647acid-MB660R-DBCO, SulfoCy5.5acid-SulfoCy5acid, SulfoCy5acid-Seta 650-DBCO, SulfoCy5acid-SeTau 647-maleimide and Seta 650-DBCO-SeTau 647-maleimide, preferably selected from the group consisting of Acridine Orange-Seta 375-NHS, Acridine Orange-MB660R acid, Acridine Orange-Alexa Fluor 350-NHS ester, Acridine Orange-PacificBlue-NHS ester, Acridine orange-sulfo Cy5 acid, Acridine orange-BODIPY, Acridine orange-Prodan, Acridine orange-Alexa Fluor 405-DBCO, Acridine orange-Cascade blue, Acridine orange-Alexa Fluor 488 acid, DRAQ5-Alexa Fluor 350-NHS ester, DRAQ5-Pacific Blue-NHS ester, DRAQ5-sulfo Cy5 acid, DRAQ5-BODIPY 500 / 510, DRAQ5-Prodan, DRAQ5-Alexa Fluor 405-DBCO, DRAQ5-Cascade blue, DRAQ5-Seta 650-DBCO, DRAQ5-SeTau 647-maleimide, DRAQ5-MB660R-acid, DRAQ5-MB660R-DBCO, DRAQ5-ATTO647 acid, Cytrak orange-Alexa Fluor 350-NHS ester, Cytrak orange-Pacific Blue-NHS ester, Cytrak orange-sulfo-Cy5 acid, Cytrak orange-BODIPY 500 / 510, Cytrak orange-Prodan, Cytrak orange-Alexa Fluor 405-DBCO, Cytrak orange-Cascade blue, Seta 375-NHS-Alexa Fluor 350-NHS ester, Seta 375-NHS-Pacific Blue-NHS ester, Seta 375-NHS-sulfo-Cy5 acid, Seta 375-NHS-BODIPY 500 / 510, Seta 375-NHS-Prodan, Seta 375-NHS-Alexa Fluor 405-DBCO, Seta 375-NHS-Cascade blue, Alexa Fluor 488 acid-Alexa Fluor 350-NHS ester, Alexa Fluor 488 acid-Pacific Blue-NHS ester, Alexa Fluor 488 acid-Sulfo Cy5 acid, Alexa Fluor 488 acid-BODIPY 500 / 510, Alexa Fluor 488 acid-Prodan, Alexa Fluor 488 acid-Alexa Fluor 405-DBCO, Alexa Fluor488 acid-Cascade blue, MB660R-acid-Alexa Fluor 350-NHS ester, MB660R-acid-Pacific Blue-NHS ester, MB660R-acid-Sulfo Cy5 acid, MB660R-acid-BODIPY 500 / 510, MB660R-acid-Prodan, MB660R-acid-Alexa Fluor 405-DBCO, MB660R-acid-Cascade blue, MB660R-acid-Seta 650-DBCO, MB660R-acid-SeTau 647-maleimide, MB660R-DBCO-Alexa Fluor 350-NHS ester, MB660R-DBCO-Pacific Blue-NHS ester, MB660R-DBCO-Sulfo Cy5 acid, MB660R-DBCO-BODIPY 500 / 510, MB660R-DBCO-Prodan, MB660R-DBCO-Alexa Fluor 405-DBCO, MB660R-DBCO-Cascade blue, MB660R-DBCO-Seta 650-DBCO, MB660R-DBCO-SeTau 647-Maleimide, ATTO647 Acid-Sulfo Cy5 Acid, ATTO647 Acid-Seta 650-DBCO, ATTO647 Acid-SeTau 647-Maleimide, ATTO647 Acid-MB660R-Acid, ATTO647 Acid- MB660R-DBCO, Sulfo Cy5.5 Acid-Sulfo Cy5 Acid, Sulfo Cy5 Acid-Seta 650-DBCO, Sulfo Cy5 Acid-SeTau 647-Maleimide and Seta 650-DBCO-SeTau 647-maleimide.

[0249] In this aspect, all embodiments relating to the method of the invention are also contemplated.

[0250] Kits and their uses The present invention further comprises: - at least one fluorescent dye as defined above, and optionally, - at least one membrane filter as defined above The present invention relates to a kit comprising:

[0251] The kit includes: - at least one container containing a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest, or materials required to obtain said at least one container, and / or - a leaflet providing instructions for using said kit It may further include.

[0252] In certain embodiments, the kit of the invention comprises: - at least one fluorescent dye as defined above, and - at least one membrane filter as defined above, and optionally a solid growth medium containing nutrients to support the growth of a microorganism or group of microorganisms of interest, or at least one container containing materials needed to obtain said at least one container, and / or a leaflet providing instructions for using said kit. Includes.

[0253] In another particular embodiment, the kit of the invention comprises: - at least one container, preferably containing at least one fluorescent dye as defined above and a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest, or the materials required to obtain said at least one container, and Optionally, - preferably at least one membrane filter as defined above, and / or - a leaflet providing instructions for using said kit Includes.

[0254] In this aspect, all embodiments relating to the method of the invention are also contemplated.

[0255] The present invention also relates to the use of a kit according to the invention for detecting or enumerating a microorganism or group of microorganisms of interest according to the method of the invention as described above. In this aspect, all embodiments relating to the method of the invention are also contemplated.

[0256] All references cited in this description are incorporated herein by reference. Other features and advantages of the present invention will become more apparent in the following examples, given for purposes of illustration and not by way of limitation. EXAMPLES

[0257] All strains, except Aspergillus braziliensis and Cutibacterium acnes, were diluted in NaCl 0.9% from previously quantified cryopreserved stocks to obtain defined amounts of organisms per experimental condition, and each bacterial preparation was filtered onto a 0.45 μm PVDF or MCE membrane (47 mm).

[0258] For A. brasiliensis and C. acnes, BioBalls® from Biomerieux (BioBall® Multishot 550 Aspergillus brasiliensis SKU number: 56001; BioBall® Multishot Propionibacterrium acnes (formerly Cutibacterium acnes, strain DSM 1897) SKU number: 416600) containing the correct number of organisms were diluted with NaCl 0.9% to obtain a defined amount of organism per experimental condition and each bacterial preparation was filtered onto a 0.45 μm PVDF or MCE membrane (47 mm).

[0259] Example 1 Thermoacidophilic Bacteria (TAB) Detection In this example, several strains of Alicyclobacillus bacteria were used, including Alicyclobacillus acidoterrestris ATCC® 49025™, Alicyclobacillus acidocaldarius ATCC® 27009™, Alicyclobacillus acidiphilus DSM 14558, Alicyclobacillus cycloheptanicus ATCC® 49028™, Alicyclobacillus hesperidum DSM 12766, Alicyclobacillus hervarius DSM 13609, and Alicyclobacillus contaminans DSM 17975.

[0260] 150 µL of a fluorescent dye selected from the group consisting of Alexa Fluor 488 acid (50 µM), Alexa Fluor 488-DBCO (50 µM), MB660R-acid (250 µM), MB660R-DBCO (250 µM) and ATTO647 acid (5 µM) was deposited on a BAT agar plate (yeast extract 2 g / l, D(+) glucose 5 g / l, calcium chloride 0.25066 g / l, magnesium sulfate 0.5 g / l, ammonium sulfate 0.2 g / l, potassium dihydrogen phosphate 3 g / l, zinc sulfate 0.00018 g / l, copper sulfate 0.00016 g / l, manganese sulfate 0.00015 g / l, sodium molybdate dihydrate 0.0003 g / l, agar 18 g / l, pH value 3.8-4.2). Each agar plate contained one drop of fluorescent dye.

[0261] A filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the plates were then incubated at 45° C. for 24 hours, except for A. acidocaldarius and A. contaminans, which were incubated at 45° C. for 48 hours.

[0262] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, light power 100%, gain 1, exposure times 100 and 400 ms).

[0263] The conditions and results tested are presented in Table 2.

[0264] [Table 2]

[0265] Example 2 Detection of acetic acid bacteria (AAB) The acetic acid bacterial strains used in this example are Acetobacter aceti ATCC® 15973™, Gluconoacetobacter liquefaciens ATCC® 14835™, Asaea thiamensis DSM 15972 and Gluconobacter oxydans ATCC® 19357™.

[0266] 150 μL of a fluorescent dye selected from the group consisting of DAPI (50 μM), Alexa Fluor 488 acid (50 μM), Alexa Fluor 488-DBCO (50 μM), MB660R-acid (250 μM), MB660R-DBCO (250 μM) and ATTO647 acid (5 μM) was deposited on a YM agar plate (glucose 10 g / L, malt extract 3 g / L, peptone 5 g / L, yeast extract 3 g / L, agar 15 g / L, pH value 6.2 ± 0.2). Each agar plate contained one drop of fluorescent dye.

[0267] The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the plates were then incubated at 25° C. for 72 hours.

[0268] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Cy5 light power 100%, gain 1, exposure time 200ms for MB660R-DBCO and ATTO647 acid, 300ms for AF488 acid, 400ms for MB660R-acid, 600ms for AF488-DBCO, for DAPI 12% UV light power, gain 1, exposure time 400ms).

[0269] The conditions and results tested are presented in Table 3.

[0270] [Table 3]

[0271] Example 3 Lactic acid bacteria (LAB) detection The lactic acid bacteria strains used in this example are Lactobacillus plantarum ATCC® 8014™, Weissella confuse ATCC® 10881™ and Lactobacillus casei ATCC® 393™.

[0272] 150 μL of a fluorescent dye selected from the group consisting of DAPI (50 μM), Alexa Fluor 488 acid (50 μM), Alexa Fluor 488-DBCO (50 μM), MB660R-acid (250 μM), MB660R-DBCO (250 μM) and ATTO647 acid (5 μM) was deposited on MRS agar plates (diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, D(+)-glucose 20 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, meat extract 5 g / L, sodium acetate 5 g / L, universal peptone 10 g / L, yeast extract 5 g / L, agar 12 g / L, pH value 5.7). Each agar plate contained one drop of fluorescent dye.

[0273] The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the plates were then incubated at 30° C. for 48 hours.

[0274] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Cy5 light power 100%, gain 1, exposure time 200ms for MB660R-DBCO and ATTO647 acid, 300ms for AF488 acid, 400ms for MB660R-acid, 600ms for AF488-DBCO, for DAPI 12% UV light power, gain 1, exposure time 100ms) except for L. casei, which was performed using 35% UV light power.

[0275] The conditions and results tested are presented in Table 4.

[0276] [Table 4]

[0277] Example 4 Anaerobic bacteria detection The anaerobic bacterial strains used in this example are Clostridium sporogenes ATCC 19404 and Cutibacterium acnes ATCC 6919.

[0278] 200 μL of DAPI (50 μM) was deposited on a TSA agar plate (tryptone 15 g / L, Soja papainic peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.3 ± 0.2) and for C. sporogenes 150 μL of a mixture of sulfo-Cy5 acid (100 μM) and MB660R-DBCO (100 μM) was deposited on a TSA agar plate and for C. acnes on a Columbia agar plate supplemented with 5% sheep blood (pancreatic digest of casein 12 g / L, pepsin digest of animal tissues 5 g / L, yeast extract 3 g / L, beef extract 3 g / L, corn starch 1 g / L, sodium chloride 5 g / L, agar 13.5 g / L, defibrinated sheep blood 5%, pH value pH 7.3 ± 0.2). Each agar plate contained one drop of fluorescent dye.

[0279] The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate. Plates containing C. sporogenes were then incubated at 35°C in anaerobic conditions for 15 h for DAPI staining to 24 h for MB660R-DBCO and Sulfo-Cy5 acid staining. Plates containing C. acnes were incubated at 35°C in anaerobic conditions for 96 h.

[0280] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX; for the mixture of sulfo-Cy5 acid and MB660R-DBCO, Cy5 light power 100%, gain 1, exposure time 200 ms; for DAPI, UV light power 12%, gain 1, exposure time 400 ms).

[0281] The conditions and results tested are presented in Table 5.

[0282] [Table 5]

[0283] Example 5 Detection of aerobic mesophilic bacteria The mesophilic bacterial strains used in this example are Pseudomonas aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633™, Escherichia coli ATCC 8739™, Staphylococcus aureus ATCC 6538™, Acinetobacter baumannii ATCC 19606, Cronobacter sakazakii ATCC 29544™, Klebsiella pneumoniae ATCC 13883™, Salmonella typhimurium ATCC 13311™, Salmonella faecalis ATCC 29212™, Salmonella sonnei ATCC 25931™, Kocuria rhizophila ATCC 9341™, Methylobacterium extroquens ATCC 25931™, and Streptococcus cerevisiae ATCC 25931™. 43645 and Burkholderia cepacia ATCC® 25608™.

[0284] Acridine orange (40 μM), Cytrak Orange (50 μM), DRAQ5 (125 μM), AF350-NHS ester (50 μM), Pacific Blue-NHS ester (10 μM), Sulfo-Cy5 acid (150 μM), Sulfo-Cy5.5 acid (50 μM), BODIPY 500 / 510 (10 μM on PVDF membrane and 150 μM on MCE membrane), Seta 375-NHS (5 μM), Prodan (15 μM on PVDF membrane and 50 μM on MCE membrane), AF405-DBCO (50 μM), Cascade Blue (1 μM), Seta 650-DBCO (100 μM), SeTau 488-NHS (50 μM), SeTau 647-maleimide (100 μM), DAPI (50 μM), Alexa Fluor 488 acid (50 μM), Alexa Fluor 150 μL of fluorescent dyes selected from the group consisting of 488-DBCO (50 μM), MB660R-acid (250 μM), MB660R-DBCO (250 μM) and ATTO647-acid (5 μM) were deposited on TSA and PCA agar plates (tryptone 5 g / L, yeast extract 2.5 g / L, dextrose 1 g / L, agar 15 g / L, pH value 7.0 ± 0.2) for Bacillus subtilis, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enzyme faecalis, Enzyme sonnei and Kocuria rhizophila, or on TSA agar plates for Methylobacterium extraquens, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Burkholderia cepacia. Each agar plate contained one drop of fluorescent dye.

[0285] The filter membrane was then deposited on top of the fluorescent dye droplets on each agar plate.The plates were then incubated at 35°C for 11 hours for Cronobacter sakazakii on PCA and TSA plates and for Klebsiella pneumoniae on TSA plates, 18 hours for Klebsiella pneumoniae on PCA plates, Bacillus subtilis, Acinetobacter baumannii, Salmonella typhimurium, Enterobacter faecalis, Enterobacter sonnei, Kocuria rhizophila on PCA and TSA plates and for Pseudomonas aeruginosa on TSA plates, 24 hours for Burkholderia cepacia on TSA plates and 96 hours for Methylobacterium extroquens on TSA plates.

[0286] After incubation, the cells were incubated with a microcolony semi-automated counter (MICA, DIAMIDEX, DAPI: UV light intensity 12%, gain 1, exposure time 200ms; MB660R-DBCO, MB660R-acid, ATTO647 acid: CY5 light intensity 100%, gain 1, exposure time 200 and 500ms; AF488 acid and AF488-DBCO: FITC light intensity 100%, gain 1, exposure time 200 and 400ms; Sulfo-Cy5 acid, Seta-650 DBCO: CY5 light intensity 100%, gain 1, exposure time 50ms and 400ms; AF350-NHS ester, AF405-DBCO: UV light intensity: 20% or 50%, gain 1, exposure time 100ms and 400ms; SeTau 647-maleimide: CY5 light intensity 100%, gain 1 Exposure times 50ms and 200ms; Prodan: 100% or 50% UV power, gain 1, exposure times 100ms and 400ms; BODIPY 500 / 510 and SeTau 488-NHS (50μM): 100% FITC power, gain 1, exposure times 50ms and 200ms; DRAQ5: 100% CY5 power, gain 1, exposure times 200ms and 400ms; Cascade Blue and Pacific Blue-NHS ester: 100% or 50% UV power, gain 1, exposure times 50ms and 200ms; Seta 375-NHS: 100% UV power, gain 1, exposure time 200ms; Sulfo-Cy5.5 acid: 20% CY5.5 power, gain 1, exposure time 50ms; Acridine orange: 100% CY5 power, gain 1, exposure time 400ms; Cytrak Orange:FITC light power 7% gain 1 exposure time 400ms was used to detect microcolonies.

[0287] The conditions and results tested are presented in Table 6.

[0288] [Table 6A]

[0289] [Table 6B]

[0290] Example 6 Yeast and mold detection The yeast and mold strains used in this example are Aspergillus brasiliensis ATCC® 16404™, Penicillium variotii ATCC 18502, Penicillium chrysogenum ATCC® 10106™, Candida albicans ATCC® 10231™, Geotrichum candidum, Saccharomyces cerevisiae ATCC® 9763™ and Zygosaccharomyces baylii DSM 70492.

[0291] MB660R-DBCO (250 μM), Alexa Fluor 488 acid (50 μM), MB660R-acid (250 μM) or ATTO647 acid (5 μM), 200 μl of Seta 375-NHS (5 μM), acridine orange (40 μM), BODIPY 150 μl of 500 / 510 (10 μM on PVDF membrane and 150 μM on MCE membrane) or 250 μl of DAPI (50 μM) were deposited on YM agar plates (10 g / L glucose, 3 g / L malt extract, 5 g / L peptone, 3 g / L yeast extract, 15 g / L agar, pH value 6.2 ± 0.2) or SDA agar plates (5 g / L pancreatic digest of casein, 5 g / L pepsin digest of animal tissues, 40 g / L dextrose, 15 g / L agar, pH value 5.6 ± 0.2) for Zygosaccharomyces baylii, Saccharomyces cerevisiae and Aspergillus brasiliensis, and on SDA agar plates for Candida albicans, Penicillium variotii and Penicillium chrysogenum. Each agar plate contained one drop of fluorescent dye. A filtration membrane was then deposited on top of the fluorescent dye drop on each agar plate. The plates were then incubated at 25° C. for 39 hours.

[0292] 150 μL of a fluorescent dye selected from the group consisting of AF350-NHS ester (50 μM), Pacific Blue-NHS ester (10 μM), Sulfo-Cy5 acid (150 μM), Prodan (15 μM on PVDF membrane and 50 μM on MCE membrane), AF405-DBCO (50 μM), Cascade Blue (1 μM), Seta 650-DBCO (100 μM), SeTau 488-NHS (50 μM), SeTau 647-maleimide (100 μM) was deposited on the SDA agar plates. Each agar plate contained one drop of fluorescent dye. A filtration membrane was then deposited on top of the fluorescent dye drop on each agar plate. The plates were then incubated at 35° C. for 21 hours.

[0293] After incubation, the cells were incubated with a microcolony semi-automated counter (MICA, DIAMIDEX, DAPI: 12% light intensity, gain 1, exposure time 200ms; MB660R-DBCO, MB660R-acid, ATTO647 acid: 100% CY5 light intensity, gain 1, exposure time 200 and 500ms; AF488 acid: 100% FITC light intensity, gain 1, exposure time 200 and 400ms; Sulfo-Cy5 acid, Seta-650 DBCO: 100% CY5 light intensity, gain 1, exposure time 50ms and 400ms; AF350-NHS ester, AF405-DBCO: UV light intensity: 50% or 20% gain 1, exposure time 100ms and 400ms; SeTau 488-NHS: 100% FITC light intensity, gain 1, exposure time 50ms and 200ms; SeTau Microcolonies were detected using the following: 647-maleimide: CY5 power 100% gain 1 exposure time 50ms and 200ms; Prodan: UV power 100% or 50% gain 1 exposure time 100ms and 400ms; Cascade Blue and Pacific Blue-NHS ester: UV power 100% or 50% gain 1 exposure time 50ms and 200ms; Seta 375-NHS: UV power 100% gain 1 exposure time 200ms; Acridine Orange: CY5 power 100% gain 1 exposure time 400ms; BODIPY 500 / 510: FITC power 100% gain 1 exposure time 50ms and 200ms).

[0294] The conditions and results tested are presented in Table 7.

[0295] [Table 7A]

[0296] [Table 7B]

[0297] A mixed culture containing C. albicans and A. brasiliensis was filtered onto a 0.45 μm PVDF or MCE membrane (47 mm). 150 μL of a fluorescent dye selected from the group consisting of AF350-NHS ester (50 μM), sulfo-Cy5 acid (150 μM), AF405-DBCO (50 μM), Seta 650-DBCO (100 μM) or SeTau 647-maleimide (100 μM) was deposited onto an SDA agar plate. Each agar plate contained one drop of fluorescent dye. A filtration membrane was then deposited on top of the fluorescent dye drop on each agar plate. The plates were then incubated at 35° C. for 21 hours. After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Sulfo-Cy5 acid, Seta-650 DBCO: CY5 light power 100% gain 1 exposure time 50ms and 400ms; AF350-NHS ester, AF405-DBCO: UV light power: 50% or 20% gain 1 exposure time 100ms and 400ms; SeTau 647-maleimide: CY5 light power 100% gain 1 exposure time 50ms and 200ms).

[0298] The results are presented in Table 8 below and in Figures 10-13.

[0299] [Table 8]

[0300] Example 7 Detection of Gram-negative bacteria in the diagnosis of UTI The Gram-negative bacterium used in this example is Escherichia coli ATCC® 8739™.

[0301] 200 μL of a mixture of MB660R-DBCO (100 μM) and Sulfo-Cy5 acid (100 μM) was deposited on Mac Conkey agar plates (peptone 20 g / L, lactose 10 g / L, bile salts 1.5 g / L, crystal violet 0.001 g / L, neutral red 0.05 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.1 + / - 0.2). Each agar plate contained one drop of the dye mixture.

[0302] The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the plates were then incubated at 35° C. for 8 hours.

[0303] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, light power 100%, gain 1, exposure time 200 ms).

[0304] In each condition, E. coli colonies were labeled and detected.

[0305] Example 8 Total number of microorganisms (bacteria, yeasts and molds) The strains used in this example are Cutibacterium acnes ATCC® 6919™, Methylobacterium extroquens ATCC® 43645™, Pseudomonas faecalis ATCC® 29212™, Aspergillus braziliensis ATCC® 16404™, Candida albicans ATCC® 10231™ and Weissella confusus ATCC® 10881™.

[0306] A mixed culture containing C. faecalis, Aspergillus brasiliensis and C. albicans was filtered onto a 0.45 μm PVDF membrane (47 mm). For the mixture, 150 μL of a mixture of MB660R-DBCO (100 μM) and sulfo-Cy5 acid (100 μM) was deposited onto a TSA or SDA agar plate. Each agar plate contained one drop of the dye mixture. The filtration membrane was then deposited on top of the fluorescent dye drop on each agar plate. The plates were then incubated for 24 h at 35 °C for TSA plates and 25 °C for SDA plates. After incubation, microcolonies were detected using a microcolony semi-automated counter (MICA, DIAMIDEX, Cy5 light power 100%, gain 1, exposure time 50 and 200 ms).

[0307] Mixed cultures containing Aspergillus braziliensis and Candida albicans and cultures of Weissella confusa were filtered onto 0.45 μm PVDF membranes (47 mm). For these cultures, 150 μL of a mixture of MB660R-DBCO (100 μM) and sulfo-Cy5 acid (100 μM) was deposited onto OSA plates (casein peptone 10 g / L, dipotassium hydrogen phosphate 3 g / L, D(+)-glucose 4 g / L, orange extract 5 g / L, yeast extract 3 g / L, agar 17 g / L, pH value 5.5 ± 0.2). Each agar plate contained one drop of the mixture of dyes. The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate. The plates were then incubated at 30 °C for 24 h. After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Cy5 light power 100%, gain 1, exposure time 200 ms).

[0308] A culture of Methylobacterium extroquens was filtered onto a 0.45 μm PVDF membrane (47 mm). For this culture, 150 μL of a mixture of MB660R-DBCO (100 μM) and sulfo-Cy5 acid (100 μM) was deposited onto a TSA or R2A agar plate (casein acid hydrolysate 0.5 g / L, dextrose 0.5 g / L, dipotassium phosphate 0.3 g / L, magnesium sulfate 0.024 g / L, proteose peptone 0.5 g / L, sodium pyruvate 0.3 g / L, starch, soluble 0.5 g / L, yeast extract 0.5 g / L, agar 15 g / L, pH value 7.2 ± 0.2). Each agar plate contained one drop of the mixture of dyes. The filtration membrane was then deposited on top of the fluorescent dye drop on each agar plate. The plates were then incubated at 25° C. for 96 hours.

[0309] A culture of Cutibacterium acnes was filtered onto a 0.45 μm PVDF membrane (47 mm). For this culture, 150 μL of a mixture of MB660R-DBCO (100 μM) and Sulfo-Cy5 acid (100 μM) was deposited onto the TSA. Each agar plate contained one drop of the dye mixture. The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate. The plates were then incubated at 35°C, 5% CO2 for 96 h.

[0310] After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, CY5 light power 100%, gain 1 exposure time 50 ms and 200 ms).

[0311] The results are presented in Table 9.

[0312] [Table 9]

[0313] Example 9 Comparison of the method of the present invention and a method without using fluorescent dyes The strains used in this example are Escherichia coli ATCC® 8739™, Aspergillus braziliensis ATCC® 16404™ and Candida albicans ATCC® 10231™.

[0314] 150 μL of fluorescent dyes selected from the group consisting of DRAQ5 (125 μM), AF350-NHS ester (50 μM), sulfo-Cy5 acid (150 μM), BODIPY 500 / 510 (10 μM), Prodan (15 μM on PVDF membrane and 50 μM on MCE membrane), AF405-DBCO (50 μM), Seta 650-DBCO (100 μM) or SeTau 647-maleimide (100 μM) were deposited on TSA agar plates for E. coli and on SDA agar plates for Aspergillus braziliensis and Candida albicans. A filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate. The plates were then incubated at 35°C for 18 h for E. coli and at 35°C for 21 h for Aspergillus braziliensis and Candida albicans. After incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Sulfo-Cy5 acid, Seta-650 DBCO: CY5 power 100% gain 1 exposure time 50ms and 400ms; AF350-NHS ester, AF405-DBCO: UV power: 50% or 20% gain 1 exposure time 100ms and 400ms; SeTau 647-maleimide: CY5 power 100% gain 1 exposure time 50ms and 200ms; Prodan: UV power 100% or 50% gain 1 exposure time 100ms and 400ms; BODIPY 500 / 510: FITC power 100% gain 1 exposure time 50ms and 200ms; DRAQ5: CY5 power 100% gain 1 exposure time 200ms and 400ms).

[0315] To compare the results obtained using the method of the present invention with those obtained using standard methods, the strains used in this example (E. coli ATCC® 8739™, Aspergillus brasiliensis ATCC® 16404™ and Candida albicans ATCC® 10231™) were filtered on a 0.45 μm MCE membrane (47 mm). The filtration membrane was then deposited on a TSA for E. coli or on a SDA agar plate for Aspergillus brasiliensis and Candida albicans. The plates were then incubated at 35° C. for 3-4 days. After incubation, colonies were counted by naked eye.

[0316] The results are presented in Table 10.

[0317] [Table 10]

[0318] As shown in this table, the method of the present invention allows for earlier detection and more accurate enumeration of microorganisms than the standard method.

[0319] Example 10 Enumerating Microorganisms Associated with Purified Water and Pharmaceutical Water Systems Water is an important part of the pharmaceutical industry. It is used, among other uses, for cleaning, as a component of aqueous sterile and non-sterile products, for hand washing, and as a steam supply to autoclaves. Because of its importance in pharmaceutical manufacturing, the microbial control of water is very important. Because water is always present, each grade of pharmaceutical water is a potential source of microbial contamination, especially if not properly managed.

[0320] There are three types of water in medicine: a) Tap water (or drinking water), b) purified water; c) WFI (Water for Injection).

[0321] Each of these is a different grade, with increasing likelihood of microbial control going down the list (i.e., stricter restrictions apply to water for injection than to tap water). Tap water is supplied by a public utility company and is of "potable water" (drinking) quality. Results from monitoring the water system are assessed in terms of heterotrophic microbial counts against predefined alert and action levels.

[0322] For the microbiological testing of water, the test method was membrane filtration using a 0.45 μm filter. The filters were placed on R2A agar and subjected to a temperature regime of 20-25°C for tap water for 14 days and 30-35°C for purified water and WFI for 5 days. The reason for these different regimes is that the European Pharmacopoeia requires GMP facilities to use the temperatures and times described. For tap water, pharmaceutical facilities can choose the incubation conditions. Choosing the optimal incubation conditions relates to a classic dilemma in microbiology regarding the optimal temperature, time and incubation medium to use. R2A is formulated with low levels of nutrients and will therefore detect a higher proportion of heterotrophic bacteria. The reasoning was that since the bacteria in the water are under nutrient-depleted conditions, they are more likely to grow in a growth medium prepared to more closely match those prevailing conditions.

[0323] A metadata study was conducted covering a 15-year review period (2000-2014) in which 54,140 samples were collected and tested, presenting a review of common culturable microorganisms recoverable from pharmaceutical water systems (Tim Sandle, September 2015. SOJ Microbiology & Infectious Diseases 3(2):1-8).

[0324] Following this metadata analysis and the recommendations of the cartridge method, a list of microorganisms was tested. The gram-negative bacteria used in this example were Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 35654, Brevundimonas diminuta ATCC 19146, Burkholderia cepacia ATCC 25608, Citrobacter freundii ATCC 8090, Edwardsiella tarda ATCC 15947, Escherichia coli ATCC 8739, Enterobacter aerogenes ATCC 35028, Ochrobacterium anthropi CIP 82.115, Klebsiella pneumoniae ATCC 13883, Methylobacterium extroquens CIP 106787, Moraxella osloensis ATCC 19976, Pantoea agglomerans ATCC 27155, Proteus mirabilis ATCC 27156, and the like. 29906, Pseudomonas aeruginosa ATCC 10145, Pseudomonas fluorescens ATCC 13525, Ralstonia picketii ATCC 27511, Salmonella typhimurium ATCC 13311, Serratia marcescens ATCC 13880, Pseudomonas sonnei ATCC 25931, Sphingomonas paucimobilis ATCC 29837, Stenotrophomonas maltophilia ATCC 13637, and Yersinia enterocolitica ATCC 9610. The gram-positive bacteria were Bacillus subtilis ATCC 6633, Pseudomonas faecalis ATCC 19433, and Staphylococcus aureus ATCC 6538. The yeasts and molds tested were Aspergillus brasiliensis ATCC® 16404™ and Candida albicans ATCC® 10231™. This selection covers at least 84% of the microbial contaminants in drinking water systems, 77% of the contaminants in purified water systems, and the most common contaminants in pharmaceutical WFI systems.

[0325] A mixture of 150 μL of MB660R-DBCO (100 μM) and sulfo-Cy5 acid (100 μM) was spread onto Reasoner's 2A agar (R2A) plates (0.5 g / L yeast extract, 0.5 g / L proteose peptone, 0.5 g / L casein hydrolysate, 0.5 g / L glucose, 0.5 g / L starch, 0.3 g / L dipotassium hydrogen phosphate, 0.024 g / L magnesium sulfate anhydrous, 0.024 g / L sodium pyruvate). 0.3 g / L, agar 15.0 g / L, final pH 7.2 ± 0.2), TSA agar plates (tryptone 15 g / L, Soja papain digested peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.3 ± 0.2) or SDA agar plates (pancreatic digest of casein 5 g / L, pepsin digest of animal tissues 5 g / L, dextrose 40 g / L, agar 15 g / L, pH value 5.6 ± 0.2). Alternatively, 150 μl of a mixture of AF350-NHS ester (150 μM) and AF405-DBCO (150 μM) was deposited on the same set of microorganisms in the same conditions. Each agar plate contained one drop of the mixture of dyes.

[0326] Each microbial culture was filtered onto a 0.45 μm PVDF or MCE membrane (47 mm). Each membrane was then deposited on top of a drop of fluorescent dye on each agar plate. The plates were then incubated at 32.5° C. for the minimum time required to enumerate all the microcolonies spiked onto the membrane using a microcolony semi-automated counter (MICA, DIAMIDEX, when MB660R-DBCO and sulfo-Cy5 acid are used: Cy5 light power 100%, gain 1, exposure time 100 and 200 ms; when AF350-NHS ester and AF405-DBCO are used: UV light power 100%, gain 1, exposure time 50 and 100 ms). The results are presented in Tables 11 and 12 below.

[0327] [Table 11]

[0328] [Table 12]

[0329] Example 11 Dyeing method using a mixture of fluorochromes Addition of fluorescent dyes to bacterial cultures prior to filtration Bacillus subtilis ATCC 6633 was diluted from a previously quantified cryopreserved stock in NaCl 0.9% to obtain a defined number of organisms per experimental condition. A mixture of MB660R-DBCO (50 μM) and Sulfo-Cy5 acid (50 μM) was added to the suspension in a final volume of 10 ml NaCl 0.9%. The resulting suspension was filtered onto a white or black MCE membrane and the organisms were grown on TSA plates (tryptone 15 g / L, Soja papain digest peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.3 ± 0.2) at 32.5 °C for 16 h.

[0330] Incorporation of fluorescent dyes in agar media A mixture composed of 150 μl of MB660R-DBCO (50 μM) and sulfo-Cy5 acid (50 μM) was streaked or added onto the surface of a TSA plate (tryptone 15 g / L, Soja papain digested peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.3 ± 0.2), mixed to melt the TSA agar medium, and then poured onto the plate. The agar plate was kept at 4° C. for 12 days. On the day of the experiment, Bacillus subtilis ATCC 6633 was diluted in NaCl 0.9% from a previously quantified cryopreserved stock to obtain a defined number of microorganisms per experimental condition. The resulting suspension was filtered onto a white MCE membrane and the microorganisms were grown on the TSA plate incorporating a mixture of fluorochromes at 32.5° C. for 16 hours.

[0331] Fluorescent dye deposited between the membrane and the agar medium Bacillus subtilis ATCC 6633 was diluted from a previously quantified cryopreserved stock in NaCl 0.9% to obtain a defined number of organisms per experimental condition. The resulting suspension was filtered onto a white or black MCE membrane. A mixture consisting of 150 μl of MB660R-DBCO (50 μM) and sulfo-Cy5 acid (50 μM) was deposited onto a TSA agar plate. The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the organisms were grown on the TSA plate at 32.5° C. for 16 hours.

[0332] For each protocol, after incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, Sulfo-Cy5 acid and MB660R-DBCO:CY5 light power 10%-20% gain 1 exposure time 50 ms and 100 ms).

[0333] Each of these methods allows for efficient staining and detection of microorganisms. The results of these experiments are illustrated in FIG.

[0334] Example 12 Detection method that avoids disconnection from agar medium In this example, several strains of Alicyclobacillus bacteria were used, including Alicyclobacillus acidoterrestris ATCC® 49025™, Alicyclobacillus cycloheptanicus ATCC® 49028™, and Alicyclobacillus hervarius DSM 13609. Strains were diluted in NaCl 0.9% from previously quantified cryopreserved stocks to obtain defined numbers of organisms per experimental condition.

[0335] Protocol 1: Detection was performed on a membrane placed on an agar medium. 150 μL of the fluorescent dye MB660R-DBCO (100 μM) was deposited on a BAT agar plate (yeast extract 2 g / l, D(+) glucose 5 g / l, calcium chloride 0.25066 g / l, magnesium sulfate 0.5 g / l, ammonium sulfate 0.2 g / l, potassium dihydrogen phosphate 3 g / l, zinc sulfate 0.00018 g / l, copper sulfate 0.00016 g / l, manganese sulfate 0.00015 g / l, sodium molybdate dihydrate 0.00030 g / l, agar 18 g / l, pH value 3.8-4.2). Each agar plate contained one drop of the fluorescent dye. Alicyclobacillus cycloheptanicus and Alicyclobacillus hervarius were filtered on a PVDF filtration membrane. The membrane was then deposited on top of the drop of fluorescent dye on each agar plate.

[0336] Protocol 2: Detection was performed directly on agar plates.

[0337] A 250 μl drop containing A. acidoterrestris diluted in purified water and 12.5 μL of the fluorescent dye MB660R-DBCO (250 μM) was streaked onto the surface of the agar medium, and the plate was then incubated at 45° C. for 24 hours.

[0338] For each protocol, after incubation, microcolonies were detected using a semi-automated microcolony counter (MICA, DIAMIDEX, light power 100%, gain 1, exposure time 100 for detection performed on membranes on agar medium; light power 100%, gain 1, exposure time 50 for direct detection on agar medium).

[0339] These two protocols allow for efficient staining and detection of microorganisms. The results of these experiments are illustrated in FIG.

[0340] Example 13 Enumerating bacteria associated with uncomplicated and complicated UTIs The most common causative agent of both uncomplicated and complicated UTIs is Escherichia coli. For agents involved in uncomplicated UTIs, E. coli is followed in prevalence by Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, Group B Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida species. For complicated UTIs, the order of prevalence of causative agents is E. coli, which is the most common, followed by Enterococcus species, Klebsiella pneumoniae, Candida species, Staphylococcus aureus, P. mirabilis, Pseudomonas aeruginosa, and GBS.

[0341] The gram-negative bacteria used in this example were E. coli ATCC® 8739™, Klebsiella pneumoniae ATCC® 13883™ and P. mirabilis ATCC® 29906™. The gram-positive bacteria were E. faecalis ATCC® 19433™ and Staphylococcus aureus ATCC® 6538™. This selection covers 89% of uncomplicated and complicated UTIs.

[0342] A mixture of 150 μL of MB660R-DBCO (100 μM) and sulfo-Cy5 acid (100 μM) was plated on TSA agar plates (tryptone 15 g / L, Soja papain digest peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH value 7.3 ± 0.2) for Gram-positive bacteria and P. mirabilis or on Columbia blood agar plates (peptone 23 g / l, starch 1 g / l, sodium chloride 5 g / l, sheep blood 50 ml / l) for 100% sucrose agar and 100% sucrose agar. , agar 14 g / l, pH value 7.3±0.2) for coliforms (E. coli and Klebsiella pneumoniae) and on TSA agar plates, Columbia blood agar plates or MacConkey agar plates (peptone 20 g / L, lactose 10 g / L, bile salts 1.5 g / L, crystal violet 0.001 g / L, neutral red 0.05 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH value 7.1+ / -0.2) for coliforms (E. coli and Klebsiella pneumoniae). Alternatively, 150 μl of DAPI (50 μM) were deposited on the same set of microorganisms in the same conditions. Each agar plate contained one drop of the mixture of dyes.

[0343] Each bacterium was diluted from a pre-quantified cryopreserved stock to obtain a defined number of organisms per experimental condition, and the resulting suspension was filtered onto a PVDF or MCE membrane.

[0344] The filtration membrane was then deposited on top of the fluorescent dye droplets on each agar plate, and the plates were then incubated at 37° C. for the minimum time required to enumerate all the microcolonies spiked onto the membrane using a semi-automated microcolony counter (MICA, DIAMIDEX, MB660R-DBCO and sulfo-Cy5 acid: Cy5 power 100%, gain 1, exposure times 100 and 200 ms; DAPI: UV power 100%, gain 1, exposure times 50 and 100 ms).

[0345] [Table 13]

[0346] As shown in Table 13, E. coli, which causes 75% of uncomplicated UTIs and 65% of complicated UTIs, can be enumerated using the methods of the present invention in less than 6 hours on TSA agar plates, less than 7.5 hours on Columbia agar plates, and less than 10 hours on MacConkey agar plates. Additionally, the most prevalent microorganisms involved in uncomplicated and complicated UTIs can be enumerated in less than 11 hours on TSA agar plates, Columbia blood agar plates, and MacConkey agar plates.

Claims

1. A method for detecting or enumerating a target microorganism or group of microorganisms, comprising: a) contacting, in a container, a sample suspected of containing said microorganism or group of microorganisms with a solid growth medium containing nutrients to support growth of said microorganism or group of microorganisms and at least one fluorescent dye; b) incubating said container for a sufficient time and under conditions sufficient to form microcolonies of said microorganism or group of microorganisms; and c) detecting or enumerating microcolonies emitting a fluorescent signal of said at least one fluorescent dye. Thereby detecting or enumerating said microorganism or group of microorganisms contained in the sample.

2. The method according to claim 1, wherein prior to step a), the microorganisms in the sample are concentrated on a membrane filter, and in step a), said membrane and microorganisms are contacted with the solid growth medium and said at least one fluorescent dye.

3. Step a) may comprise: a1) contacting a sample suspected of containing said microorganism or group of microorganisms with at least one fluorescent dye; and a2) contacting, in a container, said sample with a solid growth medium containing nutrients to support growth of said microorganism or group of microorganisms. wherein steps a1) and a2) are performed simultaneously or sequentially in any order. The method according to claim 1.

4. The method according to claim 3, wherein step a1) is performed prior to step a2).

5. The method according to claim 4, wherein prior to step a2), the microorganisms in the sample are concentrated on a membrane filter.

6. The method according to claim 1, wherein the membrane filter is made of mixed cellulose ester (MCE), polyvinylidene fluoride (PVDF), nitrocellulose, polytetrafluoroethylene, polycarbonate or nylon.

7. The method according to claim 1, wherein the membrane filter is made of MCE or PVDF.

8. The method according to claim 1, wherein the membrane filter is made of MCE.

9. The method according to claim 1, wherein the membrane filter is made of PVDF.

10. The method according to claim 1, wherein the solid growth medium is selective for said microorganism or group of microorganisms.

11. ​ The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, acridine dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof.

12. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, 4',6-diamidino-2-phenylindole (DAPI), and combinations thereof.

13. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, and combinations thereof.

14. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of MB660R, acridine orange, DRAQ5, Cytrak Orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta 650, Seta 375, Setau 647, SeTau 488, Alexa Fluor 488, ATTO 647, and DAPI, derivatives thereof, and combinations thereof.

15. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of MB660R, DRAQ5, Cytrak Orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta 650, Seta 375, Setau 647, SeTau 488, Alexa Fluor 488 and ATTO 647, derivatives thereof, and combinations thereof.

16. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of MB660R-DBCO, MB660R-acid, acridine orange, DRAQ5, Cytrak Orange, Alexa Fluor 350 NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, ATTO 647 acid and DAPI, derivatives thereof, and combinations thereof.

17. The method according to claim 1, wherein the at least one fluorescent dye is selected from the group consisting of MB660R-DBCO, MB660R-acid, DRAQ5, Cytrak Orange, Alexa Fluor 350 NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO and ATTO 647 acid, derivatives thereof, and combinations thereof.

18. The at least one fluorescent dye is selected from the group consisting of DRAQ5, Alexa Fluor 350, Pacific blue, Cy5, preferably sulfo-Cy5, BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade blue, Seta 650, Setau 647, SeTau 488, MB 660R, Alexa Fluor 488, ATTO 647 and DAPI, derivatives thereof and combinations thereof, preferably DRAQ5, Alexa Fluor 350-NHS ester, Pacific blue-NHS ester, sulfo-Cy5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade blue, Seta 650-DBCO, SeTau 488-NHS, SeTau 647-maleimide, MB 660R-acid, MB 660R-DBCO, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, ATTO 647 acid and DAPI and combinations thereof, the method according to claim 1.

19. The at least one fluorescent dye is selected from the group consisting of DRAQ5, Alexa Fluor 350, Cy5, preferably sulfo-Cy5, BODIPY 500 / 510, prodan, Alexa Fluor 405, Seta 650, Setau 647, MB 660R, Alexa Fluor 488, ATTO 647 and DAPI, derivatives thereof and combinations thereof, preferably DRAQ5, Alexa Fluor 350-NHS ester, sulfo-Cy5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Seta 650-DBCO, SeTau 647-maleimide, MB 660R-acid, MB 660R-DBCO, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, ATTO 647 acid and DAPI and combinations thereof, the method according to claim 1.

20. The target microorganism or group of microorganisms is - Belonging to thermophilic acidophilic bacteria, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus contaminans, and combinations thereof, and / or - Belonging to acetic acid bacteria, preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter, and Asaia, and combinations thereof, and / or - Belonging to lactic acid bacteria, preferably selected from bacteria belonging to the genera Lactobacillus and Weissella, and combinations thereof, and / or - Belonging to anaerobic bacteria, preferably selected from bacteria of the genera Clostridium and Cutibacterium, and combinations thereof, and / or - Belonging to aerobic mesophilic bacteria, preferably selected from the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria, and Burkholderia, and combinations thereof, and / or - Belonging to Gram-negative bacteria, preferably selected from the genera Escherichia and Pseudomonas, and combinations thereof, and / or - Belonging to the group of heterotrophic bacteria, preferably selected from bacteria belonging to the genera Acinetobacter, Erwinomonas, Brevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobactrum, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus, and combinations thereof, and / or - Belonging to the group of yeasts and molds, preferably selected from the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum, and Penicillium, and combinations thereof, the method according to claim 1.

21. The target microorganism or group of microorganisms is - Belonging to thermophilic and acidophilic bacteria, preferably selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus contaminans, and combinations thereof, and / or, - Belonging to acetic acid bacteria, preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter, and Asaia, and combinations thereof, and / or, - Belonging to lactic acid bacteria, preferably selected from bacteria belonging to the genera Lactobacillus and Weissella, and combinations thereof, and / or, - Belonging to anaerobic bacteria, preferably selected from bacteria of the genera Clostridium and Cutibacterium, and combinations thereof, and / or, - Belonging to aerobic mesophilic bacteria, preferably selected from the genera Methylobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria, and Burkholderia, and combinations thereof, and / or, - Belonging to Gram-negative bacteria, preferably selected from the genera Escherichia and Pseudomonas, and combinations thereof, and / or, - Belonging to the group of yeasts and molds, preferably selected from the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum, and Penicillium, and combinations thereof, the method according to claim 1.

22. The target microorganism or group of microorganisms preferably belongs to or comprises a microorganism belonging to the group of thermophilic and acidophilic bacteria selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus acidiphilus, Alicyclobacillus cycloheptanicus, Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus contaminans, and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of MB660R, Alexa Fluor 488, and ATTO647, and derivatives and combinations thereof, more preferably selected from the group consisting of MB660R-acid, MB660R-DBCO, Alexa Fluor 488-acid, Alexa Fluor 488-DBCO, and ATTO647-acid, and combinations thereof, the method according to claim 1.

23. The method according to claim 22, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably from the group consisting of MB660R and derivatives, more preferably from the group consisting of MB660R-acid and MB660R-DBCO, and even more preferably MB660R-DBCO.

24. The microorganism or group of microorganisms of interest is preferably selected from bacteria belonging to the genera Acetobacter, Gluconobacter, Gluconacetobacter, and Asaia and combinations thereof, more preferably selected from the group consisting of Acetobacter aceti, Gluconacetobacter liquefaciens, Gluconobacter oxydans, and Asaia siamensis and combinations thereof, belonging to or comprising a microorganism belonging to the group of acetic acid bacteria (AAB), and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647, and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid, and DAPI and combinations thereof, the method according to claim 1.

25. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably selected from the group consisting of Alexa Fluor 488, MB660R, and ATTO647 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, and ATTO647 acid and combinations thereof, the method according to claim 24.

26. The at least one fluorescent dye is selected from the group consisting of MB660R and ATTO647 and derivatives and combinations thereof, preferably selected from the group consisting of MB660R-acid, MB660R-DBCO, and ATTO647 acid and combinations thereof, more preferably selected from the group consisting of MB660R-acid and ATTO647 acid and combinations thereof, the method according to claim 24.

27. The microorganism or group of microorganisms of interest preferably belongs to or comprises a microorganism belonging to the group of lactic acid bacteria (LAB) selected from bacteria belonging to the genus Lactobacillus, the genus Weissella, and combinations thereof, more preferably selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, Weissella confusa, and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, DAPI, and combinations thereof, preferably selected from the group consisting of Alexa Fluor 488, MB660R, ATTO647, DAPI, derivatives thereof, and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, and ATTO647 acid, and combinations thereof, the method according to claim 1.

28. The at least one fluorescent dye is selected from the group consisting of Alexa Fluor 488, MB660R, DAPI, derivatives thereof, and combinations thereof, in particular selected from the group consisting of Alexa Fluor 488 acid, MB660R-acid, DAPI, and combinations thereof, preferably selected from the group consisting of xanthene-based dyes, more preferably selected from the group consisting of Alexa Fluor 488 acid and MB660R-acid, and combinations thereof, even more preferably MB660R-acid, the method according to claim 27.

29. The microorganism or group of microorganisms of interest preferably belongs to or comprises a microorganism belonging to the group of anaerobic bacteria selected from bacteria of the genus Clostridium and the genus Cutibacterium and combinations thereof, more preferably selected from the group consisting of Clostridium sporogenes and Cutibacterium acnes and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes, DAPI, and combinations thereof, preferably selected from the group consisting of MB660R, Cy5, preferably sulfo-Cy5, DAPI, derivatives thereof, and combinations thereof, more preferably selected from the group consisting of MB660R-DBCO, sulfo-Cy5 acid, DAPI, and combinations thereof, the method according to claim 1.

30. The method according to claim 29, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes and cyanine-based dyes, preferably MB660R and Cy5, more preferably selected from the group consisting of sulfo-Cy5 and its derivatives and combinations, and even more preferably selected from the group consisting of MB660R-DBCO and sulfo-Cy5 acid and their combinations.

31. The method according to claim 29, wherein the at least one fluorescent dye comprises MB660R-DBCO and sulfo-Cy5 acid.

32. The target microorganism or group of microorganisms is preferably selected from bacteria belonging to the genera Methyrobacterium, Pseudomonas, Bacillus, Escherichia, Staphylococcus, Acinetobacter, Cronobacter, Klebsiella, Salmonella, Enterococcus, Shigella, Kocuria, and Burkholderia and combinations thereof, more preferably selected from the group consisting of Methyrobacterium extorquens, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Cronobacter sakazakii, Klebsiella pneumoniae, Salmonella typhimurium, Enterococcus faecalis, Shigella sonnei, Kocuria rhizophila, and Burkholderia cepacia and combinations thereof, belonging to or including microorganisms belonging to the group of aerobic mesophilic bacteria (AMB), and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, acridine dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, and DAPI and combinations thereof, preferably selected from the group consisting of acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, BODIPY dyes, preferably BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647, and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa FluorThe method according to claim 1, selected from the group consisting of 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid, and DAPI, and combinations thereof.

33. The method according to claim 32, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and their combinations, preferably DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, more preferably sulfo-Cy5, BODIPY dyes, more preferably BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, more preferably Seta 650 or Seta 375, SeTau dyes, more preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R and ATTO647 and their derivatives and combinations, and even more preferably selected from the group consisting of DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO and ATTO647 acid and their combinations.

34. The method according to claim 32, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably, optionally, cyanine-based dyes, preferably, Cy5, in particular, sulfo-Cy5, and is more preferably selected from the group consisting of MB660R and derivatives and combinations thereof in combination with a fluorescent dye selected from the group consisting of sulfo-Cy5 acid and, optionally, MB660R-DBCO in combination with sulfo-Cy5 acid.

35. The method according to claim 1, wherein the target microorganism or group of microorganisms preferably belongs to or comprises a microorganism belonging to the group of Gram-negative bacteria selected from bacteria belonging to the genera Escherichia, Proteus, and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa and combinations thereof, and the at least one fluorescent dye may be selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, cyanine-based dyes, and DAPI and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, MB660R, and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO, and DAPI and combinations thereof.

36. The method according to claim 35, wherein the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes, and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, MB660R, and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO, and combinations thereof.

37. The microorganism or group of microorganisms of interest preferably belongs to a group of Gram-negative bacteria selected from bacteria belonging to the genera Escherichia and Pseudomonas and combinations thereof, more preferably selected from Escherichia coli, Pseudomonas aeruginosa and combinations thereof, or comprises the same, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, pyrene-based dyes, coumarin-based dyes, cyanine-based dyes and DAPI and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, Alexa Fluor 350, Alexa Fluor 405, MB660R and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO, MB660R-DBCO and DAPI and combinations thereof, according to the method of claim 1.

38. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, pyrene-based dyes, coumarin-based dyes and cyanine-based dyes and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, Alexa Fluor 350, Alexa Fluor 405 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO and MB660R-DBCO and combinations thereof, according to the method of claim 37.

39. The at least one fluorescent dye is selected from the group consisting of pyrene-based dyes, coumarin-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor 350 and Alexa Fluor 405 and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, Alexa Fluor 405-DBCO and combinations thereof, according to the method of claim 37.

40. The method according to claim 37, wherein the at least one fluorescent dye is a combination of Alexa Fluor 350-NHS ester and Alexa Fluor 405-DBCO.

41. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes, and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, MB660R, and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO, and combinations thereof, the method according to claim 37.

42. The target microorganism or group of microorganisms preferably belongs to or includes a microorganism belonging to the group of Gram-positive bacteria selected from bacteria belonging to the genus Staphylococcus and the genus Enterococcus and combinations thereof, more preferably selected from Staphylococcus aureus and Enterococcus faecalis and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes, and DAPI and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, MB660R, DAPI, and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO, DAPI, and combinations thereof, the method according to claim 1.

43. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, cyanine-based dyes, and combinations thereof, preferably selected from the group consisting of Cy5, preferably sulfo-Cy5, and MB660R, and derivatives and combinations thereof, more preferably selected from the group consisting of sulfo-Cy5 acid, MB660R-DBCO, and combinations thereof, the method according to claim 42.

44. The target microorganism or group of microorganisms is preferably selected from bacteria belonging to the genus Acinetobacter, Aeromonas, Brevundimonas, Burkholderia, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ochrobactrum, Klebsiella, Methylobacterium, Moraxella, Pantoea, Proteus, Pseudomonas, Ralstonia, Salmonella, Serratia, Shigella, Sphingomonas, Stenotrophomonas, Yersinia, Bacillus, Enterococcus, Micrococcus, Staphylococcus and combinations thereof, more preferably selected from Acinetobacter baumannii, Aeromonas hydrophila, Brevundimonas diminuta, Sepacia bacteria, Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Escherichia coli, Ochrobactrum anthropi, Klebsiella pneumoniae, Methylobacterium extorquens, Moraxella osloensis, Pantoea agglomerans, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia pickettii, Salmonella typhimurium, Serratia marcescens, Shigella sonnei, Sphingomonas paucimobilis, Stenotrophomonas maltophilia, Yersinia enterocolitica, Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus (Kocuria rhizophila), Staphylococcus aureus and combinations thereof, belonging to or containing a microorganism belonging to the group of heterotrophic bacteria, and the at least one fluorescent dye is selected from the group consisting of acridine dyes, xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squalene-based dyes, squalene rotaxane-based dyes and DAPI and combinations thereof, preferably acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.

55. A method according to claim 1, selected from the group consisting of BODIPY dyes, preferably BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, ATTO647 and DAPI and their derivatives and combinations; more preferably, acridine orange, DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, ATTO647 acid and DAPI and their combinations.

45. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, anthracene-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, and combinations thereof, preferably DRAQ5, Cytrak orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, Cy5.5, preferably sulfo-Cy5.5, BODIPY dyes, preferably BODIPY 500 / 510, prodan, Alexa Fluor 405, Cascade Blue, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, and ATTO647, and derivatives and combinations thereof, more preferably DRAQ5, Cytrak orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, sulfo-Cy5.5 acid, BODIPY 500 / 510, prodan, Alexa Fluor 405-DBCO, Cascade Blue, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, Alexa Fluor 488-DBCO, MB660R-acid, MB660R-DBCO, and ATTO647 acid, and combinations thereof, the method according to claim 44.

46. The target microorganism or group of microorganisms is preferably selected from fungi belonging to the genera Candida, Zygosaccharomyces, Aspergillus, Saccharomyces, Geotrichum, and Penicillium and combinations thereof, more preferably selected from the group consisting of Candida albicans, Zygosaccharomyces bailii, Aspergillus brasiliensis, Saccharomyces cerevisiae, Geotrichum candidum, Penicillium variotii, Penicillium chrysogenum, and combinations thereof, belongs to or includes a microorganism belonging to the group of yeasts and molds, and the at least one fluorescent dye is selected from the group consisting of acridine dyes and xanthene-based dyes, preferably selected from the group consisting of rhodamine dyes, fluorescein dyes, and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes, squaraine rotaxane-based dyes, and DAPI and combinations thereof, preferably selected from the group consisting of acridine orange, Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, prodan, Alexa Fluor 405, Cascade Blue, BODIPY 500 / 510, Seta dye, preferably Seta 650, SeTau dye, preferably SeTau 647 or SeTau 488, Alexa Fluor 488, MB660R, and DAPI and derivatives and combinations thereof, more preferably selected from the group consisting of acridine orange, Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, prodan, Alexa Fluor 405-DBCO, Cascade Blue, BODIPY 500 / 510, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid, MB660R-DBCO, and DAPI and combinations thereof, the method according to claim 1.

47. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably from the group consisting of rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, dipyrromethene-based dyes, naphthalene-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and combinations thereof, preferably Alexa Fluor 350, Pacific Blue, Cy5, preferably sulfo-Cy5, prodan, Alexa Fluor 405, Cascade Blue, BODIPY 500 / 510, Seta dyes, preferably Seta 650, SeTau dyes, preferably SeTau 647 or SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, Pacific Blue-NHS ester, sulfo-Cy5 acid, prodan, Alexa Fluor 405-DBCO, Cascade Blue, BODIPY 500 / 510, Seta 650-DBCO, SeTau 647-maleimide, SeTau 488-NHS, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof, the method according to claim 46.

48. The at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably from the group consisting of MB660R and derivatives and combinations thereof, preferably MB660R-DBCO, the method according to claim 46.

49. The target microorganism or group of microorganisms preferably comprises one or several bacteria selected from the group consisting of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobic bacteria, aerobic mesophilic bacteria, Gram-negative bacteria and Gram-positive bacteria, heterotrophic bacteria and combinations thereof, and preferably one or several microfungi selected from yeast and mold, and said at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squaraine-based dyes and squaraine rotaxane-based dyes and combinations thereof, preferably Alexa Fluor 350, Pacific blue, Cy5, preferably sulfo-Cy5, Alexa Fluor 405, Cascade blue™, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647 and SeTau 488, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, Pacific blue™-NHS ester, sulfo-Cy5 acid, Alexa Fluor 405-DBCO, Cascade blue, Seta 650-DBCO, Seta 375-NHS, SeTau 488-NHS, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof, the method according to claim 1.

50. The target microorganism or group of microorganisms preferably comprises one or several bacteria selected from the group consisting of thermoacidophilic bacteria (TAB), acetic acid bacteria (AAB), lactic acid bacteria (LAB), anaerobic bacteria, aerobic mesophilic bacteria, Gram-negative bacteria and Gram-positive bacteria and combinations thereof, and preferably one or several microfungi selected from yeast and mold, and the at least one fluorescent dye is selected from the group consisting of xanthene-based dyes, preferably rhodamine dyes, fluorescein dyes and carbopyronine-based dyes, coumarin-based dyes, cyanine-based dyes, pyrene-based dyes, squalene-based dyes and squalene rotaxane-based dyes and combinations thereof, preferably selected from the group consisting of Alexa Fluor 350, Cy5 (trademark), preferably sulfo-Cy5, Alexa Fluor 405, Seta dyes, preferably Seta 650 or Seta 375, SeTau dyes, preferably SeTau 647, Alexa Fluor 488 and MB660R and derivatives and combinations thereof, more preferably selected from the group consisting of Alexa Fluor 350-NHS ester, sulfo-Cy5 acid, Alexa Fluor 405-DBCO, Seta 650-DBCO, Seta 375-NHS, SeTau 647-maleimide, Alexa Fluor 488 acid, MB660R-acid and MB660R-DBCO and combinations thereof, even more preferably selected from the group consisting of sulfo-Cy5 acid and MB660R-DBCO and combinations thereof, the method according to claim 1.

51. The microorganism or group of microorganisms of interest belongs to or comprises a microorganism selected from the group consisting of Aspergillus brasiliensis, Candida albicans, Escherichia coli, and combinations thereof, and the at least one fluorescent dye is selected from the group consisting of Bodipy 500 / 510, Alexa Fluor 350 or a derivative thereof, preferably Alexa Fluor 350-NHS ester, Pacific blue or a derivative thereof, preferably Pacific blue-NHS ester, prodan, Alexa Fluor 405 or a derivative thereof, preferably Alexa Fluor 405-DBCO, Cascade Blue, Seta 650 or a derivative thereof, preferably Seta 650-DBCO, Setau 488 or a derivative thereof, preferably Setau 488-NHS, Setau 647 or a derivative thereof, preferably Setau 647-maleimide, and any combination thereof, the method according to claim 1.

52. The method according to claim 2, wherein before step c) and after step b), the membrane filter is removed from the solid growth medium, and in step c), the microcolonies thereon are detected and / or counted.

53. The method according to claim 1, wherein in step c), a fluorescence microscope or a solid-phase hemacytometer is used to detect and / or count the microcolonies emitting a fluorescence signal.

54. Use of the method according to claim 1 in determining whether a sample contains at least one microorganism of interest, in determining whether a sample is sterile, or in determining the bioburden of a sample.

55. A kit for detecting and / or counting a microorganism or group of microorganisms of interest according to the method according to any one of claims 1 to 54, the kit comprising: (i) - preferably, at least one fluorescent dye as defined in any one of claims 11 to 19, and optionally, - preferably, at least one membrane filter as defined in any one of claims 6 to 9 and / or - at least one container containing a solid growth medium containing nutrients to support the growth of the microorganism or group of microorganisms of interest or the materials required to obtain said at least one container, and / or - A leaflet providing instructions for using the kit, or (ii)- Preferably, at least one container containing at least one fluorescent dye defined in any one of claims 11 to 19, and a solid growth medium containing nutrients to support the growth of the target microorganism or group of microorganisms, or the materials necessary to obtain said at least one container, and Optionally, - Preferably, at least one membrane filter defined in any one of claims 6 to 9, and / or - A leaflet providing instructions for using the kit A kit comprising.

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