Automated microbiological laboratory for quantitative and qualitative microbiological analysis

Abstract

The present invention provides an integrated automated system for quantitative and / or qualitative microbiological assessment and / or analyte assessment of a sample using in situ generated culture devices. The system comprises: an integrated automated system comprising a sterile medium and / or buffer reagent, culture device parts, and automated components for medium and / or buffer treatment, test sample treatment, culture device assembly, microbe counting and / or detection, all operations being completed in a sterile environment; one or more processors; and a memory including computer executable instructions that, if executed by one or more processors, cause the integrated automation system to determine that an encoded test sample loaded into the system is to be quantitatively and / or qualitatively evaluated, and processing and microbiologically evaluating the encoded test sample using correspondingly one or more specified in-situ generation culture devices for quantitative evaluation, thereby eliminating the need to sterilize, transport, store and / or unseal the culture devices made off-site prior to inoculation.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 439,562, entitled "AUTOMATED MICROBIOLOGICAL LABORATORY FOR QUANTITATIVE AND QUALITATIVE MICROBIOLOGY", filed on January 17, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical field

[0003] Aspects of the present invention generally relate to an integrated automated system that receives a sample and optionally extracts the sample, prepares a culture medium and in - situ assembles a culture device in a compact structure, inoculates the culture medium / culture device in - situ, and then the culture medium / culture device can be incubated and read to quantitatively determine the bioburden of the sample. In additional aspects, the system also provides or alternatively provides qualitative sample assessment (e.g., pathogenic bacteria, spoilage bacteria, food microbiome, allergens, genetically modified organisms (GMOs), toxins, ingredients, etc.), for example, using gene - based or immunochemical detection methods, etc. Background art

[0004] In microbiological testing of the microbial load of a sample, typically a portion of the sample is extracted and plated onto various microbial culture media by the pour - plate method, spread - plate method, or spiral - plate method, each of which allows one or more biota to grow into colonies. After appropriate incubation, the colonies are counted, the bioburden is calculated, and the results are reported based on CFU / g, CFU / ml, or CFU / unit product, or CFU / cm² or CFU / in².

[0005] The most commonly used microbial culture plates are Petri dishes, but alternatives to Petri dishes have been introduced to the market. For example, miniaturized alternatives include microbial membranes, Petri films, peel - plates, etc. These devices are centrally manufactured, irradiated for sterilization, and then globally distributed to various testing laboratories.

[0006] Some companies have also developed automated systems that load the extracted samples onto machines, which automatically perform the plating of various culture media according to the requirements of the Laboratory Information Management System (LIMS). Although such automated systems have significantly reduced labor costs, each automated system is only designed for pre-assembled microbial culture plates of specific sizes and types (the inventory status of which is affected by the supply chain and product shelf life). Although some of these systems can produce petri dishes during the automated process, these assembled petri dishes are problematic in a sense: they are bulky and require the addition of agar-based media at elevated temperatures, often resulting in the media solidifying in the delivery tubes. These systems also require a water bath to maintain the media at an elevated temperature.

[0007] There is an urgent need for automated systems with sample extraction capabilities that have the following characteristics: in-situ preparation of culture media and assembly and inoculation of compact culture devices (e.g., thin microbial membranes, culture bags, etc.); integrated and efficient selective development and quantitative analysis of cultured microorganisms; and no need for high-temperature media or a water bath to avoid problems with pipeline blockage.

[0008] There is a further need for such automated systems to, in addition or alternatively, provide qualitative laboratory testing capabilities, including but not limited to testing for pathogenic bacteria, spoilage bacteria, food microbiome, allergens, genetically modified organisms, toxins, ingredients, etc. by detecting analytes after extraction. Summary of the Invention

[0009] The present invention provides an integrated automated system that allows for automated full-process quantitative and / or qualitative analysis of samples. The automated system receives and extracts samples, prepares culture media and in-situ assembles culture devices in a compact structure, inoculates the culture media / culture devices, and then can incubate and read the culture media / culture devices to determine the bioburden of the samples and / or qualitatively detect microorganisms in the samples.

[0010] These integrated automated systems can provide various components or modules for achieving media mixing, dispensing, printing, sample extraction, dilution, aliquoting and inoculation, in-situ assembly of culture devices, incubation, image recognition and colony counting, and / or detection of target organisms, etc. Various forms of in-situ assembled culture devices can be used for quantitative analysis. In each case, the automated system will receive the original sample extract in an appropriate container (the container is attached with a barcode, e.g., generated by LIMS), the system will perform appropriate sample dilution operations, and inoculate appropriate volumes of each dilution into the in-situ assembled culture devices.

[0011] Preferred integrated system versions include an integrated incubation module and a reader module, and / or a detection module, which can automatically incubate the inoculated culture device and / or the inoculated enrichment culture, then read and / or detect, and report data (e.g., report to the LIMS system).

[0012] The embodiments of the present disclosure may be described in view of the following terms:

[0013] Automated microbiology laboratory for quantitative and qualitative microbiological analysis

[0014] 1. A system for performing qualitative and / or quantitative microbiological assessment, comprising:

[0015] An integrated automated system, including sterile culture medium and / or buffer reagents, parts of a culture device, and automated components for culture medium and / or buffer processing, test sample processing, culture device assembly, microbial counting and / or detection, all operations being completed in a sterile environment; one or more processors; and a memory, the memory including computer-executable instructions (e.g., LIMS instructions), which if executed by the one or more processors, cause the integrated automated system to determine that a coded test sample loaded into the system is to be quantitatively and / or qualitatively evaluated, and

[0016] For quantitative assessment

[0017] a) Use the parts of the culture device to in-situ assemble a specified type of microbial culture device for culturing and quantitative processing of the coded test sample;

[0018] b) As part of the in-situ assembly (e.g., during or after the in-situ assembly), inoculate the specified type of culture device with an appropriate amount of the coded test sample or its extract and / or diluent; and

[0019] c) Attribute the inoculated culture device to the coded test sample, wherein the assembly and inoculation are completed in-situ by the integrated automated components of the system in the sterile environment, thus eliminating the need for sterilization, transportation, storage, and / or opening of culture devices made in a different location before inoculation; and / or

[0020] For qualitative assessment,

[0021] A) Add a certain amount of culture medium or concentrated culture medium to at least a part of the coded test sample or its extract to provide a coded enrichment culture; and / or

[0022] A1) Dilute a part of the coded test sample or its extract by adding a certain amount of appropriate analyte extraction buffer.

[0023] 2. The system as described in clause 1, wherein executing the computer-executable instructions further causes the system to:

[0024] For quantitative assessment,

[0025] d) Place the inoculated and assigned culture device at a certain temperature for incubation for a period of time, the temperature and time being suitable to promote the growth of colonies corresponding to one or more target microorganisms;

[0026] e) Use the counting component to count the colonies in situ; and

[0027] f) Store and / or transmit the count data attributed to the encoded test sample; and / or

[0028] For qualitative assessment, executing the computer-executable instructions further causes the system to:

[0029] B) Place the enrichment culture in an incubator at a certain temperature for incubation for a period of time, the temperature and time being suitable to promote the growth of one or more target microorganisms to a detectable level (and preferably evenly distributed throughout the culture), thereby providing an enriched sample,

[0030] C) Use at least one aliquot of the enriched sample to perform in situ detection of the one or more target microorganisms by the detection component; and

[0031] D) Store and / or transmit the detection data attributed to the encoded test sample for storage and / or reporting and / or analysis; and / or

[0032] B1) Use the extraction component of the system to place the analyte sample of A1) in a suitable analyte extraction module / component for analyte extraction;

[0033] C1) In the detection module / component of the system, use a part of the extracted analyte of B1) to detect one or more extracted analytes; and

[0034] D1) Store and / or transmit the C1) detection data attributed to the encoded test sample for storage and / or reporting and / or analysis.

[0035] 3. The system as described in clause 2, wherein placing the inoculated and assigned culture device in an incubation environment in d), and / or placing the enrichment culture in an incubation environment in B), includes: performing in situ incubation using an incubator integrated into the system, and / or performing off-site incubation using a non-integrated incubator.

[0036] 4. The system as described in clause 2 or 3, wherein:

[0037] In e), in situ counting of the colonies includes using an integrated imaging device and an image analysis program; and / or

[0038] In C), in situ detection of the one or more target organisms includes performing in situ detection using a suitable detection assay; and / or

[0039] In C1), in situ detection of the one or more extracted analytes includes performing in situ detection using a suitable detection assay.

[0040] 5. The system as described in clause 4, wherein the detection assay in C) is based on one or more methods selected from RNA, DNA, and immunochemistry; and / or

[0041] wherein the detection assay in C1) is based on one or more methods selected from ELISA and / or lateral flow chromatography.

[0042] 6. The system as described in any one of clauses 1 to 5, wherein:

[0043] For quantitative assessment,

[0044] When the encoded test sample loaded into the system is an original sample, executing the computer-executable instructions further causes the system to:

[0045] Mix or homogenize the encoded test sample with an appropriate amount of a specified buffer and / or medium to provide an extraction sample;

[0046] Take a sub-part of the extraction sample; and

[0047] Dilute the taken part (e.g., continuously and / or in parallel if specified) to provide an extraction sample or a dilution thereof for inoculating the assembled culture device; and / or

[0048] When the encoded test sample loaded into the system is an extraction sample, executing the computer-executable instructions further causes the system to:

[0049] Take a sub-part of the extraction sample; and

[0050] Dilute the taken part (e.g., continuously and / or in parallel if specified) to provide an extraction sample or a dilution thereof for inoculating the assembled culture device; and / or

[0051] For qualitative assessment,

[0052] When the encoded test sample loaded into the system is an original sample, executing the computer-executable instructions further causes the system to:

[0053] Prior to A), mix or homogenize the encoded test sample with an appropriate amount of a specified buffer to provide an extraction sample.

[0054] 7. The system according to any one of clauses 1 to 6, wherein the computer-executable instructions include one or more of LIMS instructions, PLC instructions, firmware, and programming instructions and logic.

[0055] 8. The system according to any one of clauses 1 to 6, wherein, for quantitative assessment, the in-situ assembled microbial culture device of the specified type includes at least one culture device selected from the group consisting of: a microbial membrane card, a culture dish containing a gel- and / or gum-based culture medium, and a culture bag.

[0056] 9. The system according to clause 8, wherein the in-situ assembled microbial culture device is a culture dish, and wherein, for the in-situ assembly, executing the computer-executable instructions further causes the system to:

[0057] Remove the lid of the pre-loaded culture dish having a base and a lid;

[0058] Introduce an appropriate amount of a sterile mixture containing one or more culture media plus one or more gellable polymers and / or gums;

[0059] Introduce an appropriate amount of the encoded test sample, or an extract and / or a dilution thereof;

[0060] Place the lid on the base of the culture dish; and

[0061] Before gelling, mix the introduced sterile mixture with the sample, its extract, or dilution.

[0062] 10. The system according to clause 9, wherein introducing the sterile mixture is synchronous with introducing the encoded test sample or an extract and / or a dilution thereof.

[0063] 11. The system according to clause 9 or 10, wherein the one or more gellable polymers and / or gums in the introduced sterile mixture do not gel at ambient temperature in the absence of one or more cations, and wherein, before introducing the sterile mixture and the sample, its extract, or dilution, executing the computer-executable instructions further causes the system to:

[0064] Cover the base with a base gel layer containing the one or more cations, the cations being capable of diffusing from the base gel layer into the introduced sterile mixture and the sample, its extract or diluent.

[0065] 12. The system according to clause 11, wherein the one or more gellable polymers and / or gums in the introduced sterile mixture include pectin and / or alginate, and wherein the one or more cations of the base gel layer include divalent or trivalent cations.

[0066] 13. The system according to clause 11 or 12, wherein the base gel layer comprises agar, gelatin, silica gel or carrageenan.

[0067] 14. The system according to any one of clauses 11 to 13, wherein the one or more cations include Ca 2+ 。

[0068] 15. The system according to any one of clauses 9 to 14, wherein the sterile mixture is introduced in liquid form, or wherein the one or more gellable polymers and / or gums in the introduced sterile mixture are initially introduced as a powder coating, and when the liquid medium and / or the encoded test sample or its extract and / or diluent are introduced, the powder absorbs the liquid and forms a gel on the base gel layer.

[0069] 16. The system according to clause 8, wherein the in-situ assembled microbial culture device is a microbial membrane device having a gasket card and a top cover, and wherein for the in-situ assembly, executing the computer-executable instructions further causes the system to:

[0070] Apply a sterile culture medium to the surface of the gasket card;

[0071] Introduce an appropriate amount of the encoded test sample, or its extract and / or diluent, to contact with the culture medium to form a mixture; and

[0072] Place the top cover on the gasket card; and

[0073] Press the placed top cover to distribute the mixture over a predetermined test area of the gasket card.

[0074] 17. The system according to clause 16, wherein the surface of the gasket card defines a reservoir for receiving the culture medium and the encoded test sample or its extract and / or diluent, and the surface area of the reservoir defines the predetermined test area.

[0075] 18. The system as described in clause 16 or 17, wherein, before or after coating the sterile culture medium onto the surface of the gasket card, executing the computer-executable instructions further causes the system to:

[0076] Coat an adhesive, gel, wax or grease in a pattern to define the predetermined area where the mixture is to be distributed.

[0077] 19. The system as described in clause 18, wherein the adhesive includes a pressure-sensitive adhesive.

[0078] 20. The system as described in any one of clauses 15 to 19, wherein before placing the top cover, executing the computer-executable instructions further causes the system to:

[0079] Introduce a certain amount of concentrated culture medium, which is diluted by the introduced coded test sample or its extract and / or diluent.

[0080] 21. The system as described in any one of clauses 18 to 20, wherein the adhesive, gel, wax or grease is coated before coating the sterile culture medium.

[0081] 22. The system as described in any one of clauses 16 to 21, wherein coating the sterile culture medium onto the surface of the gasket card includes:

[0082] Printing the sterile culture medium onto the surface of the gasket card; and / or otherwise dispensing the sterile culture medium onto the surface of the gasket card and then drying it.

[0083] 23. The system as described in any one of clauses 16 to 22, wherein the top cover is pre-coated with a gelling agent powder on the surface, and the gelling agent powder contains or does not contain dried culture medium or culture medium components.

[0084] 24. The system as described in clause 8, wherein the in-situ assembled microbial culture device is a culture bag, which has or is configured to have an opening.

[0085] 25. The system as described in clause 24, wherein for the in-situ assembly, executing the computer-executable instructions further causes the system to:

[0086] Introduce an appropriate amount of a sterile mixture containing one or more culture media plus one or more coagulable polymers and / or gums through the opening;

[0087] Introduce an appropriate amount of the coded test sample, or its extract and / or diluent, through the opening;

[0088] Mix the introduced sterile mixture with the sample, its extract or diluent within the culture bag;

[0089] Roll, press or otherwise shape the culture bag to distribute the mixed components before gelling, thereby providing a desired gel thickness and surface area; and

[0090] Close the opening before and / or after the mixing and / or the rolling.

[0091] 26. The system according to clause 24, wherein the sterile mixture is introduced in liquid form, or wherein one or more of the culture media and one or more of the coagulable polymers and / or gums in the introduced sterile mixture are initially introduced in powder form, and when the encoded test sample or its extract and / or diluent is introduced, the powder absorbs liquid and forms a gel.

[0092] 27. The system according to clauses 25 and 26, wherein one or more of the coagulable polymers and / or gums in the introduced sterile mixture do not coagulate at ambient temperature in the absence of one or more cations, and wherein the inner surface of the culture bag is pretreated with one or more cations that can diffuse into the introduced sterile mixture and the sample, its extract or diluent.

[0093] 28. The system according to clause 27, wherein before introducing the sterile mixture and the sample, its extract or diluent, the execution of the computer-executable instructions further causes the system to:

[0094] Pretreat the inner surface of the culture bag with one or more cations that can then diffuse into the introduced sterile mixture and the sample, its extract or diluent. Description of the Drawings

[0095] Those skilled in the art should understand that the drawings described below are for illustrative purposes only. These drawings are not intended to limit the scope of the present disclosure in any way.

[0096] Figure 1 The process of dispensing a pressure-sensitive adhesive onto a microbial membrane culture device liner card in a circular and linear pattern at an assembly station is illustrated by a non-limiting example of the present invention.

[0097] Figure 2 The process of synchronously dispensing a sample and a culture medium into the test area of the microbial membrane liner card shown is illustrated by a non-limiting example of the present invention. Figure 1

[0098] Figure 3 ​The process of laminating the top membrane onto the Figure 2 allocated samples and culture media shown is illustrated by non - limiting examples of the present invention.

[0099] Figure 4 The process of adding a sterilized basal medium (not shown) containing a polymer via a nozzle at an assembly station to cover the inner bottom surface of a culture dish is illustrated by non - limiting examples of the present invention. The aforementioned polymer coagulates in the presence of one or more of cations, gums, water, and appropriate salts containing these cations.

[0100] Figure 5 The process of tilting the Figure 4 culture dish base at an assembly station at a large enough angle and for a long enough time to allow excess (un - adhered) gel to drain and collect, and while tilting, aspirating the collected gel via a pipette tip 408, thereby leaving a thin layer of formed gel coating (not shown, i.e., the basal gel layer) on the culture dish base is illustrated by non - limiting examples of the present invention.

[0101] Figure 6 The process of stacking multiple culture dish bases with adhered basal gel layers on an integrated turntable for rapid cooling is illustrated by non - limiting examples of the present invention.

[0102] Figure 7 The process of synchronously adding an appropriate amount of sample / diluent and a sterile mixture containing one or more culture media plus one or more coagulable polymers to the culture dish base at an assembly station is illustrated by non - limiting examples of the present invention.

[0103] Figure 8 The process of opening a culture bag at an assembly station to allow the introduction of a powdered or liquid / pasty culture medium containing a gelling agent and a sample / diluent for inoculation through the opening is illustrated by non - limiting examples of the present invention.

[0104] Figure 9 The process of Figure 8 after gently mixing and labeling the culture bag, passing it through a rolling mechanism with an appropriate gap to evenly distribute the liquid sample and culture medium inside the bag, so as to have an appropriate gel thickness for subsequent gelling is illustrated by non - limiting examples of the present invention.

[0105] Figure 10 The process of showing a flowchart of the implementation path of computer - executable instructions for performing sample processing and quantitative analysis within an integrated automated laboratory system, which is used for the quantitative assessment of microbiological samples and other samples, is illustrated by non - limiting examples of the present invention.

[0106] Figure 11A flowchart showing an implementation path of computer-executable instructions for performing sample processing and quantitative and / or qualitative analysis within an integrated automated laboratory system is illustrated by non-limiting examples of the present invention. The system is for the quantitative and / or qualitative assessment of microbiological samples and other samples. Detailed Description

[0107] Aspects of the present invention overcome the deficiencies of existing microbiological testing methods.

[0108] The present invention provides a process flow and an integrated automated system for a quantitative and / or qualitative microbiological testing system, the system having an in-situ preparation function for microbiological culture devices, such as petri dishes, microbiological membranes, and culture bags, for microbiological culture. The integrated automated system receives an original sample (stored in a suitable container) with a sample identifier and / or an extracted sample with a sample identifier as a starting point. After reading the sample identifier, the system communicates with, for example, a laboratory information management system (LIMS) and receives test instructions including dilution instructions: for quantitative assessment, instructions for plating each dilution with one or more different media and one or more different culture devices based on the required test; and / or for qualitative assessment, instructions for inoculating an enrichment culture for the detection of target organisms.

[0109] To extract the sample in an automated manner, the following operations are performed on each original sample: introducing an appropriate buffer or medium, mixing, and taking subsamples when needed to complete serial dilution.

[0110] Microbiological membrane (film). A first exemplary embodiment of a culture device includes an in-situ assembled microbiological membrane. For performing quantitative assessment using such a microbiological membrane, for example, a container containing the extracted sample or a dilution container is then moved to another station (the plating station) of the automated system, where an appropriate amount of each sample / dilution is plated onto a corresponding microbiological membrane culture device, which is manufactured in-situ by an independent module (the membrane assembly module) within the same automated system. For example, based on LIMS instructions, a bottom card is picked up and placed at the microbiological membrane assembly station. The bottom card may be made of a piece of cardboard or other suitable material, and at least one side thereof is provided with (e.g., formed by coating or otherwise containing) a water-impermeable barrier layer.

[0111] Reference Figure 1, the bottom card 102 is moved into the assembly station 100 with the water-impermeable barrier layer side 104 facing up (e.g., facing upward). Here, in a preferred aspect, a layer of, for example, an adhesive (e.g., glue), gel, wax, or grease, etc., can be deposited in a certain shape (e.g., geometric shape) via the nozzle 106, and this shape defines the outer boundary of a certain shape (test area) that will accommodate the culture medium. Figure 1 It shows that the pressure-sensitive adhesive is dispensed as an annular pattern 108 that defines the test area 110, and an optional independent linear pattern (strip) 112 on the bottom card (liner card) 102 for sealing purposes. Subsequently, the culture medium can be deposited (e.g., printed) on the bottom card (within the boundary / test area 110 defined by the adhesive / glue / gel / wax / grease). In an alternative embodiment, the culture medium can be deposited without depositing the defining boundary or before depositing the defining boundary. After depositing the culture medium, or synchronously therewith, an appropriate amount of sample or sample diluent is added to the bottom card 102 on which the culture medium is deposited or being deposited.

[0112] Reference Figure 2 , it shows that the sample and the culture medium (not shown) are synchronously dispensed into the test area 110 of the microbial membrane liner card 102, where the sample is dispensed through the pipette tip 114 and the culture medium is dispensed through the nozzle 116. In this example, the dispensing of the sample and the culture medium is carried out at the assembly station 200, which can be the same as or different from the assembly station 100.

[0113] Subsequently, as Figure 3 shown, the top film 118 is covered and placed on the deposited sample / culture medium (not shown). In this example, placing the top film 118 is carried out at the assembly station 300, which can be the same as or different from the assembly station 100 and / or 200. The top film 118 can be made of a plastic or non-plastic based material, and one side of it (e.g., the bottom surface in contact with the sample / culture medium) may be coated with a dry gelling agent containing / without a dry culture medium or culture medium components (e.g., it can contain nutrients, buffers, chromogens, pH indicators, screening agents, growth factors, etc.), or may not be coated with the aforementioned dry gelling agent. In the next step, also as Figure 3As shown, the top film 118 is pressed by the pressing head 120. The edge 122 of the pressing head 120 combines with the deposited annular pattern 108 to define the area on which the sample will diffuse. In this example, the pressing by the pressing head 120 is also carried out at the assembly station 300, which may be the same as or different from the assembly stations 100 and / or 200. Alternatively, the pressing can be carried out at a station different from the placement of the top film 118. The pressing process causes the sample to diffuse and further mix over the entire surface of the test area 110, and also allows the deposited adhesive (such as glue), gel, wax or grease (specifically the pressure-sensitive adhesive annular pattern 108 in this example) to combine with the gasket card 102 and the top film 118 to form a barrier layer to prevent the aqueous sample from leaking out of the test area 110. When there is any gelling agent on the surface of the top film 118, it will absorb the coated sample and culture medium mixture, and the resulting gel allows the capture of any gas that may be generated due to microbial growth, and this gas may have diagnostic value. The deposited pressure-sensitive adhesive linear strip 112 provides an additional anchoring structure for sealing or resealing the top film 116.

[0114] The assembled microbial membrane is labeled (by sample code), and then it is guided / transported or moved to an integrated or non-integrated incubator and incubated at a temperature appropriate for each test.

[0115] Petri dish. Exemplary alternative embodiments of the culture device include in-situ assembled Petri dishes. For quantitative assessment using Petri dishes, for example, a container containing the extracted sample or a dilution container is moved to a station (e.g., the plating station) of the integrated automated system, where an appropriate amount of each sample / diluent is plated on the corresponding Petri dish, which is manufactured in-situ by an independent module (Petri dish assembly module) within the same automated system.

[0116] As Figure 4 shown, based on, for example, LIMS instructions, a pre-loaded Petri dish (including the base 402 plus the lid (not shown)) will be moved to the Petri dish assembly station 400, where the lid (not shown) is removed. A sterilized basal medium containing a polymer (not shown) is added via the nozzle 404 to cover the inner bottom surface 406 of the base 402, and the aforementioned polymer coagulates in the presence of one or more of cations, gums, water, and appropriate salts containing these cations (e.g., a hot pectin mixture can be used, together with Ca +2 ). Removing the lid and adding the sterilized basal medium can both be carried out at the station 400, or adding the basal medium can be carried out at a different station within the Petri dish assembly module.

[0117] As Figure 5As shown, the culture dish base 402 is then tilted at a large enough angle and held for a long enough time to allow excess (unadhered) gel to drain and collect. While tilting, the collected gel is aspirated via the pipette tip 408, leaving a thin layer of formed gel coating (not shown, i.e., the base gel layer) on the culture dish base 402. In this example, both tilting and aspiration are performed at the assembly station 400, but alternatively, they can be performed at different assembly stations.

[0118] As Figure 6 shown, multiple culture dish bases 402 with adhered base gel layers can be stacked on the integrated turntable 600 for rapid cooling.

[0119] As Figure 7 shown, the culture dish base 402 is then moved to another assembly station 700 where an appropriate amount of sample / diluent is added on top of the base gel layer via the pipette head 502, while an appropriate amount of sterile mixture is added via the nozzle 504. The sterile mixture contains one or more culture media plus one or more coagulable polymers and / or gums that do not coagulate at ambient temperature in the absence of one or more cations (e.g., pectin can be used which will coagulate at ambient temperature in the presence of a suitable cation (e.g., Ca +2 )). One or more cations (e.g., Ca + 2+) can diffuse from the base gel layer into the introduced sterile mixture and sample, thus promoting the formation of, for example, a pectin gel layer on top of the base gel layer. Then the culture dish lid (not shown) is placed on the culture dish base 402, the assembled culture dish is labeled (according to the sample code), gently mixed, transferred to a culture stack, and then the culture stack is transferred to an incubator (preferably an integrated incubator) and incubated at a specified temperature for a specified time suitable for promoting the growth of colonies corresponding to one or more target microorganisms.

[0120] Culture bag. Additional alternative exemplary culture device embodiments include in-situ assembled culture bags. For quantitative assessment using a culture bag, a container containing the extracted sample or a dilution container is moved to a station (e.g., the culture bag plate inoculation station) of the integrated automated system where an appropriate amount of each sample / diluent is plate inoculated using a culture bag that is in-situ assembled by an independent module (culture bag assembly module) within the same automated system.

[0121] As Figure 8As shown, based on, for example, LIMS instructions, a pre-loaded culture bag 802 (e.g., a plastic bag of appropriate size, with or without a closing mechanism) is transferred to the culture bag assembly station 800, where the culture bag 802 is opened or configured to have a closable opening. In either case, it allows the introduction of a powdered or liquid / pasty culture medium containing a gelling agent and the sample / diluent to be inoculated through this opening. For inoculation, the sample / diluent can be added to the culture bag 802 before, after, or simultaneously with the addition of the culture medium and / or gelling agent. The sample can be introduced via a pipette tip 804, and the culture medium can be introduced via a nozzle 806. After sample inoculation, the culture bag 802 can be closed (or not closed), gently mixed, and labeled, and then as Figure 9 shown, through a rolling mechanism 900 or a double-plate pressing device (in any case, an appropriate gap is set to evenly distribute the liquid). If the culture bag 802 is not closed before the sample and the culture medium are mixed inside the bag, it is closed after mixing. In either case, subsequent gelling is required. Then the culture bag 802 is transferred to an incubator (e.g., operating in a first-in, first-out manner), preferably an integrated incubator, and incubated at a specified temperature for a specified time, which is suitable for promoting the growth of colonies of one or more target microorganisms.

[0122] Incubator. Regarding any culture device implementation (including those discussed above) processed in an integrated automated system, the incubator can be integrated or not integrated into the integrated automated system. Preferably, an integrated temperature-controlled culture chamber is used, and the inoculated culture device is placed therein and incubated at a certain temperature suitable for promoting the growth of selective microorganisms (e.g., colonies of the target organism). The volume of the culture chamber is preferably large enough to incubate all the culture devices in a single production batch (e.g., the cumulative device output during a 24-hour production cycle). Alternatively, the inoculated culture device is transferred to an external culture chamber for off-site incubation.

[0123] Reader Module. After appropriate incubation (e.g., as determined by each LIMS test protocol), the culture device (e.g., microbial membrane, petri dish, culture bag, etc.) is removed from the incubator and transferred into the reader module (e.g., including an integrated imaging device and an image analysis program), where each culture device (e.g., microbial membrane) is read and data is transmitted to, for example, the LIMS system. After reading, the culture device is transferred into a storage device (integrated or non-integrated) for storage until quality control is completed and a report is issued, and then disposed of in an appropriate manner. For example, the color and / or gas production of the colonies to be counted are analyzed and entered into the LIMS system. The incubated culture device can be removed from the culture chamber and positioned by a robotic arm or other suitable instrument according to the first-in, first-out principle. For system versions that incubate the inoculated device in an external (non-integrated) culture chamber, the positioning of the culture device for image analysis will be programmed accordingly.

[0124] Sterilization. Ideally, the system is of a fully enclosed structure and operates under positive pressure maintained by HEPA-filtered air. All components, culture media, buffers, microbial membrane cards, parts, conduits, tips, etc. are pre-sterilized before being loaded into the automated system. The automated system can be equipped with an integrated in-situ cleaning (CIP) system for conduit components or ultraviolet lamps can be deployed to maintain a sterile environment (e.g., before equipment startup and during sanitation cycles).

[0125] Workstation / Module. The integrated automated system includes multiple functional modules or workstations. The integrated automated system can, for example, include a culture medium preparation unit / workstation, a sample extraction and dilution unit / workstation, a culture medium and sample positioning and mixing unit / workstation, a culture device assembly unit / workstation, an integrated or non-integrated temperature-controlled culture chamber, an image analysis-based colony counting unit / workstation (e.g., digital), and / or a detection workstation for qualitative detection of target microorganisms or other analytes.

[0126] Quantitative Detection Process. Figure 10 Show an exemplary high-level flowchart that shows the computer-executable instruction implementation path for performing sample processing and quantitative analysis within an integrated automated laboratory system for the quantitative assessment of microbiological samples and other samples.

[0127] For the quantitative application detection process, the automated system can, for example, use functional modules or workstations to quantitatively process samples according to the following exemplary steps: (1) Dissolve the sterilized culture medium dry powder mixture in water and then add it (optionally together with a gelling agent) to one or more designated versions of the in-situ assembled microbial culture device (e.g., microbial membrane card, culture bag, or petri dish); (2) Extract the coded sample, add it to the designated culture device with attribution information (e.g., petri dish, microbial membrane, and / or culture bag, etc.) after dilution (if specified), and the addition of the sample or its dilution can optionally be synchronized with the addition of the culture medium to facilitate mixing; (3) For the microbial membrane card version, an optionally gelling agent-coated thin film top cover can be assembled on the upper surface of the base film (base card or bottom card); (4) Place the assembled post-inoculation culture device in a temperature-controlled chamber (incubator) and incubate for a designated period; (5) Analyze the microbial colonies formed on the culture device; preferably through an integrated digital image-based intelligent recognition and counting module / program, which reads the analysis results and reports / stores them to, for example, the LIMS component of the integrated automated system.

[0128] Example 1 (below) describes the quantitative detection process of the integrated automated system, where the microbial membrane card serves as the culture device, assembled and inoculated in-situ in a sterile environment for microbiological quantitative analysis.

[0129] Example 2 (below) describes the quantitative detection process of the integrated automated system, where the culture bag serves as the culture device, assembled and inoculated in-situ in a sterile environment for microbiological quantitative analysis.

[0130] Example 3 (below) describes the quantitative detection process of the integrated automated system, where the petri dish serves as the culture device, assembled and inoculated in-situ in a sterile environment for microbiological quantitative analysis.

[0131] Qualitative detection process. In a microbiology laboratory, some test samples (e.g., food samples or non-food samples, etc.) not only require quantitative determination of the quantity of various microbial populations (e.g., counting of yeasts and molds, coliforms, fecal coliforms, common Escherichia coli (E. coli), Enterobacteriaces, lactic acid bacteria, Staphylococcus, Bacillus cereus, etc.), but also need to conduct or alternatively implement qualitative detection of pathogenic and / or spoilage bacteria (qualitative microbiological analysis). Additional qualitative detection processes include, but are not limited to, genetic tests for the presence or absence of specific genes / genetic markers (e.g., product certification, GMO testing, allergen testing, species identification, ingredient discrimination, spoilage characteristic analysis, etc.).

[0132] Figure 11 Shows an exemplary high-level flowchart that demonstrates the computer-executable instruction implementation path for performing sample processing and quantitative and / or qualitative analysis within an exemplary integrated automated laboratory system for the quantitative and / or qualitative assessment of microbiological samples and other samples.

[0133] For a qualitative application detection process that includes detecting pathogenic or spoilage bacteria, for example, the automated system can use functional modules or workstations to qualitatively process the sample according to the following exemplary steps: (1) Add concentrated culture medium to the coded sample or its extract to provide an enriched culture with attribution information (if multiple analyses are specified and the media used are incompatible, the sample / extract can be appropriately aliquoted to provide more than one attributed enriched culture); (2) Guide / transfer the enriched culture to an incubator (preferably an integrated incubator) and incubate for a specified time to provide an attributed enriched culture; (3) Guide / move the attributed enriched culture to a detection module (preferably an integrated detection module); (4) Take an aliquot (usually a trace amount) from each coded enriched culture and transfer the sample to the corresponding attributed reagent tube / plate according to the detection method; and (5) Perform a detection test on the contents of the attributed reagent tube (e.g., a detection test based on nucleic acid (e.g., DNA, RNA) or immuno-chemistry), and report / store the results to, for example, the LIMS component of the integrated automated system.

[0134] For a qualitative detection process that includes genetic tests for the presence or absence of specific genes / genetic markers (e.g., product authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage characterization, etc.), the automated system can use functional modules or workstations to qualitatively process the sample according to the following exemplary steps: (1) Take a portion of the homogenized sample and dilute it with an appropriate analyte extraction buffer; (2) Then, depending on the type of analyte, guide / transfer the diluted sample to an appropriate extraction module in the system (e.g., for nucleic acid extraction, transfer to a bead milling disruption module / component; for polypeptide / protein antigen extraction, transfer to an oscillating heating module / component; etc.) for analyte extraction; (3) Subsequently, guide / transfer a portion of the extracted analyte to an appropriate detection module in the system (e.g., for nucleic acid, transfer to an amplification module / component (e.g., PCR / isothermal amplification module); for polypeptide / protein antigen, transfer to an ELISA and / or lateral flow module / component; etc.), and report / store the results to, for example, the LIMS component of the integrated automated system.

[0135] Example 4 (described below) describes an integrated automated microbiology laboratory system implementation having a quantitative detection process (e.g., quantitative analysis of pathogenic or spoilage bacteria) and / or a qualitative detection process, such as a qualitative detection process, such as a genetic test for the presence or absence of a specific gene / genetic marker (e.g., product authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage characterization, etc.).

[0136] These integrated automated systems are synchronized with the process flow by in-situ preparation and assembly of culture devices (e.g., petri dishes, microbiological membranes, and culture bags, etc.) on demand, and sample testing and quantitative analysis in the integrated on-site system, thus eliminating the need for steps such as manufacturing devices at non-integrated and / or remote locations, sending them for irradiation sterilization, transporting them to the detection site, and storing them before use at the detection site, thereby addressing the unmet needs in the prior art. Instead, the sterilized components are assembled in a sterile integrated environment, eliminating the need for further sterilization, transportation, storage, and / or opening of culture devices manufactured off-site before inoculation. In terms of the combination of qualitative / quantitative, the integrated system uses in-situ assembled culture devices to provide integrated incubation (for qualitative testing) and / or quantitative analysis (e.g., colony counting).

[0137] Example

[0138] The following non-limiting working examples are provided to further illustrate the specific embodiments of the invention disclosed herein.

[0139] Example 1

[0140] (Microbiological membrane card in-situ assembly for use in the integrated automated system as described herein)

[0141] Certain aspects of the invention provide an integrated automated system including sterile culture media and / or buffer reagents, culture device parts, disposable and / or reusable dispensing tips, and automated components for media and / or buffer handling, test sample handling, culture device assembly, microbial counting, and / or detection, all operations being completed in a sterile environment; one or more processors; and a memory including computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programming instructions and logic), which if executed by one or more processors cause the integrated automated system (e.g., after loading each sample in an appropriate amount into a suitable container with appropriate encoded instructions (barcodes, etc.)) to perform the automated steps of the following method:

[0142] Microbial membrane card. Certain exemplary microbial membrane card embodiments can include one or more bottom liner cards, an optional intermediate confinement layer, and a top lid optionally coated with a gelling agent film. For example, in the integrated automated systems described herein, using sterile ingredients, dry culture medium is dissolved in water. A macromolecule-based thickening agent can be added to regulate the flow behavior of the culture medium solution. The culture medium solution is coated (it can be printed) onto the central reservoir area of the liner card (substrate film), which, as described above, can have a layer (e.g., an adhesive such as a pressure-sensitive adhesive), gel, wax, or grease deposited in a certain geometry to define the outer boundary of the test area that will hold the culture medium. The encoded sample suspension, which has been extracted and diluted as required, is dispensed into the central reservoir area of the substrate film. The culture medium and the sample can be introduced sequentially or simultaneously. The film top lid can be pre-cut and stacked in the integrated automated system. The film top lid can be pre-coated on one side (e.g., the bottom surface) with an adhesive and / or a gelling agent powder that contains or does not contain dry culture medium or culture medium components (e.g., can include nutrients, buffers, chromogens, pH indicators, screening agents, growth factors, etc.). A robotic arm picks up the coated film top lid and assembles it with the inoculated substrate film. Subsequently, the assembly is pressed together so that the sample is distributed over the test area between the substrate film and the film top lid. After attaching the attribution information label, the assembled and inoculated coded film is sent to an incubator (preferably an integrated incubator), and the appropriate temperature is set and the incubation time is specified for incubation.

[0143] Example 2

[0144] (The culture bag is assembled in situ for use in the integrated automated systems described herein)

[0145] Certain aspects of the present invention provide an integrated automated system that includes sterile culture medium and / or buffer reagents, parts of a culture device, disposable and / or reusable dispensing tips, and automated components for culture medium and / or buffer processing, test sample processing, culture device assembly, microbial counting, and / or detection, all operations being performed in a sterile environment; one or more processors; and a memory that includes computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programming instructions and logic), which, if executed by one or more processors, cause the integrated automated system (e.g., after each sample is loaded in an appropriate amount into a suitable container with appropriate encoded instructions (such as barcodes)) to perform the automated steps of the following method:

[0146] Culture bag. Exemplary embodiments of the culture device for selection include plastic bags of appropriate size, with or without a closing mechanism. For example, in an integrated automated system as described herein, such culture bags are transferred to an assembly station. The positioned culture bag is opened, or its configured closable opening is utilized. In either case, it allows the introduction of a powdered or liquid / pasty culture medium containing a suitable gelling agent, as well as the introduction of a sample / diluent. After sample inoculation, the culture bag is closed, gently mixed, labeled, and passed through a roller pressing mechanism or a double-plate pressing device. In any case, an appropriate gap is set to evenly distribute the liquid, and then the bag is closed (if it was not closed before the sample and the culture medium were mixed inside the bag). Subsequently, the culture bag is transferred to an incubator, preferably an integrated incubator (e.g., operating in a first-in, first-out manner).

[0147] Example 3

[0148] (Petri dishes are assembled in situ and used in the integrated automated system as described herein)

[0149] Certain aspects of the present invention provide an integrated automated system, including sterile culture media and / or buffer reagents, parts of the culture device, disposable and / or reusable dispensing tips, and automated components for culture medium and / or buffer handling, test sample handling, culture device assembly, microbial counting, and / or detection, all operations being completed in a sterile environment; one or more processors; and a memory that includes computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programming instructions and logic). If these computer-executable instructions are executed by one or more processors, then the integrated automated system (e.g., after each sample is loaded in an appropriate amount into a suitable container with appropriate coded instructions (such as barcodes)) performs the automated steps of the following method:

[0150] Petri dish. Additional alternative embodiments of the culture device include Petri dishes. For example, in an integrated automated system as described herein, Petri dishes are pre-loaded in the system. The Petri dish is transferred to an assembly station where the lid is removed. A sterilized basal medium containing a polymer is added to cover the base, thereby providing a thin layer of formed gel coating (basal gel layer) on the Petri dish base. The aforementioned polymer coagulates in the presence of one or more of cations, gums, water, and appropriate salts containing these cations. Subsequently, the Petri dish is transferred to another assembly station where an appropriate amount of sample is added on top of the basal gel layer, and at the same time, an appropriate amount of sterile mixture is added. The sterile mixture contains one or more culture media plus one or more coagulable polymers (such as pectin) and / or gums. The coagulable polymer and / or gum coagulate in the absence of one or more cations (e.g., Ca +2In the case of [[ID=]], there will be no condensation at the ambient temperature. One or more cations can diffuse from the base gel layer into the introduced sterile mixture and the sample, thereby promoting the formation of a gel layer above the base gel layer. Then, a lid is placed on the petri dish, the petri dish is labeled (according to the sample coding), gently mixed, transferred into a culture stack rack, and then the culture stack rack is transferred into an incubator (preferably an integrated incubator) and incubated at a specified temperature for a specified time, which is suitable for promoting the growth of colonies corresponding to one or more target microorganisms.

[0151] Example 4

[0152] (Providing an integrated automated microbiology laboratory for quantitative and qualitative microbiological analysis and other qualitative analysis)

[0153] Overview. In a microbiology laboratory, some test samples need to be subjected to qualitative microbiological analysis (such as detection of pathogenic bacteria, etc.) and quantitative determination of the quantities of various microbial groups, where the microbial groups include, for example, yeasts and molds, coliforms, fecal coliforms, ordinary Escherichia coli, Enterobacteriaceae, lactic acid bacteria, Staphylococcus, Bacillus cereus, etc.

[0154] Certain aspects of the present invention provide an integrated automated system for quantitative and / or qualitative microbiological analysis (or other qualitative analysis of samples), including sterile culture media and / or buffer reagents, culture device parts, disposable and / or reusable dispensing tips, and automated components for culture media and / or buffer handling, test sample handling, culture device assembly, microbial counting and / or microbial detection, and / or detection of other analytes, all operations being completed in a sterile environment; one or more processors; and a memory that includes computer-executable instructions (such as LIMS instructions, PLC instructions, firmware, and programming instructions and logic), which, if executed by one or more processors, cause the integrated automated system (such as after appropriately loading each sample into a suitable container with appropriate coding instructions (barcodes, etc.)) to perform the automated steps of the following exemplary method:

[0155] A: Quantitative and qualitative analysis detection process

[0156] 1. Sample collection device: For example, a stomacher bag or a wide-mouth bottle;

[0157] 2. Sample arrival and registration: The sample submission form is received electronically. Each sample is labeled with a barcode / has a unique identification code;

[0158] 3. Load the sample onto the sample carrier and send it into the machine;

[0159] 4. When each sample identifier is read, the LIMS loads the analysis request into the automated system;

[0160] 5. For example, remove the lid of the sample container or open the sample bag, add an appropriate buffer to each sample container / sample bag based on the sample weight, close the collection device, and pass the sample through a homogenizer;

[0161] 6. Homogenize the sample;

[0162] 7. For quantitative analysis, remove the lid / open the sample container, take a portion of the sample for dilution (e.g., serial and / or parallel dilution for quantitative testing, transfer it into the quantitative detection process using an in-situ assembled culture device as discussed in detail elsewhere in this document);

[0163] 8. For qualitative analysis, place an aliquot of the initially homogenized sample in an appropriate container and then direct it to the qualitative module / detection process (e.g., pathogenic bacteria, spoilage bacteria, etc.).

[0164] B. Pathogenic or spoilage bacteria detection process

[0165] B1. To follow the qualitative detection process (such as the pathogenic or spoilage bacteria detection process), add concentrated medium to an aliquot of the homogenized sample for enrichment culture. If more than one analysis of the sample is required and the required media are incompatible, sub-sample the sample (e.g., into two parts) and add the appropriate concentrated medium to each part separately;

[0166] B2. After adding the concentrated medium, direct / move the enriched sample container towards the incubator (culture module) and follow the qualitative (e.g., pathogenic or spoilage bacteria) detection process;

[0167] B3. Place the enriched sample container in an appropriate incubator and incubate the sample at an appropriate temperature (e.g., in the range of 25°C to 45°C) for an appropriate period of time (e.g., in the range of 4 hours to 48 hours);

[0168] B4. At the end of the incubation period, automatically transfer the enriched sample to the test / detection module;

[0169] B5. Then, open the sample container, take out an appropriate amount of the enriched sample, and perform detection by a suitably selected detection method (e.g., nucleic acid (e.g., DNA / RNA); immunoassay, etc.). For example, for a nucleic acid-based detection method, transfer an appropriate aliquot of the enriched sample to an assigned reagent tube / plate for nucleic acid detection assays (e.g., a nucleic acid (e.g., DNA / RNA) extraction tube). Then add reagents, mix the tube, place it on the magnetic separation module, where the supernatant is removed, and then successively perform washing, magnetic separation, and supernatant removal (these steps can be repeated). After the last wash, add an appropriate buffer to bring / extract the nucleic acid into the solution. Subsequently, transfer a portion of the extract to an amplification tube containing an appropriate amplification reaction buffer and reagents. Then transfer the amplification tube containing the buffer and reagents to the PCR / isothermal amplification module of the system, which performs and reads the amplification reaction and reports the results to, for example, the LIMS component of the integrated automated system. In an alternative embodiment, an appropriate amount of the enriched sample can be directly added to the amplification reaction tube and then placed in an amplification module (PCR / isothermal amplification module), etc.

[0170] C. Genetic Test Detection Process

[0171] C1. To follow a qualitative detection process, such as genetic tests for the presence or absence of specific genes / genetic markers, etc. (e.g., product authentication, GMO testing, allergen testing, species identification, ingredient discrimination, spoilage characterization, etc.), appropriate assays / tests are implemented by the integrated automated system. For example, tests based on the use of magnetic beads and amplification can be implemented.

[0172] C2. In such a test method, for example, take a portion of the homogenized sample, dilute it with extraction buffer in a sample tube, and then transfer the mixture to the bead milling disruption module / component of the integrated system, where coated magnetic beads are added to the mixture in the tube. Then mix the diluted sample in the tube with the magnetic beads, place the tube on a magnetic rack, and remove the supernatant. Add wash buffer to wash the magnetic beads, place the tube on the magnetic rack, and remove the supernatant, and then suspend the magnetic beads in an appropriate volume of buffer. For each preparation of such a test sample, transfer approximately 1 to 5 microliters of the magnetic bead suspension to an appropriate amplification tube containing an appropriate amplification reaction buffer and reagents. Then transfer the amplification tube containing the buffer and reagents to the PCR / isothermal amplification module of the system, which performs and reads the amplification reaction and reports the results to, for example, the LIMS component of the integrated automated system.

[0173] C3. For tests that require immunological testing, for example, an appropriate extraction buffer is added to an aliquot of the initial homogenized sample or its dilution in a sample tube, and then the sample tube is directed to the oscillating heating module / component of the integrated automated system. After an appropriate stay in the oscillating heating module, aliquots of each sample are transferred to a system analysis module with ELISA plates and / or lateral flow device components, and then reagent addition, incubation, washing are automatically performed, and finally the results are read and reported to, for example, the LIMS component of the integrated automated system.

Claims

1. A system for performing qualitative and / or quantitative microbial assessment, comprising: An integrated automated system, including sterile culture media and / or buffer reagents, parts of a culture device, and automated components for culture media and / or buffer handling, test sample handling, culture device assembly, microbial counting, and / or detection, with all operations being completed in a sterile environment; And One or more processors; And a memory, the memory including computer-executable instructions (e.g., LIMS instructions), which, if executed by the one or more processors, cause the integrated automated system to determine that a coded test sample loaded into the system requires quantitative and / or qualitative assessment, and For quantitative assessment, a) In-situ assemble a specified type of microbial culture device using the parts of the culture device for culturing and quantitatively processing the coded test sample; b) As part of the in-situ assembly (e.g., during or after the in-situ assembly), inoculate the specified type of culture device with an appropriate amount of the coded test sample or its extract and / or diluent; And c) Attribute the inoculated culture device to the coded test sample, wherein the assembly and inoculation are completed in-situ by the integrated automated components of the system in the sterile environment, thus eliminating the need for sterilization, transportation, storage, and / or opening of culture devices made elsewhere before inoculation; and / or For qualitative assessment, A) Add a certain amount of culture media or concentrated culture media to at least a part of the coded test sample or its extract to provide a coded enrichment culture; and / or A1) Dilute a part of the coded test sample or its extract by adding a certain amount of a suitable analyte extraction buffer.

2. The system according to claim 1, wherein executing the computer-executable instructions further causes the system: For quantitative assessment, d) Incubate the inoculated and attributed culture device at a certain temperature for a period of time suitable for promoting the growth of colonies corresponding to one or more target microorganisms; e) Count the colonies in-situ using the counting component; and f) Store and / or transmit the counting data attributed to the coded test sample; and / or For qualitative assessment, executing the computer-executable instructions further causes the system: B) Incubate the enrichment culture in an incubator at a certain temperature for a period of time suitable for promoting the growth of one or more target microorganisms to a detectable level (and preferably evenly distributed throughout the culture), thereby providing an enriched sample; C) Use at least one aliquot of the enriched sample to perform in-situ detection of the one or more target microorganisms by means of the detection component; And D) Store and / or transmit the detection data attributed to the coded test sample for storage and / or reporting and / or analysis; and / or B1) Use the extraction component of the system to place the analyte sample of A1) in a suitable analyte extraction module / component for analyte extraction; C1) In the detection module / component of the system, detect one or more extracted analytes using a part of the extracted analyte of B1); and D1) storing and / or transmitting C1) detection data attributable to the encoded test sample for storage and / or reporting and / or analysis.

3. The system according to claim 2, wherein placing the inoculated and assigned culture device in an incubation environment in d), and / or placing the enriched culture in an incubation environment in B), includes: Performing in-situ incubation using an incubator integrated into the system, and / or performing off-site incubation using a non-integrated incubator.

4. The system according to claim 2, wherein: In e), in-situ counting of the colonies includes using an integrated imaging device and an image analysis program; and / or In C), in-situ detection of the one or more target organisms includes performing in-situ detection using a suitable detection assay; and / or In C1), in-situ detection of the one or more extracted analytes includes performing in-situ detection using a suitable detection assay.

5. The system according to claim 4, wherein the detection assay in C) is based on one or more methods selected from RNA, DNA, and immunochemistry; and / or wherein the detection assay in C1) is based on one or more methods selected from ELISA and / or lateral flow chromatography.

6. The system according to claim 1, wherein: For quantitative assessment, When the encoded test sample loaded into the system is an original sample, executing the computer-executable instructions further causes the system to: Mix or homogenize the encoded test sample with an appropriate amount of a specified buffer and / or medium to provide an extraction sample; Take a sub-part of the extraction sample; and If specified, dilute the taken part to provide an extraction sample or a dilution thereof for inoculating the assembled culture device; and / or When the encoded test sample loaded into the system is an extraction sample, executing the computer-executable instructions further causes the system to: Take a sub-part of the extraction sample; and If specified, dilute the taken part to provide an extraction sample or a dilution thereof for inoculating the assembled culture device; and / or For qualitative assessment, When the encoded test sample loaded into the system is an original sample, executing the computer-executable instructions further causes the system to: Before A), mix or homogenize the encoded test sample with an appropriate amount of a specified buffer to provide an extraction sample.

7. The system according to claim 1, wherein the computer-executable instructions include one or more of LIMS instructions, PLC instructions, firmware, and programming instructions and logic.

8. The system according to claim 1, wherein For quantitative assessment, the specified type of in-situ assembled microbial culture device includes at least one culture device selected from the group consisting of a microbial membrane card, a petri dish containing a gel- and / or gum-based medium, and a culture bag.

9. The system according to claim 8, wherein the in-situ assembled microbial culture device is a petri dish, and wherein for the in-situ assembly, executing the computer-executable instructions further causes the system to: Remove the lid of a pre-loaded petri dish having a base and a lid; Introduce an appropriate amount of a sterile mixture containing one or more media plus one or more coagulable polymers and / or gums; Introduce an appropriate amount of the encoded test sample, or its extract and / or diluent; Cover the base of the petri dish with the lid; and Before gelling, mix the introduced sterile mixture with the sample, its extract, or diluent.

10. The system according to claim 9, wherein the introduction of the sterile mixture and the introduction of the encoded test sample, or its extract and / or diluent, are carried out synchronously.

11. The system according to claim 9, wherein the one or more coagulable polymers and / or gums in the introduced sterile mixture do not coagulate at ambient temperature in the absence of one or more cations, and wherein, Before introducing the sterile mixture and the sample, its extract, or diluent, the execution of the computer-executable instructions further causes the system to: Cover the base with a base gel layer containing the one or more cations, and the cations can diffuse from the base gel layer into the introduced sterile mixture and the sample, its extract, or diluent.

12. The system according to claim 11, wherein the one or more gellable polymers and / or gums in the introduced sterile mixture include pectin and / or alginate, and wherein the one or more cations of the base gel layer include divalent or trivalent cations.

13. The system according to claim 11, wherein the base gel layer comprises agar, gelatin, silica gel, or carrageenan.

14. The system according to claim 11, wherein the one or more cations comprise Ca 2+ .

15. The system according to claim 9, wherein the sterile mixture is introduced in liquid form, or wherein the one or more gellable polymers and / or gums in the introduced sterile mixture are initially introduced as a powder coating, and when introducing the liquid medium and / or the encoded test sample, or its extract and / or diluent, the powder absorbs the liquid and forms a gel on the base gel layer.

16. The system according to claim 8, wherein the in-situ assembled microbial culture device is a microbial membrane device having a gasket card and a top cover, and for the in-situ assembly, the execution of the computer-executable instructions further causes the system to: Coat a sterile medium onto the surface of the gasket card; Introduce an appropriate amount of the encoded test sample, or its extract and / or diluent, to contact the medium to form a mixture; and Place the top cover on the gasket card; and Press the placed top cover to distribute the mixture over a predetermined test area of the gasket card.

17. The system according to claim 16, wherein the surface of the gasket card defines a reservoir for receiving the medium and the encoded test sample, or its extract and / or diluent, and the surface area of the reservoir defines the predetermined test area.

18. The system according to claim 16, wherein, Before or after coating the sterile medium onto the surface of the gasket card, the execution of the computer-executable instructions further causes the system to: Coat an adhesive, gel, wax, or grease in a pattern to define the predetermined area where the mixture is distributed.

19. The system according to claim 18, wherein the adhesive includes a pressure-sensitive adhesive.

20. The system according to claim 15, wherein before placing the top cover, executing the computer-executable instructions further causes the system to: introduce a certain amount of concentrated culture medium, which is diluted by the introduced encoded test sample or its extract and / or diluent.

21. The system according to claim 18, wherein the adhesive, gel, wax or grease is coated before coating the sterile culture medium.

22. The system according to claim 16, wherein coating the sterile culture medium onto the surface of the pad card includes: printing the sterile culture medium onto the surface of the pad card; and / or otherwise dispensing the sterile culture medium onto the surface of the pad card, followed by drying.

23. The system according to claim 16, wherein the top cover is pre-coated with a gelling agent powder on the surface, and the gelling agent powder contains or does not contain a dried culture medium or culture medium components.

24. The system according to claim 8, wherein the in-situ assembled microbial culture device is a culture bag, which has or is configured to have an opening.

25. The system according to claim 24, wherein with respect to the in-situ assembly, executing the computer-executable instructions further causes the system to: introduce an appropriate amount of a sterile mixture containing one or more culture media plus one or more gellable polymers and / or gums via the opening; introduce an appropriate amount of the encoded test sample, or its extract and / or diluent, via the opening; mix the introduced sterile mixture with the sample, its extract or diluent inside the culture bag; roll, press or otherwise shape the culture bag to distribute the mixed components before gelling, so as to provide a desired gel thickness and surface area; and close the opening before and / or after the mixing and / or the rolling.

26. The system according to claim 24, wherein the sterile mixture is introduced in liquid form, or wherein the one or more culture media and the one or more gellable polymers and / or gums in the introduced sterile mixture are initially introduced in powder form, and when the encoded test sample or its extract and / or diluent is introduced, the powder absorbs the liquid and forms a gel.

27. The system according to claim 25, wherein the one or more gellable polymers and / or gums in the introduced sterile mixture do not gel at ambient temperature in the absence of one or more cations, and wherein the inner surface of the culture bag is pretreated with the one or more cations, which can diffuse into the introduced sterile mixture and the sample, its extract or diluent.

28. The system according to claim 27, wherein before introducing the sterile mixture and the sample, its extract or diluent, executing the computer-executable instructions further causes the system to: Pretreat the inner surface of the culture bag with the one or more cations, which can then diffuse into the introduced sterile mixture and the sample, its extract or diluent.

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