Attenuated-background microbiological nutrient media and methods of using the same

Abstract

To provide nutrient media with reduced background.SOLUTION: The present invention provides a liquid or solid or semi-solid nutrient medium comprising at least one dye and / or pigment, wherein the nutrient medium supports the growth of microorganisms, wherein the at least one dye and / or pigment substantially reduces background of the medium, and wherein the solid or semi-solid nutrient medium further comprises a gelling agent.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] In many industries, especially in the food, beverage, healthcare, electronics, and pharmaceutical industries, it is essential to rapidly analyze samples for the degree of contamination by microorganisms (such as bacteria, yeast, or mold). In particular, pharmaceutical and biopharmaceutical companies are required to test clean rooms and sterile products for the presence of microbiological contaminants by using nutrient media and attempting to culture some microorganism. However, most nutrient media have background that interferes with optical detection methods. Therefore, there is a need for nutrient media with reduced background. Summary of the Invention [Problem to be solved by the invention]

[0002] There is a need for a nutrient medium with reduced background. [Means for solving the problem]

[0003] The invention features a microbiological nutrient medium that reduces background, for example, a microbiological nutrient medium that contains a dye and / or dye that allows for more rapid detection and / or higher detection sensitivity of microorganisms in a sample. The invention features a liquid, solid, or semi-solid nutrient medium that contains at least one dye and / or dye. The solid or semi-solid medium also includes a gelling agent. The nutrient medium supports the growth of microorganisms, and the at least one dye and / or dye substantially reduces the background of the medium.

[0004] In one aspect, the invention features a liquid, solid, or semi-solid nutrient medium comprising at least one dye and / or dye, where the nutrient medium supports the growth of a microorganism, where the at least one dye and / or dye substantially reduces background of the medium, and where the solid or semi-solid nutrient medium further comprises a gelling agent.

[0005] In one embodiment, the nutrient medium is disposed in a container, such as a dish, plate, or slide. In another embodiment, the nutrient medium is proud relative to the container. In another embodiment, the nutrient medium is rounded. In one embodiment, the nutrient medium comprises a flat portion. In another embodiment, the nutrient medium further comprises a sloping peripheral portion.

[0006] In one embodiment, the at least one dye and / or pigment causes the medium to become black. In another embodiment, the at least one dye and / or pigment substantially reduces a red background, an orange background, a yellow background, a green background, a blue background, or a purple background in the nutrient medium due to exposure to white light. In another embodiment, the at least one dye and / or pigment substantially reduces a background in the nutrient medium due to exposure to blue light. In one embodiment, the at least one dye and / or pigment provides a peak optical density in the nutrient medium of at least 0.004, such as at least 0.022, 0.046, 0.071, 0.097, 0.125, 0.155, 0.187, 0.222, 0.260, 0.301, 0.347, 0.398, 0.456, 0.523, 0.602, 0.699, 0.824, 1, 1.30, 2, 3, 4, 5, or 6.

[0007] In one embodiment, the nutrient medium comprises a pigment. In another embodiment, the pigment comprises carbon particles or iron oxide particles. In another embodiment, the carbon particles have an average diameter of less than 500 nm. In another embodiment, the carbon particles have an average diameter of about 100 nm to about 400 nm. In one embodiment, the invention provides carbon particles comprising carbon black.

[0008] In another embodiment, the nutrient medium comprises a dye. In another embodiment, the dye comprises an azo dye. In another embodiment, the azo dye is Acid Black 71, Acid Black 194, Acid Black 2, Reactive Black 5, Direct Black 36, or Nyanza Black.

[0009] In one embodiment, the nutrient medium further comprises a polymer that binds at least one dye and / or pigment, hi another embodiment, the polymer comprises polydiallyldimethylammonium chloride (polyDADMAC), starch, cellulose, chitosan, β-cyclodextrin, a calix[4]arene-based polymer, or a p-tert-butylcalix[4]arene-based polymer.

[0010] In one embodiment, the nutrient medium further comprises a free radical scavenger selected from the group consisting of, for example, N-acetyl-L-cysteine, sodium ascorbate, thiourea, nicotinamide, pyridoxine, sodium pyruvate, D-mannitol, 2-oxo-4-thiomethylbutyric acid (OBTA), 3-(methylthio)propionaldehyde (methional), dimethylsulfoxide (DMSO), tert-butyl alcohol, benzoate, catalase, NADH peroxidase, thiol peroxidase, bacterioferritin comigratory protein, alkyl hydroperoxide reductase, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrin.

[0011] In one embodiment, the microorganism is, for example, a species of the genus Acinetobacter, Aspergillus, Bacillus, Burkholderia, Campylobacter, Candida, Clostridium, Corynebacterium, Delftia, Dermacoccus, Escherichia, Exserohilum, Helicobacter, Kocuria, Legionella, Listeria, Methylobacterium, Micrococcus, or the like. cus, Mycobacterium, Paenibacillus, Penicillium, Propionibacterium, Pseudomonas, Ralstonia, Salmonella, Selenomonas, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Vibrio, and Yersinia.

[0012] In one embodiment, the gelling agent is selected from the group consisting of agar, gellan, sodium alginate, xanthan gum, guar gum, gelatin, agarose, polyacrylamide, and a polysaccharide produced by Rhizobium sp. (CNCM number: I-1809).

[0013] In another embodiment, the nutrient medium comprises nutrients selected from the group consisting of Trypticase Soy Broth (TSM), Fluid Thioglycollate Broth (FTM), Schaedler's medium, Lysogeny / Lennox (LB) broth, Mannitol Yeast Extract Peptone Broth, Terrific Broth II, Super Optimal Broth (SOC) with Catabolite Repression Medium, Thornton's medium, Lowenstein-Jensen medium, Sabouraud dextrose medium, D / E Neutral Medium, Loeffler's medium, Nalidixic Acid and Colimycin (ANC) medium, and Artificial Seawater Medium. In one embodiment, the nutrient medium comprises nutrients derived from bismuth sulfite agar, Chapman agar, Mueller-Hinton agar (MHA), blood agar, MacConkey agar, Trypticase Soy Agar (TSA), Sabouraud Dextrose Agar (SDA), Reasoner's 2A Agar (R2A), Mueller-Miller Tellurite Agar, or Tinsdale Tellurite Agar.

[0014] In another embodiment, the nutrient medium is a solid or semi-solid. In one embodiment, the nutrient medium is a liquid. In another embodiment, the nutrient medium is disposed within a porous matrix.

[0015] In one embodiment, the nutrient medium has a pH value of about pH 3.0 to about pH 12.0.

[0016] In one aspect, the invention provides a method for detecting microbial growth by contacting a surface of a nutrient medium with a sample under suitable conditions to allow microbial growth and optically detecting the surface to determine any microbial growth. In one embodiment, the contacting comprises contacting a membrane containing the sample with the surface. In another embodiment, the contacting comprises contacting another surface with the surface. In another embodiment, the contacting comprises allowing air to contact the surface.

[0017] In one embodiment, the microorganism belongs to a genus selected from the group consisting of Acinetobacter, Aspergillus, Bacillus, Burkholderia, Campylobacter, Candida, Clostridium, Corynebacterium, Delftia, Dermacoccus, Escherichia, Exelohilum, Helicobacter, Kocuria, Legionella, Listeria, Methylobacterium, Micrococcus, Mycobacterium, Paenibacillus, Penicillium, Propionibacterium, Pseudomonas, Ralstonia, Salmonella, Selenomonas, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Vibrio, and Yersinia.

[0018] In another embodiment, optically detecting includes detecting fluorescence.

[0019] definition The term "about" as used herein refers to a numerical value that is ±10% of the stated numerical value.

[0020] The term "background" as used herein refers to the light scattered, reflected, or emitted from a growth medium in response to incident light in the absence of microorganisms.

[0021] The term "dye" as used herein refers to a colored substance that is soluble in water.

[0022] The term "label," as used herein, refers to an exogenous substance that is optically detectable.

[0023] The term "optical density" as used herein refers to the attenuation of light per cm (Log(I / I)) exhibited by a dye and / or pigment. "Peak optical density" as used herein refers to the optical density at the absorption maximum.

[0024] The term "pigment" as used herein refers to a colored substance that is insoluble in water.

[0025] The term "substantially reduce" as used herein refers to a background reduction of at least 50% relative to a nutrient medium that does not contain the dye and / or pigment as described herein. In particular, the background reduction can be at least 60, 65, 70, 75, 80, 85, 90, 95, or 99%. The reduction can occur over one or more wavelengths, for example, over a range of wavelengths. [Brief description of the drawings]

[0026] [Figure 1A] Photographs of colonies grown on different growth media: RODAC plates (Becton Dickinson) (left), the medium of the present invention (center), and TSA medium covered with a black membrane (cassette from Rapid Micro Biosystems, Inc., Lowell, Mass.) (right). Figure 1A shows D. nishinomiyaensis, Figure 1B shows C. albicans, and Figure 1C shows M. luteus. [Figure 1B] This is a continuation of Figure 1A. [Figure 1C] This is a continuation of Figure 1B. [Diagram 2]1 is a series of photographs showing microbial colonies grown on the media of the present invention (top row) and on TSA medium with a membrane (bottom row). A. brasiliensis, B. subtilis, C. albicans, P. aeruginosa, and S. aureus were grown on the media. [Diagram 3] 1 is a graph showing background fluorescence levels for cassettes of Nanoshel® carbon black and TSA with lecithin and polysorbate 80 (TSA-LP80) (black medium) and a control TSA-LP80 cassette with membrane (control medium). Background fluorescence levels of media were measured after storage in a refrigerator or at room temperature for the indicated periods of time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention provides a nutrient medium for optical detection of microorganisms and a method of using the same. The nutrient medium of the present invention comprises dyes and / or pigments that reduce the background of the medium, making it useful for optical detection, for example by visual or automated detection. The medium is advantageous in increasing the shelf life and stability under various storage temperatures, and / or reducing the time to detect certain microorganisms, and / or detecting microorganisms with low growth rates, and / or increasing the recovery rate from different surfaces for microbiological monitoring.

[0028] The nutrient medium of the present invention is useful in detecting microorganisms, for example in the growth or sensitivity of microorganisms. The dyes and / or pigments substantially reduce the background of the medium, enhancing the ability to detect microorganisms.

[0029] Microorganisms can be detected by any suitable optical method, for example by visual inspection, fluorescence, phosphorescence, luminescence, absorption, light scattering or reflection. Detection can be detection of innate optical properties of the microorganism, such as autofluorescence, bioluminescence, colorimetry, phosphorescence, light scattering, absorption or reflection, or can be derived from a label, such as a fluorescent label or a chemiluminescent label. In one embodiment, detection is detection of blue light induced autofluorescence, for example as employed in the GrowthDirect® automated detection system.

[0030] External illumination may or may not be employed in the detection. In certain modalities, a specific wavelength or wavelength range of external illumination may be required, for example, for the detection of fluorescence or phosphorescence. In other modalities, non-specific wavelengths may be employed, for example, for the detection of reflection. In other modalities, external illumination is not required, for example, for the detection of bioluminescence or chemiluminescence. Those skilled in the art can select suitable light sources (if used), such as LED lights, lasers, and gas discharge lamps, for the detection method. Broad spectrum lamps may also be employed with suitable spectral filters. Signals from microorganisms may be in the range spanning the UV-NIR range, typically in the visible range.

[0031] The nature of the dyes and / or pigments employed may vary depending on the desired optical detection method. For example, dyes or pigments may be employed that reduce background from a broad spectral region across the visible range. Alternatively, the dyes and / or pigments may reduce background from a narrower spectral region that corresponds to a specific signal from the microorganism, or a combination of the signal and the incident light. For example, the dyes and / or pigments may absorb or scatter the incident light to prevent excitation of components of the nutrient medium. Alternatively, the dyes and / or pigments may absorb or scatter light emitted by the nutrient medium in response to the incident light. A combination of such mechanisms is also envisaged by the present invention.

[0032] The dyes and / or pigments, or combinations thereof, may, for example, substantially absorb or scatter across the entire visible range and therefore appear black, or the dyes and / or pigments, or combinations thereof, may substantially reduce background in one or more of the following spectral ranges: red background (625-740 nm), orange background (590-625 nm), yellow background (565-590 nm), green background (495-565 nm), blue background (400-495 nm), or purple background (380-400 nm).

[0033] The dyes and / or pigments are present in an amount sufficient to substantially reduce the desired background. Typically, the optical density (OD) of the dyes and / or pigments in the nutrient medium is between 0.004 and 6, such as at least 0.022, 0.046, 0.071, 0.097, 0.125, 0.155, 0.187, 0.222, 0.260, 0.301, 0.347, 0.398, 0.456, 0.523, 0.602, 0.699, 0.824, 1, 1.30, 2, 3, 4, or 5, or up to 6, 5, 4, 3, 2, 1.3, or 1, such as between 1 and 6.

[0034] Dyes, pigments, and additional components Any suitable dye, pigment, or combination thereof may be employed in the nutrient medium. The dye and / or pigment reduces background and does not substantially interfere with the growth of the microorganism to be detected. Preferably, the dye and / or pigment does not migrate to the surface being tested. The dye and / or pigment may be of any suitable color, such as black, red, orange, yellow, green, blue, or purple. For example, the dye and / or pigment may make the medium visually black. Examples of pigments include nanoparticles, such as carbon nanoparticles from carbon black, and iron oxide. Exemplary sizes of nanoparticles are less than 500 nm, e.g., between 100 and 400 nm, e.g., about 1 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 50 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 195 nm, about 200 nm, about 205 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 295 nm, about 300 nm, about 305 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 395 nm, about 400 nm, about 405 nm, about 410 nm, about 425 nm, about 450 nm, about 495 nm, or about 499 nm.In another embodiment, the particles are from about 100 nm to about 400 nm, e.g., from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 100 nm to about 150 nm, from about 100 nm to about 125 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, about 150 nm to about 175 nm, about 200 nm to about 400 nm, about 200 nm to about 375 nm, about 200 nm to about 350 nm, about 200 nm to about 325 nm, about 200 nm to about 300 nm, about 200 nm to about 250 nm, about 300 nm to about 400 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm, or about 375 nm to about 400 nm. In another embodiment, the carbon particles can be carbon black, graphitized carbon black, graphitic carbon, turbostratic carbon, black carbon, soot, biochar, carbon nanotubes, graphene, carbon nanofibers, or other light absorbing carbonaceous particles.Representative concentrations of nanoparticles are about 0.25% (w / v) to about 2.5% (w / v), for example, about 0.25% (w / v) to about 0.85% (w / v), about 0.25% (w / v) to about 1.5% (w / v), about 0.30% (w / v) to about 0.75% (w / v), about 0.30% (w / v) to about 2% (w / v), about 0.35% (w / v) to about 0.65% (w / v), about 0.35% (w / v) to about 1.75% (w / v), about 0.40% (w / v) to about 0.75% (w / v), Approximately 0.40% (w / v) ~ approximately 2% (w / v), approximately 0.45% (w / v) ~ approximately 0.85% (w / v), approximately 0.45% (w / v) ~ approximately 1.5% (w / v), approximately 0.50% (w / v) ~ approximately 0.75% (w / v), approximately 0.50% (w / v)~about 1.5%(w / v), about 0.50%(w / v)~about 2%(w / v), about 0.55%(w / v)~about 0.90%(w / v), about 0.55%(w / v)~about 1.75%(w / v), about 0.60%(w / v)~about 0.95%( 0.60% (w / v) ~ 2% (w / v), 0.65% (w / v) ~ 1% (w / v), 0.65% (w / v) ~ 1.75% (w / v), 0.70% (w / v) ~ 1% (w / v), 0.70% (w / v) v) ~ approx. 1.7% (w / v), approx. 0.75% (w / v) ~ approx. 1.5% (w / v), approx. 0.75% (w / v) ~ approx. 2% (w / v), approx. 0.80% (w / v) ~ approx. 1.2% (w / v), approx. 0.80% (w / v) ~ approx. 1.8% (w / v) ), about 0.85% (w / v) to about 1.5% (w / v), about 0.85% (w / v) to about 2% (w / v), about 0.90% (w / v) to about 1.5% (w / v), about 0.90% (w / v) to about 1.75% (w / v), about 0.95% (w / v) to about 1.5% (w / v), about 0.95% (w / v) to about 2% (w / v), about 1% (w / v) to about 1.8% (w / v), about 1.5% (w / v) to about 2% (w / v), or about 1.75% (w / v) to about 2% (w / v).

[0035] Suitable dyes include azo dyes such as Acid Black 71, Acid Black 194, Reactive Black 5, Direct Black 36, and Nianza Black. Another example is Acid Black 2. Combinations of various dyes and pigments are also contemplated. In certain embodiments, the dyes and / or pigments do not change color with changes in pH. In other embodiments, the dyes and / or pigments are not digested by microbial enzymes.

[0036] It is believed that certain dyes or pigments are prone to migrate to the surface when in contact with the nutrient medium of the present invention. Therefore, the nutrient medium may include a polymer that binds the dye and / or pigment to reduce or prevent migration. An exemplary polymer is polydiallyldimethylammonium chloride (polyDADMAC). Other polymers include sugar-based polymers (oligopolysaccharides), such as starch, cellulose, chitosan, β-cyclodextrin, calix[4]arene-based polymers, and p-tert-butylcalix[4]arene-based polymers.

[0037] The nutrient medium may also be sterilized by various methods, such as autoclaving or gamma irradiation. Gamma irradiation may induce free radicals in the medium. Thus, the nutrient medium of the present invention may also include a free radical scavenger. Examples of free radical scavengers include N-acetyl-L-cysteine, sodium ascorbate, thiourea, nicotinamide, pyridoxine, sodium pyruvate, D-mannitol, 2-oxo-4-thiomethylbutyric acid (OBTA), 3-(methylthio)propionaldehyde (methional), dimethylsulfoxide (DMSO), tert-butyl alcohol, benzoate, and enzymes such as catalase, NADH peroxidase, thiol peroxidase, bacterioferritin symbiotic protein, alkyl hydroperoxide reductase, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrin.

[0038] Gelling Agents and Matrices The nutrient medium may include a gelling agent. Non-limiting examples of gelling agents include agar, gellan, sodium alginate, xanthan gum, guar gum, gelatin, agarose, polyacrylamide, Eladium™ (a polysaccharide produced by Rhizobium species (CNCM number: I-1809)), coagulated eggs, coagulated serum, and combinations thereof. The amount of gelling agent in the solid composition of the present invention depends on the type of gelling agent.

[0039] Alternatively or additionally, the nutrient medium may be absorbed or gelled within a matrix, such as a porous matrix, such as a foam pad, a sponge, a porous plastic, such as Porex, or paper.

[0040] Growth Medium Composition Any suitable nutrients can be included in the nutrient medium of the present invention. The nutrients selected depend on the nature of the microorganism to be detected. Non-limiting examples of nutrients include those contained in Trypticase Soy Medium (TSM), Fluid Thioglycolate Medium (FTM), Schaedler's Medium, Lysogeny / Lennox (LB) broth, Mannitol Yeast Extract Peptone Broth, Terrific Broth II, Super Optimal Broth (SOC) with Catabolite Repression Medium, Nalidixic Acid and Colimycin (ANC) Medium, Lowenstein-Jensen Medium, Sabouraud Dextrose Medium, D / E Neutral Medium, and Loeffler's Medium. The nutrient may also be derived from any one of Thornton's medium, Trypticase Soy Agar (TSA), Sabouraud Dextrose Agar (SDA), Riesner's 2A Agar (R2A), Mueller-Hinton Agar (MHA), Bismuth Sulfite Agar, Chapman Agar, Blood Agar, MacConkey Agar, Mueller-Miller Tellurite Agar, and Tinsdale Tellurite Agar. Further suitable media may be found in WO 2015 / 164827.

[0041] The nutrient medium of the present invention may have any pH value, including acidic, alkaline, or neutral pH. Exemplary pH values ​​of the nutrient medium are about pH 3.0 to about pH 12.0, for example, about pH 3.0 to about pH 11.0, about pH 3.5 to about pH 10.0, about pH 4.0 to about pH 9.5, about pH 4.5 to about pH 9.0, about pH 5.0 to about pH 8.5, about pH 5.5 to about pH 8.0, about pH 5.5 to about pH 7.5, about pH 5.5 to about pH 6.5, about pH 6.0 to about pH 7.0, about pH 6.5 to about pH 7.5, about pH 6.8 to about pH 7.3, about pH 7.0 to about pH 8.0, about pH 7.0 to about pH 8.5, about pH 7.0 to about pH 9.0, about pH 7.0 about pH to about pH 9.5, about pH 7.0 to about pH 10.0, about pH 7.0 to about pH 11.0, about pH 7.0 to about pH 12.0, about pH 8.0 to about pH 9.0, about pH 8.0 to about pH 10.0, about pH 8.0 to about pH 11.0, about pH 8.0 to about pH 12.0, about pH 9.0 to about pH 10.0, about pH 9.0 to about pH 11.0, about pH 9.0 to about pH 12.0, about pH 10.0 to about pH 11.0, about pH 11.0 to about pH 12.0, about pH 3.0 to about pH 4.0, about pH 4.0 to about pH 5.0, or about pH 5.0 to about pH 6.0.

[0042] Optional additives may be included in the nutrient medium of the present invention. Such additives may improve the physicochemical properties of the medium without significantly adversely affecting the growth characteristics of the nutrient medium. Non-limiting examples of additives include buffers (e.g., Tris and / or citric acid), chelating agents (e.g., EDTA), antioxidants, detergents, surfactants (e.g., polysorbate 20), emulsifiers, bactericidal neutralizers (e.g., lecithin, polysorbate 80, histidine, thiosulfate, bisulfite, glycine, divalent cations (e.g., Mg 2+ or Ca 2+ ), and / or thioglycolates), micelle forming agents, micelle inhibitors, and / or polypropylene glycol, polyethylene glycol, and / or carboxymethylcellulose. The culture medium may further comprise antibiotics, e.g., for susceptibility or resistance testing or to select for resistant cells.

[0043] Form of nutrient mediumThe nutrient medium can be in any suitable form or shape. In particular, the nutrient medium can be provided in dry form, where a liquid, e.g. water, is added to form a gel or broth. Alternatively, the nutrient medium can be provided in gel form. In gel form, the nutrient medium is typically contained within or on top of a container, e.g. a plate, dish, slide, or other flat surface, e.g. with a bottom and a lid. The nutrient medium is overhanging relative to the container, e.g. a plate or dish, i.e. it extends above the bottom (and any side walls present) so that it can contact the surface, or it has a recess in the bottom (e.g. below the end of the side walls). Exemplary thicknesses of the solid or semi-solid medium inside or on top of the container are from about 100 μm to about 2 cm, for example, from about 100 μm to about 1.5 cm, from about 100 μm to about 1 cm, from about 150 μm to about 1.5 cm, from about 200 μm to about 1 mm, from about 200 μm to about 9 mm, from about 200 μm to about 2 cm, from about 250 μm to about 1.5 mm, from about 250 μm to about 7.5 mm, from about 300 μm to about 1 mm, from about 300 μm to about Approximately 1.5cm, approximately 400μm ~ approximately 2mm, approximately 400μm ~ approximately 5mm, approximately 400μm ~ approximately 2cm, approximately 500μm ~ approximately 1mm, approximately 500μm ~ approximately 7mm, approximately 500μm ~ approximately 1.5cm, approximately 600μm ~ approx. 9mm, approx. 600μm ~ approx. 2cm, approx. 700μm ~ approx. 1.5cm, approx. 750μm ~ approx. 7.5mm, approx. 800μm ~ approx. 2.5mm, approx. 800μm ~ approx. 1.75cm, approx. 90 0μm~about 9mm, about 900μm~about 2cm, about 1mm~about 5mm, about 1mm~about 7mm, about 1mm~about 1cm, about 2mm~about 6mm, about 2mm~about 1.5cm, about 3mm~about 9mm , about 3mm to about 1.75cm, about 4mm to about 1cm, about 4mm to about 1.5cm, about 4mm to about 2cm, about 5mm to about 1.2cm, about 5mm to about 1.8cm, about 6mm to about 1.5cm, about 6 mm to about 2 cm, about 7 mm to about 1.2 cm, about 7 mm to about 1.9 cm, about 8 mm to about 1.4 cm, about 9 mm to about 1.75 cm, about 1 cm to about 1.4 cm, about 1 cm to about 1.8 cm, about 1.1 cm to about 1.6 cm, about 1.2 cm to about 1.7 cm, about 1.3 cm to about 1.9 cm, about 1.4 cm to about 2 cm, about 1.5 cm to about 2 cm, or about 1.7 cm to about 2 cm. The medium may have a flat or rounded raised shape, or may include a flat portion, for example, 1 to 15 cm in diameter.If the nutrient medium includes a flat portion, the end of the flat portion may be vertical, i.e., 90 degrees, or the medium may include a peripheral portion around the flat portion, in which case the peripheral portion is oblique, i.e., inclined at less than 90 degrees, for example, up to 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 degrees. The surface of the medium may be concave or convex. A concave surface may facilitate contact with other surfaces. The medium may be in the form of a liquid, which may be loaded into a matrix, for example when it includes a dye, or may be employed in a dish or culture tube.

[0044] The dye and / or pigment may be uniformly mixed throughout the nutrient medium or may be present in a surface layer, for example, a layer (e.g., 0.1-10 mm) of nutrient medium containing the dye and / or pigment may be applied to a base layer of nutrient medium that does not contain the dye and / or pigment.

[0045] microorganisms Microorganisms include bacteria, fungi, protists, and archaea. Non-limiting examples of genera of microorganisms (e.g., bacteria or fungi) that can be cultured using the nutrient medium of the present invention include the genus Acinetobacter (e.g., Acinetobacter lwofii), the genus Aspergillus (e.g., Aspergillus brasiliensis or Aspergillus fumigates), the genus Bacillus (e.g., Bacillus clausii, Bacillus idriensis, Bacillus licheniformis, or Bacillus subtilis), the genus Burkholderia (e.g., Burkholderia pseudomallei), the genus Campylobacter (e.g., Campylobacter jejuni), the genus Bacillus subtilis, the genus Bacillus spp. (e.g., Bacillus subtilis ... jejuni), Candida spp. (e.g. Candida albicans), Clostridium spp. (e.g. Clostridium botulinum or Clostridium perfringens), Corynebacterium spp. (e.g. Corynebacterium tuberculostearicum or Corynebacterium xerosis), Delftia spp. (e.g. Delftia acidovorans), Dermacoccus spp. (e.g. Dermacoccus nishinomiyaensis), Escherichia spp. (e.g. Escherichia coli), Exserohilum spp. (e.g. Exserohilum rostratum), rostratum), Helicobacter (e.g., Helicobacter pylori), Kocuria (e.g., Kocuria rhizophila), Legionella (e.g., Legionella pneumophila), Listeria (e.g., Listeria ivanovii),ivanovii, Listeria grayi, or Listeria monocytogenes), Methylobacterium spp. (e.g. Methylobacterium radiotolerans), Micrococcus spp. (e.g. Micrococcus luteus), Mycobacterium spp. (e.g. Mycobacterium tuberculosis, or Mycobacterium leprae), Paenibacillus spp. (e.g. Paenibacillus glucanolyticus), Penicillium spp. (e.g. Penicillium chrysogenum, or Penicillium notatum), notatum), Propionibacterium spp. (e.g. Propionibacterium acnes), Pseudomonas spp. (e.g. Pseudomonas aeruginosa), Ralstonia spp. (e.g. Ralstonia pickettii), Salmonella spp. (e.g. Salmonella enterica serotype Typhi), Selenomonas spp. (e.g. Selenomonas noxia), Serratia spp. (e.g. Serratia marcescens), Shigella spp. (e.g. Shigella flexneri), Staphylococcus spp. (e.g. Staphylococcus aureus, Staphylococcus epidermidis), epidermidis, or Staphylococcus hominis), Stenotrophomonas spp. (e.g., Stenotrophomonas maltophilia), Streptococcus spp. (e.g., Streptococcus pyogenes), Streptomyces spp. (e.g., Streptomyces halsteadii),halstedii), Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, or Vibrio vulnificus), and Yersinia (e.g., Yersinia enterocolitica).

[0046] The nutrient medium may also be used to propagate intracellular viruses, such as lytic viruses (which form plaques in the lawn of the host organism).

[0047] use Exemplary areas of use for the nutrient media of the present invention include general cell culture and laboratory fluids, air, soil, surfaces, industrial or clinical samples, pharmaceutical products (sterile or non-sterile), food products, beverage products, cosmetics, personal care products, or nutritional supplement products for microbial bioburden. The nutrient media may also be employed in other assays, such as clinical assays for blood or other infectious diseases and assays for antibiotic resistance. Samples that may be assayed using the nutrient medium of the present invention include, but are not limited to, industrial samples (e.g., raw materials, intermediate materials, or finished materials in the manufacture of foods, beverages, or dietary supplements; raw materials, intermediate materials, or finished materials in the manufacture of medical devices or in vitro diagnostic devices; chemical products; industrial surfaces; equipment; or mechanisms), pharmaceuticals and reagents used in the preparation of pharmaceutical products (e.g., raw materials, intermediate materials, or finished materials in the manufacture of pharmacological, cosmetic, personal care, blood, or other products for topical or internal use in humans or animals), biological samples, environmental samples (e.g., water samples (e.g., natural water bodies (e.g., rivers, lakes, ponds, and oceans), wastewater, and treated water sources (e.g., municipal water supplies, etc.)), air samples (passive or active), soil samples, and surface samples). Surfaces that may be tested include equipment, materials, and equipment used in the manufacture, packaging, or storage of items (e.g., pharmaceuticals); equipment, materials, and equipment used in research; clothing, bedding, and other fabrics (e.g., for health care providers or patients); and equipment, materials, and equipment used in medical treatment (e.g., hospitals, clinics, and physicians' offices).

[0048] The sample may be placed on the nutrient medium by any suitable method. Such methods include, for example, direct contact with a surface or passive or active air sampling. Furthermore, the method may include contact with any suitable device, such as a loop, needle, or swab. The sample may also be added to the nutrient medium in liquid form. A membrane through which the sample is filtered may also be placed on the nutrient medium of the present invention (dyes and / or pigments may be placed around the edge of the membrane or may penetrate the membrane to reduce detectable background).

[0049] For example, the nutrient medium of the present invention may be employed in detecting microorganisms by colony counting. Microorganism growth on the nutrient medium of the present invention is easier to optically detect, for example, visually or automatically, due to the reduced background. The easier optical detection of microbial growth on the nutrient medium may reduce the time to result (TTR) for certain microorganisms (see, for example, Table 1). The nutrient medium may be particularly useful for rapid microbiological detection, for example, in microcolony detection. For example, the nutrient medium of the present invention may be employed in detecting colonies having a diameter of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 μm, for example, up to 1 mm.

[0050] [Table 1]

[0051] [Example] The following examples are intended to illustrate the present invention and are not intended to impose limitations thereon in any way.

[0052] [Example 1] Comparison of microbial growth on the nutrient medium of the present invention with growth on media without dyes and / or pigments FIG. 1A to FIG. 1C are Rapid Micro Biosystems cassette with black filter on TSA containing lecithin and polysorbate 80 without added pigments or dyes (TSA-LP80) (right); Nutrient medium according to an embodiment of the present invention, namely TSA-LP80 (center) containing 1% mesoporous carbon black nanopowder pigment (graphitized, particle size less than 500 nm) (Sigma Aldrich #699624); and BD RODAC™ nutrient medium (left) without added dyes and / or pigments In USP <1227> Representative colonies are shown for different microbial species cultured according to.

[0053] Figure 1A shows the growth of D. Nishinomiyaensis, Figure 1B shows the growth of C. albicans, and Figure 1C shows the growth of M. luteus.

[0054] Representative images of microbial colonies grown on the media of the present invention (top row) and on Rapid Micro Biosystems cassettes containing TSA medium with membrane (bottom row) are shown in Figure 2. A. brasiliensis, B. subtilis, C. albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were grown on both media.

[0055] [Example 2] Comparison of Recovery Rates of Microorganisms Grown on the Nutrient Media of the Invention and RODAC™ Nutrient Media Table 2 lists the microorganisms cultured, including their individual colony forming unit (CFU) counts, as well as the percent recovery of CFU obtained from embodiments of the invention compared to BD RODAC™ Nutrient Media. For all microorganisms cultured, greater than 70% recovery rate was achieved in the nutrient media of the invention compared to BD RODAC™ Nutrient Media. Each test was performed in six replicates.

[0056] [Table 2]

[0057] [Example 3] Comparison of recoveries of microbial growth using nutrient media in contact tests. Table 3 presents the recovery of B. subtilis sampled on different surface types. The number of CFUs obtained from each surface type is displayed for RODAC™ nutrient medium (RODAC), cassettes containing TSA-LP80, and black medium cassettes containing 1% w / v Nanoshel® carbon mesoporous nanopowder (particle size less than 100 nm) (present invention). The CFU recovery (%) of the RMB standard cassettes versus RODAC™ nutrient medium and the recovery (%) of the black medium cassettes versus RODAC™ nutrient medium are shown. The black medium cassettes demonstrated recovery rates of greater than 85% compared to RODAC™ nutrient medium for all surfaces tested.

[0058] [Table 3]

[0059] [Example 4] Comparison of background fluorescence levels between black medium cassettes and control cassettes after storage at different temperatures Background fluorescence levels of black medium cassettes (Nanoshel® carbon black and TSA with lecithin and polysorbate 80 (TSA-LP80)) and control TSA-LP80 cassettes were measured after storage in a refrigerator (4° C.) or at room temperature for the indicated periods (i.e., 0 weeks to 11 months) (FIG. 3). Control medium stored at room temperature exhibited higher background fluorescence at subsequent time points (e.g., 3, 5, 9, and 11 months) compared to the 1-week storage; medium of the present invention did not exhibit a significant increase in background fluorescence even after 11 months of storage at room temperature.

Claims

1. A liquid or solid or semi-solid nutrient medium comprising at least one dye and / or pigment, wherein the nutrient medium supports the growth of a microorganism, the at least one dye and / or pigment substantially reduces the background of the medium, and the solid or semi-solid nutrient medium further comprises a gelling agent.

2. 13. The nutrient medium of claim 1 disposed in a container.

3. 3. The nutrient medium of claim 2, which protrudes relative to the container.

4. 4. The nutrient medium according to claim 3, which is rounded and raised.

5. 4. The nutrient medium of claim 3, comprising a flat portion.

6. 6. The nutrient medium of claim 5, further comprising a sloped peripheral portion.

7. 2. The nutrient medium of claim 1, wherein at least one dye and / or pigment imparts a black color to the medium.

8. 2. The nutrient medium of claim 1, wherein at least one dye and / or pigment substantially reduces a red background, an orange background, a yellow background, a green background, a blue background, or a purple background upon exposure to white light.

9. 2. The nutrient medium of claim 1, wherein at least one dye and / or pigment substantially reduces background upon exposure to blue light.

10. 2. The nutrient medium of claim 1, wherein the at least one dye and / or pigment provides a peak optical density of from 0.004 to 6.

11. 2. The nutrient medium of claim 1, wherein the medium comprises a pigment.

12. 12. The nutrient medium of claim 11, wherein the pigment comprises carbon particles or iron oxide particles.

13. 13. The nutrient medium of claim 12, wherein the carbon particles have an average diameter of less than 500 nm.

14. 13. The nutrient medium of claim 12, wherein the carbon particles have an average diameter of about 100 nm to about 400 nm.

15. 13. The nutrient medium of claim 12, wherein the carbon particles comprise carbon black.

16. 2. The nutrient medium of claim 1, wherein the medium comprises a dye.

17. 17. The nutrient medium of claim 16, wherein the dye comprises an azo dye.

18. 18. The nutrient medium of claim 17, wherein the azo dye is Acid Black 71, Acid Black 194, Acid Black 2, Reactive Black 5, Direct Black 36, or Nianza Black.

19. 10. The nutrient medium of claim 1, further comprising a polymer that binds at least one dye and / or pigment.

20. 20. The nutrient medium of claim 19, wherein the polymer comprises polydiallyldimethylammonium chloride (polyDADMAC), starch, cellulose, chitosan, β-cyclodextrin, a calix[4]arene-based polymer, or a p-tert-butylcalix[4]arene-based polymer.

21. 10. The nutrient medium of claim 1, further comprising a free radical scavenger.

22. 22. The nutrient medium of claim 21, wherein the free radical scavenger is selected from the group consisting of N-acetyl-L-cysteine, sodium ascorbate, thiourea, nicotinamide, pyridoxine, sodium pyruvate, D-mannitol, 2-oxo-4-thiomethylbutyric acid (OBTA), 3-(methylthio)propionaldehyde (methional), dimethylsulfoxide (DMSO), tert-butyl alcohol, benzoate, catalase, NADH peroxidase, thiol peroxidase, bacterioferritin comigratory protein, alkyl hydroperoxide reductase, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrin.

23. 2. The nutrient medium of claim 1, wherein the microorganism is a bacterium, a fungus, a protist, or an archaea.

24. The microorganisms are Acinetobacter, Aspergillus, Bacillus, Burkholderia, Campylobacter, Candida, Clostridium, Corynebacterium, Delftia, Dermacoccus, Escherichia, Exserohilum, Helicobacter, Kocuria, Legionella, Listeria, Methylobacterium, Micrococcus.

24. The nutrient medium of claim 23, wherein the bacterium belongs to a genus selected from the group consisting of Mycobacterium, Paenibacillus, Penicillium, Propionibacterium, Pseudomonas, Ralstonia, Salmonella, Selenomonas, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Vibrio, and Yersinia.

25. 2. The nutrient medium of claim 1, wherein the gelling agent is selected from the group consisting of agar, gellan, sodium alginate, xanthan gum, guar gum, gelatin, agarose, polyacrylamide, and a polysaccharide produced by Rhizobium sp. (CNCM number: I-1809).

26. 2. The nutrient medium of claim 1, further comprising nutrients selected from the group consisting of Trypticase Soy Medium (TSM), Fluid Thioglycollate Medium (FTM), Schaedler Medium, Lysogeny / Lennox (LB) broth, Mannitol Yeast Extract Peptone Broth, Terrific Broth II, Super Optimal Broth with Catabolite Repressor (SOC) medium, Thornton Medium, Lowenstein-Jensen Medium, Sabouraud Dextrose Medium, D / E Neutral Medium, Loeffler Medium, Nalidixic Acid and Colimycin (ANC) Medium, and Artificial Seawater Medium.

27. 10. The nutrient medium of claim 1, further comprising nutrients derived from bismuth sulfite agar, Chapman agar, Mueller-Hinton agar (MHA), blood agar, MacConkey agar, trypticase soy agar (TSA), Sabouraud dextrose agar (SDA), Reasoner's 2A agar (R2A), Mueller-Miller tellurite agar, or Tinsdale tellurite agar.

28. The nutrient medium according to any one of claims 1 to 27, wherein the nutrient is solid or semi-solid.

29. A nutrient medium according to any one of claims 1 to 27, which is liquid.

30. 30. The nutrient medium of claim 29, disposed within a porous matrix.

31. The nutrient medium according to any one of claims 1 to 30, having a pH value of from about pH 3.0 to about pH 12.

0.

32. 1. A method for detecting microbial growth, comprising: (a) contacting a surface of the nutrient medium of claim 28 with a sample under suitable conditions; and (b) Optically detecting a surface to determine any growth of microorganisms. The method includes:

33. 33. The method of claim 32, wherein the microorganism belongs to a genus selected from the group consisting of Acinetobacter, Aspergillus, Bacillus, Burkholderia, Campylobacter, Candida, Clostridium, Corynebacterium, Delftia, Dermacoccus, Escherichia, Exelohilum, Helicobacter, Kocuria, Legionella, Listeria, Methylobacterium, Micrococcus, Mycobacterium, Paenibacillus, Penicillium, Propionibacterium, Pseudomonas, Ralstonia, Salmonella, Selenomonas, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Streptomyces, Vibrio, and Yersinia.

34. 33. The method of claim 32, wherein optically detecting comprises detecting fluorescence.

35. 33. The method of claim 32, wherein step (a) comprises contacting a membrane containing the sample with the surface.

36. 33. The method of claim 32, wherein step (a) comprises contacting an exterior surface with the surface.

37. 33. The method of claim 32, wherein step (a) comprises contacting air with the surface.

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