An automated microbiology laboratory for quantitative microbiology using in situ prefabricated culture devices.

Abstract

An integrated, automated system for quantitative and / or qualitative microbiological and / or analyte evaluation of samples using in situ production and culture devices is provided. The system includes an integrated automation system including sterile media and / or buffer reagents, culture device parts, and automated components for media and / or buffer handling, test sample handling, culture device assembly, and microbial enumeration and / or detection, all within a sterile environment; one or more processors; and memory containing computer-executable instructions that, when executed by the one or more processors, cause the integrated automation system to determine that a sample should be quantitatively and / or qualitatively evaluated, and, in response, process and microbiologically evaluate the coded test sample using one or more designated in situ production and culture devices for quantitative evaluation, thereby eliminating the need to sterilize, transport, store, and / or open the ex situ-manufactured culture device prior to inoculation thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 439,561, filed January 17, 2024, entitled "AUTOMATED MICROBIOLOGICAL LABORATORY FOR QUANTITATIVE MICROBIOLOGY USING IN SITU ASSEMBLED CULTURE DEVICES," the disclosure of which is incorporated herein by reference in its entirety.

[0002] Aspects of the present invention generally relate to an integrated, automated system that can receive and optionally extract a sample, prepare a culture medium, assemble a culture device in situ in a compact format, inoculate the culture medium / culture device in situ, and then incubate and read the culture medium / culture device to quantitatively measure the bioburden of the sample. In additional aspects, the system also or alternatively provides a qualitative assessment of the sample (e.g., pathogens, spoilage organisms, food microbiome, allergens, GMOs, toxins, ingredients, etc.), such as using genetic or immunochemical-based detection methods. [Background technology]

[0003] Microbiological testing of a sample for microbial bioburden typically involves extracting a portion of the sample and plating it onto various microbiological media by pour-plating, smear-plating, or spiral-plating, each of which allows one or more organisms to grow and form colonies. After appropriate incubation, the colonies are then counted and the bioburden is calculated and reported based on cfu / gram, cfu / ml, or cfu / product unit, or square centimeters / square inch.

[0004] The most commonly used microbiological plate is the Petri dish, but alternatives to the Petri dish have been introduced onto the market. For example, miniaturized alternatives include microfilm, Petrifilm, and peel plates. These devices are manufactured centrally, sent for irradiation, and then distributed to laboratories around the world.

[0005] Additionally, a few companies have developed automated systems in which extracted samples are loaded into a machine, which then automatically plates them onto various media as required by the Laboratory Information Management System (LIMS). While such automated systems significantly reduce labor costs, each automated system is designed for specific sizes and types of pre-assembled microbiology plates, the availability of which is affected by the supply chain and product shelf life. While some of these systems create Petri dishes during the automated process, assembled Petri dishes are problematic in that they are bulky and require the addition of agar-based media at high temperatures, which typically solidify within the transfer tube. These systems also require a water bath to maintain the media at high temperatures.

[0006] There is a need for an automated system that is integrated with sample extraction capabilities, in situ medium preparation, assembly and inoculation of compact culture devices (e.g., thin microfilms, Petri pouches, etc.), and efficient and selective visualization and quantification of cultured microorganisms, without the need for high temperature media or water baths, and that avoids clogging issues.

[0007] There is a further need for automated systems that perform analyte extraction and subsequent detection to additionally or alternatively provide qualitative clinical testing, including but not limited to testing for pathogens, spoilage organisms, food microbiome, allergens, GMOs, toxins, ingredients, etc. Summary of the Invention

[0008] SUMMARY OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION An integrated automated system is provided that is capable of automating comprehensive quantitative and / or qualitative analysis of a sample, and is capable of receiving and extracting the sample, producing media, assembling the culture device in situ in a compact format, inoculating the media / culture device, and then incubating and reading to measure the bioburden of the sample and / or qualitatively detecting microorganisms in the sample.

[0009] An integrated automation system can provide various parts or modules for media mixing, dispensing, printing, sample extraction, dilution, dispensing, and inoculation, in situ culture device assembly, incubation, image recognition and colony counting, and / or target microorganism detection. Multiple versions of in situ prefabricated culture devices can be used for quantification. In each case, for example, the automation system receives the primary sample extract in an appropriate container with a barcode, e.g., issued by a LIMS, makes the appropriate sample dilutions, and inoculates the appropriate volume of each dilution into the in situ prefabricated culture device(s).

[0010] A preferred version of the integrated system consists of an integrated incubation and reader module and / or detection module that can automatically incubate the inoculated culture device and / or the inoculated enrichment culture, and then read and / or detect and report the data (e.g., to a LIMS system).

[0011] Embodiments of the present disclosure can be described in light of the following provisions.

[0012] Clause 1. A system for microbiological evaluation using an in situ production culture device, comprising: an integrated automation system including sterile media and / or buffer reagents, culture device parts, and automated components for media and / or buffer handling, sample handling, culture device assembly, and microbial enumeration, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions; which, when executed by the one or more processors, cause the integrated automation system to perform the following operations: a) Determine that coded test samples loaded into the system should be quantitatively evaluated; b) Using the culture apparatus components, assemble in situ a microbiological culture apparatus of the specified type for culturing and quantitatively processing coded test samples; c) inoculating a culture device of the designated type with an appropriate amount of the coded test sample, or extracts and / or dilutions thereof, either as part of or after in situ assembly; and d) A system for attributing an inoculated culture device to a coded test sample, wherein assembly and inoculation are performed in situ in a sterile environment by integrated automated components of the system, thereby eliminating the need to sterilize, transport, store, and / or open an ex-situ manufactured culture device prior to its inoculation.

[0013] Clause 2. The system of clause 1, wherein execution of the computer-executable instructions further causes the system to: e) subjecting the inoculated attribution culture device to incubation at a temperature and time suitable to promote the growth of colonies corresponding to one or more target microorganisms; f) counting colonies using an in situ counting component; and g) A system that stores and / or transmits (e.g., to a LIMS or similar program) enumeration data attributed to coded test samples for storage and / or reporting and / or analysis.

[0014] Clause 3. The system of clause 2, wherein in e), inducing incubation includes incubating the inoculated attribution culture device in situ using an incubator integrated into the system, and / or incubating the inoculated attribution culture device ex situ using a non-integrated incubator.

[0015] The system of clause 2 or 3, wherein in clause 4.f), counting colonies in situ comprises using an integrated imaging device and image analysis program.

[0016] Clause 5. The system according to any one of clauses 1 to 4, If the coded test sample loaded into the system is a primary sample, execution of the computer executable instructions further causes the system to: Mixing or homogenizing the coded test sample with a suitable amount of a designated buffer and / or medium to provide an extracted sample; Taking a portion of the extracted sample; and Diluting the collected portion, if specified (e.g., serially and / or in parallel), and providing the extracted sample or dilution thereof for inoculation into the prefabricated culture device; and / or If the coded test sample loaded into the system is an extracted sample, execution of the computer executable instructions further causes the system to: Taking a portion of the extracted sample; and A system in which the collected portion is diluted (serial and / or parallel), if specified, to provide the extracted sample or its dilution for inoculation into the assembled culture device.

[0017] Clause 6. The system of any one of clauses 1-5, wherein the computer-executable instructions include one or more of LIMS instructions, PLC instructions, firmware, and programmed instructions and logic.

[0018] Clause 7. A system according to any one of clauses 1 to 6, wherein the in situ assembled microbial culture device of the specified type comprises at least one selected from the group consisting of microfilm cards, Petri dishes containing gel and / or gum-based media, and Petri pouches.

[0019] Clause 8. The system of clause 7, wherein the in situ assembled microbial culture device is a Petri dish, and for in situ assembly, execution of the computer-executable instructions further causes the system to perform the following operations: Remove the lid of a pre-loaded Petri dish with a base and lid; introducing an appropriate amount of a sterile mixture containing one or more media and one or more solidifying polymers and / or gums; introducing an appropriate amount of coded test sample, or extracts and / or dilutions thereof; Place the lid on the base of the Petri dish; and A system that mixes the sample, its extract, or dilution with an introduced sterile mixture before gelation.

[0020] Clause 9. A system according to clause 8, wherein the introduction of the sterilisation mixture and the introduction of the coded test sample, or extracts and / or dilutions thereof, are simultaneous.

[0021] Clause 10. The system of clause 8 or 9, wherein one or more coagulable polymers and / or gums in the introduced sterilization mixture do not coagulate at room temperature in the absence of one or more cations, and prior to introducing the sterilization mixture and the sample, extract, or diluent thereof, execution of the computer-executable instructions further causes the system to: The system comprises a base gel layer covering the base containing one or more cations, the one or more cations being diffusible from the base gel layer upon introduction of the sterilization mixture and the sample, extract thereof, or dilution.

[0022] Clause 11. The system of clause 10, wherein the one or more solidifying polymers and / or gums in the introduced sterilization mixture comprise pectin and / or alginate, and the one or more cations of the base gel layer comprise divalent or trivalent cations.

[0023] Clause 12. The system of clause 10 or 11, wherein the base gel layer comprises agar, gelatin, silica gel, or carrageenan.

[0024] Article 13. One or more cations are Ca 2+ 13. The system of any one of clauses 10 to 12, comprising:

[0025] Clause 14. A system according to any one of clauses 8 to 12, wherein the sterilization mixture is introduced in liquid form or wherein one or more of the solidifying polymers and / or gums in the introduced sterilization mixture are initially introduced as a coating in powder form, which upon introduction of the culture medium and / or coded test sample, or extracts and / or diluents thereof in liquid form, absorbs and forms a gel over the base gel layer.

[0026] Clause 15. The system of clause 7, wherein the in situ assembled microbial culture device is a microfilm device having a backing card and a top cover, and for in situ assembly, execution of the computer executable instructions causes the system to further perform the following operations: Apply sterile medium to the surface of the backing card; introducing an appropriate amount of the coded test sample, or extract and / or dilution thereof, into contact with the medium to form a mixture; placing the top cover over the backing card; and The system stamps the top cover in place to distribute the mixture over the desired test area of ​​the backing card.

[0027] Clause 16. The system of clause 16, wherein the surface of the backing card defines a reservoir for containing the culture medium and the coded test sample, or an extract and / or dilution thereof, the reservoir having a surface area defining the desired test area.

[0028] Clause 17. The system of clause 15 or 16, wherein, before or after applying the sterile medium to the surface of the backing card, execution of the computer-executable instructions further causes the system to: A system for applying adhesive, gel, wax, or grease in a pattern that defines a desired area to distribute the mixture.

[0029] Clause 18. The system of clause 17, wherein the adhesive comprises a pressure sensitive glue.

[0030] Clause 19. The system of any one of clauses 15 to 18, wherein, prior to placing the top cover, execution of the computer-executable instructions further causes the system to: The system introduces a volume of enrichment medium that is diluted with the introduced coded test sample, or extracts and / or diluents thereof.

[0031] Clause 20. A system according to any one of clauses 17 to 19, wherein an adhesive, gel, wax or grease is applied prior to the sterilization medium.

[0032] Clause 21. The system according to any one of clauses 15 to 20, wherein applying the sterilized medium to the surface of the backing card comprises: Printing a sterile medium onto the surface of a backing card; and / or otherwise dispensing the sterile medium onto the surface of the backing card and then drying.

[0033] Clause 22. A system according to any one of clauses 15 to 21, wherein the surface of the top cover is pre-coated with a gelling agent powder, with or without dry medium or medium components.

[0034] Clause 23. The system described in clause 7, wherein the in situ assembled microbial culture device is a Petri pouch having or configured to have an opening.

[0035] Clause 24. The system of clause 23, wherein for in-situ assembly, execution of the computer-executable instructions further causes the system to: introducing an appropriate amount of a sterile mixture containing one or more media and one or more solidifying polymers and / or gums through the opening; introducing an appropriate amount of the coded test sample, or extracts and / or dilutions thereof, through the opening; mixing the introduced sterile mixture with the sample, its extract, or dilution in the pouch; Prior to gelling, the pouch is rolled, pressed, or otherwise formed to distribute the mixed ingredients and achieve the desired gel thickness and surface area; and A system for closing openings before or after mixing and / or rolling.

[0036] Clause 25. A system according to clause 24, wherein the sterilization mixture is introduced in liquid form, or wherein one or more culture media and one or more solidifying polymers and / or gums in the introduced sterilization mixture are initially introduced in powder form, which culture media and solidifying polymers and / or gums absorb and form a gel upon introduction of the coded test sample, or extracts and / or diluents thereof.

[0037] Clause 26. A system according to clauses 24 and 25, wherein one or more solidifying polymers and / or gums in the introduced sterilization mixture do not solidify at room temperature in the absence of one or more cations, and the inside of the Petri pouch has been pretreated with one or more cations that are diffusible into the introduced sterilization mixture and the sample, its extract, or diluent.

[0038] Clause 27. The system of clause 26, wherein, prior to introducing the sterilization mixture and the sample, extract thereof, or dilution, execution of the computer-executable instructions further causes the system to: A system in which the inside of the Petri pouch is pretreated with one or more cations, the one or more cations being diffusible into the introduced sterilization mixture and the sample, extract thereof, or dilution.

[0039] Clause 28. The system of clause 27, wherein the one or more cations comprises a divalent or trivalent cation.

[0040] Article 29. One or more cations are Ca 2+ 29. The system of claim 28, comprising: Those skilled in the art will appreciate that the drawing(s) described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0041] [Figure 1] As a non-limiting example of the present invention, pressure sensitive adhesive is shown dispensed in ring and line patterns onto the backing card of a microfilm incubation device in the assembly position. [Figure 2] As a non-limiting example of the present invention, the microfilm backing card shown in FIG. 1 shows sample and media being dispensed simultaneously into the test area. [Figure 3]As a non-limiting example of the present invention, FIG. 2 shows stamping a top film over the dispensed sample and medium. [Figure 4] As a non-limiting example of the present invention, a sterilized base medium (not shown) containing a polymer that solidifies in the presence of one or more of cations, gums, water, and suitable salts containing cations is shown being added through a nozzle to cover the inner base surface of a Petri dish in an assembled position. [Figure 5] As a non-limiting example of the present invention, in the assembled position, the Petri dish base of FIG. 4 is shown tilted to a sufficient angle and for a sufficient time to drain and pool excess (non-adherent) gel, and during tilting, the suction nozzle 408 aspirates the pooled gel, leaving a thinly formed gel coating (not shown) on the Petri dish base (base gel layer). [Figure 6] As a non-limiting example of the present invention, multiple Petri dish bases containing adhered base gel layers are shown stacked on an integrated carousel for rapid cooling. [Figure 7] As a non-limiting example of the present invention, a Petri dish base is shown to which, in the assembly position, an appropriate amount of sample / diluent is added simultaneously with the addition of an appropriate amount of a sterile mixture containing one or more culture media and one or more solidifying polymers. [Figure 8] As a non-limiting example of the present invention, a Petri pouch is shown in an assembly position where the pouch is opened and a powder or liquid / paste medium containing a gelling agent can be introduced through the opening, and a sample / diluent for inoculation can be introduced. [Figure 9] As a non-limiting example of the present invention, the Petri pouch in Figure 8 is shown being gently mixed, labeled, and then passed through rollers with an appropriate gap to distribute the liquid sample and medium within the pouch, which then gels to the appropriate gel thickness. [Figure 10] As a non-limiting example of the present invention, a flow diagram illustrating the implementation of computer-executable instructions for sample processing and quantitative analysis within an automated integrated laboratory system for quantitative evaluation of microbiological and other samples is provided. [Figure 11] As a non-limiting example of the present invention, a flow diagram illustrating the implementation of computer-executable instructions for sample processing and quantitative and / or qualitative analysis within an automated integrated laboratory system for quantitative and / or qualitative evaluation of microbiological and other samples is provided. DETAILED DESCRIPTION OF THE INVENTION

[0042] Aspects of the present invention overcome the deficiencies of current methods for microbiological testing.

[0043] A process and an integrated automation system for quantitative and / or qualitative microbiological testing systems are provided that in situ manufacture microbiological culture devices (e.g., Petri dishes, microfilm, Petri pouches) for microbiological culture. The integrated automation system starts by receiving a primary sample having a sample ID and / or an extracted sample having a sample ID in an appropriate container. After reading the sample ID, the system communicates with, for example, a Laboratory Information Management System (LIMS) to receive test instructions, including dilution instructions, and, for quantitative evaluation, instructions for plating each dilution onto one or more different media and one or more different culture devices based on the required tests, and / or, for qualitative evaluation, instructions for inoculating enrichment cultures for detection of target microorganisms.

[0044] To extract samples in an automated manner, each primary sample is extracted by introducing the appropriate buffer or medium, mixing, and taking subsamples for serial dilutions as needed.

[0045] Microfilm (thin film). A first exemplary incubation device embodiment includes an in situ assembled microfilm. For quantitative evaluation using such microfilm, for example, the container containing the extracted sample or the diluent container is then moved to another position (plating position) in the automation system, where an appropriate amount of each sample / diluent is plated onto a respective microfilm incubation device fabricated in situ in another module (thin film assembly module) of the same automation system. For example, based on LIMS instruction(s), a bottom card is removed and placed at the microfilm assembly position. The card may be made of cardboard or other suitable material having a water-impermeable barrier on at least one side (e.g., coated with or otherwise including a water-impermeable barrier).

[0046] Referring to FIG. 1 , a card 102 with its water-impermeable barrier side 104 facing up (e.g., facing upward) is moved to an assembly position 100, where, in a preferred embodiment, a layer of, for example, adhesive (e.g., glue), gel, wax, or grease can be deposited via a nozzle 106 in a shape (e.g., geometric shape) that defines the outer boundary of a shape (test area) that will contain a culture medium. FIG. 1 shows a pressure-sensitive adhesive being dispensed onto the bottom card (backing card) 102 as a ring 108 that defines a test area 110 and as an optional separate line (ribbon) 112 for sealing purposes. Culture medium can then be deposited (e.g., printed) onto the 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 or before the defining boundary is deposited. After or simultaneously with the deposition of the medium, an appropriate amount of sample or sample diluent is added to the card 102 on which the medium has been or is being deposited.

[0047] 2, sample and media (not shown) are shown being simultaneously dispensed onto test area 110 of microfilm backing card 102, with sample being dispensed from pipette tip 114 and media being dispensed from nozzle 116. In this example, sample and media dispensing occurs at assembly position 200, which may be the same or different from assembly position 100.

[0048] Thereafter, as shown in FIG. 3 , a top film 118 is placed over and on top of the deposited sample / medium (not shown). In this example, the top film 118 is placed at assembly position 300, which may be the same or different from assembly positions 100 and / or 200. The top film 118 can be made of a plastic-based or non-plastic-based material(s) and may be coated on one side (e.g., the bottom side that contacts the sample / medium) with or without a dry medium or dry gelling agent(s) with or without medium components (e.g., nutrients, buffers, chromogens, pH indicators, selection agents, growth factors, etc.). In a next step, as shown in FIG. 3 , a stamp 120 is stamped onto the top film 118, and an edge 122 of the stamp 120, in combination with the deposition ring 108, defines the area over which the sample will be spread. In this example, stamping with the stamp 120 also occurs at assembly position 300, which may be the same or different from assembly positions 100 and / or 200. Alternatively, stamping can be performed at a location different from the placement of the top film 118. The stamping process spreads and further mixes the sample over the surface of the test area 110, and the deposited adhesive (e.g., glue), gel, wax, or grease (in this example, the pressure-sensitive adhesive ring 108) combines with the backing card 102 and top film 118 to form a barrier that prevents the aqueous sample from leaking out of the test area 110. The optional gelling agent(s), if present on the surface of the top film 118, absorbs the applied sample and media mixture, and the resulting gel can trap any gases that may be generated as a result of microbial growth, where the gases may have diagnostic value. The linear ribbon 112 with the pressure-sensitive adhesive deposited thereon provides an additional anchor for sealing or resealing the top film 116.

[0049] The assembled microfilms are labeled (coded according to the specimen) and introduced / transported or moved into an integrated or non-integrated incubator at the appropriate temperature for each test.

[0050] Petri dishes. An alternative exemplary culture device embodiment includes in situ assembled Petri dishes. For quantitative evaluation using Petri dishes, for example, extracted samples or diluent containers are moved to a location (plating position) in the integrated automation system where appropriate amounts of each sample / diluent are plated onto respective Petri dishes fabricated in situ in another module (Petri dish assembly module) of the same automation system.

[0051] As shown in Figure 4, based on instructions, for example, from a LIMS, a pre-loaded Petri dish (base 402 and lid (not shown)) is moved to a Petri dish assembly location 400, where the lid (not shown) is removed. A sterilized base medium (not shown) containing a polymer that solidifies in the presence of one or more of cations, gums, water, and a suitable salt containing cations is added through nozzle 404 to cover the inner base surface 406 of base 402 (e.g., Ca +2 (The heated pectin mixture can be used in conjunction with the lid removal.) The removal of the lid and the addition of the sterilized base medium can both occur at position 400 or can occur at another location within the Petri dish assembly module.

[0052] 5, the Petri dish base 402 is then tilted to a sufficient angle and time to drain and pool the excess (non-adhered) gel, and during tilting, the suction nozzle 408 aspirates the pooled gel, leaving a thin gel coating (not shown) on the Petri dish base 402 (base gel layer). In this example, tilting and aspiration are both performed at assembly position 400, but could alternatively be performed at different assembly positions.

[0053] As shown in FIG. 6, multiple Petri dish bases 402 containing adhered base gel layers may be stacked on an integrated carousel 600 for rapid cooling.

[0054] As shown in FIG. 7, the dish base 402 is then moved to another assembly position 700, where an appropriate amount of sample / diluent is added via pipette tip 502 onto the base gel layer, while simultaneously dispensing via nozzle 504 one or more media and one or more solidifying polymers and / or gums that do not solidify at ambient temperature in the absence of one or more cations (e.g., Ca). +2 Pectin that solidifies at ambient temperature in the presence of suitable cations such as ammonium hydroxide may be used), and an appropriate amount of the sterilized mixture is added. +2 ) diffuses from the base gel layer into the introduced sterilization mixture and sample, for example, forming a pectin gel over the base gel layer. A Petri dish lid (not shown) is then placed on the dish base 402, and the assembled dish is labeled (coded according to the sample), gently mixed, and then moved to an incubation stack and then to an incubator (preferably an integrated incubator) set at a temperature and time suitable to promote the growth of colonies corresponding to one or more target microorganisms.

[0055] Petri Pouches. Additional exemplary culture device embodiments include in situ assembled Petri pouches. For quantitative evaluation using Petri pouches, for example, containers containing extracted samples or diluent containers are moved to a location (e.g., a pouch plating location) of the integrated automation system where appropriate amounts of each sample / diluent are plated using Petri pouches assembled in situ in another module (Petri Pouch Assembly Module) of the same automation system.

[0056] As shown in FIG. 8 , based on instructions, for example, from a LIMS, a pre-loaded Petri pouch 802 (e.g., an appropriately sized plastic bag with or without closure mechanism(s)) is moved to a Petri pouch assembly location 800, where the pouch 802 is either opened or provided with a closable opening through which a powder or liquid / paste medium containing a gelling agent can be introduced, and a sample / diluent for inoculation can be introduced. For inoculation, the sample / diluent may be added to the pouch 802 before, after, or simultaneously with the medium and / or gelling agent. Sample introduction can be via a pipette tip 804, and medium introduction can be via a nozzle 806. After sample inoculation, the Petri pouch 802 may be closed (or not), gently mixed, labeled, and passed through a roller 900 or two plates, in either case with an appropriate gap to distribute the liquid, as shown in Figure 9, after which the pouch 802 is closed if not closed prior to mixing the sample and medium within the pouch, and in either case gelation occurs thereafter. The pouch 802 is then moved to an incubator (e.g., first-in, first-out), preferably an integrated incubator, set at a temperature and time suitable to promote the growth of colonies corresponding to one or more target microorganisms.

[0057] Incubator. For any culture device embodiment processed by an integrated automation system, including those described above, the incubator may or may not be integrated into the integrated automation system. Preferably, an integrated temperature-controlled incubation chamber is used to place and incubate the inoculated culture device at a temperature suitable to promote growth of the selected microorganism (e.g., colonies of the target organism). The volume of the incubation chamber is preferably sufficient to incubate a production lot of devices (e.g., a cumulative production of devices accumulated over a 24-hour production lot). Alternatively, the inoculated culture device is transferred to an external chamber for ex situ incubation.

[0058] Reader Module. After an appropriate incubation (e.g., as determined by each LIMS test protocol), the culture devices (e.g., microfilm, Petri dishes, Petri pouches, etc.) are retrieved from the incubator and transferred to a reader module (e.g., including an integrated imaging device and image analysis program), where each culture device (e.g., microfilm) is read and the data is transmitted, e.g., to a LIMS system. After reading, the culture devices are transferred to a storage device (integrated or non-integrated) for storage until QC completion and report issuance, after which they are disposed of in an appropriate manner. For example, colonies to be counted are analyzed for color and / or gas production, which are reported to the LIMS system. Incubated culture devices can be positioned by a robotic arm or other suitable equipment on a first-in, first-out basis from the incubation chamber. In versions of the system incubating inoculated devices in an external (non-integrated) chamber, the positioning of the culture devices for image analysis is programmed accordingly.

[0059] Sterilization. Optimally, the system is completely sealed and under HEPA-filtered positive pressure. All ingredients, media, buffers, microfilm cards, components, tubing, tips, etc. are pre-sterilized before loading into the automated system. The automated system may have an integrated clean-in-place (CIP) system for tubing components and may also be equipped with UV light to maintain a sterile environment (e.g., before unit startup and during sanitation cycles).

[0060] Stations / Modules. An integrated automation system includes multiple functional modules or stations. An integrated automation system may include, for example, a media preparation section / station, a sample extraction and dilution section / station, a media and sample positioning and mixing section / station, a culture device assembly section / station, an integrated or non-integrated temperature-controlled incubation chamber, an image analysis-based colony counting section / station (e.g., digital), and / or a detection station for qualitative detection of target microorganisms or other analytes.

[0061] Quantitative Track: Figure 10 shows an exemplary high-level flow diagram illustrating the implementation of computer-executable instructions for sample processing and quantitative analysis within an automated integrated laboratory system for quantitative evaluation of microbiological and other samples.

[0062] For the quantitative application track, the automated system can, for example, use functional modules or stations to quantitatively process samples in the following exemplary steps: (1) Dissolve a sterilized, dry powdered medium mixture in water and place it on one or more specified version(s) of an in situ assembled microbial culture device: for example, a microfilm card, a Petri pouch, or a Petri dish (peri (2) the coded sample is extracted, diluted if specified, and added to the designated attribution culture device(s) (e.g., Petri dishes, microfilm, and / or Petri pouches, etc.), and the addition of the sample or its dilution can optionally be simultaneous with the addition of the culture medium to facilitate mixing; (3) in the case of a microfilm card version, a thin film cover optionally coated with a gelling agent can be assembled on top of the base film (base card or bottom card); (4) the assembled inoculated culture device is incubated in a temperature-controlled chamber (incubator) for a specified time; (5) the microbial colonies formed on the culture device are analyzed; preferably, they are analyzed and read by an integrated digital image-based smart recognition and counting module / program, and the results are reported / stored, for example, in a LIMS component of the automated integrated system.

[0063] Example 1 (hereinafter) describes a quantitative track of an automated integrated system in which microfilm cards serve as culture devices, which are assembled and inoculated in situ in a sterile environment for microbial quantification.

[0064] Example 2 (hereinafter) describes a quantitative track of an automated integrated system in which Petri pouches serve as culture devices, which are assembled and inoculated in situ in a sterile environment for microbial quantification.

[0065] Example 3 (hereinafter) describes a quantitative track of an automated integrated system in which Petri dishes serve as culture devices, which are assembled and inoculated in situ in a sterile environment for microbial quantification.

[0066] Qualitative Track. In microbiology laboratories, some test samples (e.g., food or non-food samples) are subjected to quantitative measurement (enumeration) of the number of various microbial groups (e.g., yeasts and molds, coliforms, fecal coliforms, generic E. coli, Enterobacteriaces, Lactobacillus, Staphylococcus, B. cerus, etc.) and / or alternatively, qualitative pathogen and / or spoilage detection(s) (qualitative microbiology). Additional qualitative tracks include, but are not limited to, genetic testing for the presence of specific genes / genetic markers (e.g., authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage profiles, etc.).

[0067] FIG. 11 shows an exemplary high-level flow diagram illustrating the implementation of computer-executable instructions for sample processing and quantitative and / or qualitative analysis within an exemplary automated integrated laboratory system for quantitative and / or qualitative evaluation of microbiological and other samples.

[0068] For a qualitative application track involving pathogen or spoilage detection, for example, the automated system may use functional modules or stations to qualitatively process samples in the following exemplary steps: (1) add enrichment media to the coded sample or extract thereof to obtain an assigned enrichment culture (if multiple analyses are specified and the media are incompatible, the sample / extract can be appropriately divided to obtain multiple assigned enrichment cultures); (2) induce / transfer the enrichment culture to an incubator (preferably an integrated incubator) and incubate for a specified time to obtain an assigned enrichment culture; (3) induce / transfer the assigned enrichment culture to a detection module (preferably an integrated detection module); (4) take an aliquot (typically a small amount) from each coded enrichment culture and transfer the sample to each assigned reagent tube / plate, depending on the detection method; and (5) perform a detection assay (e.g., a nucleic acid (e.g., DNA, RNA) or immunochemistry-based detection assay) on the contents of the assigned reagent tube and report / store the results, such as in a LIMS component of the automated integrated system.

[0069] For qualitative tracks involving genetic testing for the presence of specific genes / genetic markers, etc. (e.g., authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage profiles, etc.), the automated system can use functional modules or stations to qualitatively process samples in the following exemplary steps: (1) dilute a portion of the homogenized sample with an appropriate analyte extraction buffer; (2) then direct / transfer the diluted sample to the appropriate extraction module of the system for analyte extraction (e.g., transfer to a bead-beating module / component for nucleic acid extraction, a shaking / heating module / component for polypeptide / protein antigen extraction, etc.); (3) direct / transfer a portion of the extracted analyte to the appropriate detection module of the system (e.g., transfer to an amplification (e.g., PCR / isothermal) module / component for nucleic acids, an ELISA and / or lateral flow module / component for polypeptide / protein antigens, etc.), with the results reported / stored, for example, in a LIMS component of the automated integrated system.

[0070] Example 4 (hereinafter) describes an embodiment of an automated integrated microbiology laboratory system having a quantitative track (e.g., pathogen or spoilage quantification) and / or a qualitative track, such as genetic testing for the presence of specific genes / genetic markers (e.g., authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage profile, etc.).

[0071] Automated and integrated systems and processes address unmet needs by manufacturing and assembling culture devices (such as petri dishes, microfilm, and petri pouches) in situ as needed for sample testing and quantification in an integrated, on-site system, thereby avoiding device manufacturing at a non-integrated and / or remote facility, irradiation, shipping to an assay facility, and storage at the assay facility prior to use. Rather, sterilized components are assembled in a sterile, integrated environment, eliminating the need for further sterilization, transportation, storage, and / or opening of ex-situ manufactured culture devices prior to inoculation. In combined qualitative / quantitative aspects, the integrated system provides integrated incubation (for qualitative testing) and / or quantification (e.g., colony counting) using in situ assembled culture devices. [Example]

[0072] The following non-limiting examples are provided to further illustrate certain embodiments of the invention disclosed herein.

[0073] Example 1 (Microfilm cards are assembled in situ for use in the automated integrated system described herein) Certain aspects of the present invention provide an integrated automation system including sterile media and / or buffer reagents, incubation device parts, disposable and / or permanent dispensing tips, and automated components for media and / or buffer handling, test sample handling, incubation device assembly, and microbial enumeration and / or detection, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programmed instructions and logic) that, when executed by the one or more processors, cause the integrated automation system (e.g., loading appropriate amounts of each sample into appropriate containers and providing appropriate coded instructions (e.g., barcodes)) to perform the automated steps of the following method: Microfilm card. Certain embodiments of the microfilm card may include one or more bottom backing card(s), an optional intermediate confining layer, and an optional top cover coated with a thin film of gelling agent. For example, in the automated integrated system described herein, the dry culture medium is dissolved in water using sterile components. A polymer-based thickener can be added to adjust the flow behavior of the culture medium solution. The culture medium solution is applied (or printed) into a central reservoir of the backing card (base film), which, as described above, has a layer of adhesive (e.g., pressure-sensitive adhesive), gel, wax, or grease deposited in a geometric shape that defines the outer boundary of the test area containing the culture medium. The extracted and diluted (if specified) coded sample suspension is dispensed into the central reservoir of the base film. The culture medium and sample may be introduced sequentially or simultaneously. The thin film top cover may be pre-cut and stacked within the integrated automated system. The thin film top cover may be pre-coated on one side (e.g., bottom) with adhesive and / or gelling agent powder, with or without dried medium or medium components (e.g., nutrients, buffers, chromogens, pH indicators, selection agents, growth factors, etc.). A robotic arm picks up the coated thin film cover and assembles it with the inoculated base film. The assembly is then stamped to distribute the sample over the test area between the base film and the thin film cover, an attribution label is applied, and the assembled inoculated, coded thin film is sent to an incubator, preferably an integrated incubator, set at the appropriate temperature and with a specified incubation time.

[0074] Example 2 (Petri pouches are assembled in situ for use in the automated integrated system described herein) Certain aspects of the present invention provide an integrated automation system including sterile media and / or buffer reagents, incubation device parts, disposable and / or permanent dispensing tips, and automated components for media and / or buffer handling, test sample handling, incubation device assembly, and microbial enumeration and / or detection, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programmed instructions and logic) that, when executed by the one or more processors, cause the integrated automation system (e.g., loading appropriate amounts of each sample into appropriate containers and providing appropriate coded instructions (e.g., barcodes)) to perform the automated steps of the following method: Petri pouch. An alternative exemplary culture device embodiment includes an appropriately sized plastic bag with or without closure mechanism(s). For example, in the automated integrated system described herein, such a culture bag is advanced to an assembly position. The positioned bag is either open or has a closable opening, which in either case allows for the introduction of a powder or liquid / paste medium containing a suitable gelling agent, and the introduction of a sample / diluent. After sample inoculation, the Petri pouches are closed, gently mixed, labeled, passed through rollers or two plates, in either case with an appropriate gap for dispersing the liquid, and the pouches are closed (if not closed before mixing the sample and medium in the pouches), and then moved into an incubator, preferably an integrated incubator (e.g., first-in, first-out).

[0075] Example 3 (Petri dishes are assembled in situ and used in the automated integrated system described herein) Certain aspects of the present invention provide an integrated automation system including sterile media and / or buffer reagents, incubation device parts, disposable and / or permanent dispensing tips, and automated components for media and / or buffer handling, test sample handling, incubation device assembly, and microbial enumeration and / or detection, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programmed instructions and logic) that, when executed by the one or more processors, cause the integrated automation system (e.g., loading appropriate amounts of each sample into appropriate containers and providing appropriate coded instructions (e.g., barcodes)) to perform the automated steps of the following method: Petri Dish. An additional alternative culture device embodiment includes a Petri dish. For example, in the automated integrated system described herein, the Petri dish is pre-loaded into the system. The dish is moved to an assembly position where the lid is removed. A sterilized base medium containing a polymer that solidifies in the presence of one or more of cations, gums, water, and a suitable salt containing cations is added to cover the base, forming a thin gel coating (base gel layer) on the base of the Petri dish. The plate is then moved to another assembly position where a thin gel coating (base gel layer) is added on top of the base gel layer containing one or more media and one or more cations (e.g., Ca). +2 An appropriate amount of sample is simultaneously added with an appropriate amount of sterilized mixture containing one or more solidifying polymers (e.g., pectin) and / or gums that will not solidify at ambient temperature in the absence of cations. One or more cations diffuse from the base gel layer into the introduced sterilized mixture and sample to form a gel over the base gel layer. A lid is then placed on the plate, the plate is labeled (coded according to the sample), gently mixed, and moved to an incubation stack, and then to an incubator (preferably an integrated incubator) set at a temperature and time suitable to promote the growth of colonies corresponding to one or more target microorganisms.

[0076] Example 4 (An automated integrated microbiology laboratory for quantitative and qualitative microbiology, as well as other qualitative analyses, will be provided) Overview: In microbiology laboratories, some test samples are subjected to qualitative microbiology (e.g., pathogen detection(s)), as well as quantitative determination of the number of various microbial groups, such as yeasts and molds, coliforms, fecal coliforms, generic E. coli, Enterobacteriaces, Lactobacillus, Staphylococcus, and B. cerus, for example.

[0077] Certain aspects of the present invention provide an integrated automation system for quantitative and / or qualitative microbiology (or other qualitative analysis of samples) comprising sterile media and / or buffer reagents, incubation device parts, disposable and / or permanent dispensing tips, and automated components for media and / or buffer handling, test sample handling, incubation device assembly, and microbial enumeration and / or microbial detection and / or detection of other analytes, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions (e.g., LIMS instructions, PLC instructions, firmware, and programmed instructions and logic) that, when executed by the one or more processors, cause the integrated automation system (e.g., loading appropriate amounts of each sample into appropriate containers and providing appropriate coded instructions (e.g., barcodes)) to perform the automated steps of the following exemplary method(s):

[0078] A: Quantitative and Qualitative Analysis Track 1. A specimen collection device: for example, a stomacher bag or a wide-mouth jar; 2. Specimen Arrival and Registration: The specimen submission form is received electronically. The specimen is barcoded or has a unique identification code. 3. Load the sample onto the sample holding tray and feed into the machine. 4. As each sample identifier is read, the LIMS loads the analysis request into the automation system; 5. For example, remove the cap from the sample container or open the sample bag, add the appropriate buffer based on the sample weight, close the collection device, and pass the sample through the homogenizer. 6. Sample homogenization; 7. For quantitative analysis, remove / open the cap of the sample container and take a portion of the sample, dilute it (e.g., serially / or in parallel) for quantitative testing, and move it to a quantitative track using the in situ assembled incubation device detailed elsewhere herein); 8. For qualitative analysis, direct a portion of the initial homogenized sample in an appropriate container to the qualitative module / track (e.g., pathogens, spoilage organisms, etc.).

[0079] B. Pathogen or spoilage track(s) B1. To qualitatively track pathogens or spoilage organisms, enrich a portion of the homogenized sample with an enrichment medium. If multiple analyses are desired for a sample and the required medium is not compatible, split the sample (e.g., into two portions) and add the appropriate enrichment medium to each portion;

[0080] B2. After adding enrichment medium, guide / transfer the enrichment sample container(s) to the incubator (incubation module) to track qualitative tracks such as pathogens or spoilage bacteria;

[0081] B3. Place the enrichment sample container in an appropriate incubator and incubate the sample at an appropriate temperature (e.g., in the range of 25-45°C) for an appropriate time (e.g., in the range of 4-48 hours);

[0082] B4. At the end of the incubation period, the enrichment sample(s) are automatically transferred to the testing / detection module;

[0083] B5. The sample container is then opened, and an appropriate amount of enriched sample is removed and detected by the appropriate detection method selected (e.g., nucleic acid (e.g., DNA / RNA); immunochemistry, etc.). For example, in the case of a nucleic acid-based detection method, the appropriate enriched sample is transferred to the appropriate reagent tube / plate for the nucleic acid detection assay (e.g., nucleic acid (e.g., to DNA / RNA) extraction tube). Reagents are then added, the tube is mixed, and placed in a magnetic separation module where the supernatant is removed, followed by washing, magnetic separation, and supernatant removal (these steps may be repeated). After the final wash, an appropriate buffer is added to dissolve / extract the nucleic acids into solution. A portion of the extract is then transferred to an amplification tube containing the appropriate amplification reaction buffer and reagents. The amplification tube with buffer and reagents is then transferred to the PCR / isothermal amplification module of the system, which performs and reads the amplification reaction and reports the results, for example, to a LIMS component of an automated integrated system. In an alternative embodiment, the appropriate amount of enriched sample may be added directly to an amplification reaction tube, which is then placed in a PCR / isothermal amplification module, etc.

[0084] C. Genetic Testing Track C1. To follow qualitative tracks such as genetic testing for the presence of specific genes / genetic markers etc. (e.g. authentication, GMO testing, allergen testing, species identification, ingredient identification, spoilage profile etc.), appropriate assays / tests are performed by integrated automated systems. For example, tests based on the use of magnetic beads and amplification can be performed.

[0085] C2. In such a testing approach, for example, a portion of the homogenized sample is diluted with extraction buffer in a sample tube, and the mixture is transferred to a bead-beating module / component of the integrated system, where coated magnetic beads are added to the mixture in the tube. The diluted sample and beads are then mixed in the tube, the tube is placed on a magnet, and the supernatant is removed. A wash buffer is then added to wash the beads, the tube is placed on a magnet, the supernatant is removed, and the beads are then suspended in an appropriate volume of buffer. For each such test sample preparation, approximately 1 to 5 microliters of suspended beads are transferred to each appropriate amplification tube containing the appropriate amplification reaction buffer and reagents. The amplification tubes with buffer and reagents are then transferred to the system's PCR / isothermal amplification module, which performs and reads the amplification reaction and reports the results, for example, to a LIMS component of the automated integrated system.

[0086] For example, in a test requiring an immunoassay, an appropriate extraction buffer is added to a portion of the original homogenized sample or a dilution thereof in a sample tube, which is then directed to the shaking / heating module component of the integrated automation system. After an appropriate residence time in the shaking / heating module, a portion of each sample is transferred to the analysis module of the system, which contains an ELISA plate and / or lateral flow device component, followed by automated addition of reagents, incubation, washing, and reading and reporting of results to, for example, the LIMS component of the automated integrated system.

Claims

1. 1. A system for microbiological evaluation using an in situ production and culture device, comprising: An integrated automation system comprising sterile media and / or buffer reagents, culture device parts, and automated components for media and / or buffer handling, sample handling, culture device assembly, and microbial enumeration, all within a sterile environment; and one or more processors; and memory containing computer-executable instructions that, when executed by the one or more processors, cause the integrated automation system to perform the following operations: a) determining that coded test samples loaded into the system should be quantitatively evaluated; b) using said culture device components to assemble in situ a microbiological culture device of the designated type for culturing and quantitatively processing said coded test sample; c) inoculating the designated type of culture device with an appropriate amount of the coded test sample, or extracts and / or dilutions thereof, either as part of the in situ assembly or after the in situ assembly; and d) attributing the inoculated culture device to the coded test sample, wherein the assembly and inoculation is performed in situ in the sterile environment by the integrated automated components of the system, thereby eliminating the need to sterilize, transport, store, and / or open an ex-situ manufactured culture device prior to its inoculation.

2. 10. The system of claim 1, wherein execution of the computer-executable instructions further causes the system to perform the following actions: e) subjecting the inoculated culture device to incubation at a temperature and time suitable to promote the growth of colonies corresponding to one or more target microorganisms; f) counting the colonies using the counting component in situ; and g) The system stores and / or transmits (e.g., to a LIMS or similar program) the enumeration data attributed to the coded test samples for storage and / or reporting and / or analysis.

3. 3. The system of claim 2, wherein in e), inducing incubation comprises incubating the inoculated attribution culture device in situ using an incubator integrated into the system and / or incubating the inoculated attribution culture device ex situ using a non-integrated incubator.

4. 3. The system of claim 2, wherein in step f), counting the colonies in situ comprises using an integrated imaging device and image analysis program.

5. 10. The system of claim 1, If the coded test sample loaded into the system is a primary sample, execution of the computer-executable instructions further causes the system to: mixing or homogenizing the coded test sample with a suitable amount of a designated buffer and / or medium to provide an extracted sample; taking a portion of the extracted sample; and Diluting the extracted portion, if specified, and providing the extracted sample or a dilution thereof for inoculation into the prefabricated culture device; and / or If the coded test sample loaded into the system is an extracted sample, execution of the computer-executable instructions further causes the system to: taking a portion of the extracted sample; and The system diluting the collected portion, if specified, and providing the extracted sample or a dilution thereof for inoculation into the assembled culture device.

6. The system of claim 1 , wherein the computer-executable instructions include one or more of LIMS instructions, PLC instructions, firmware, and programmed instructions and logic.

7. 10. The system of claim 1, wherein the specified type of in situ assembled microbial culture device includes at least one selected from the group consisting of microfilm cards, Petri dishes containing gel and / or gum-based media, and Petri pouches.

8. 10. The system of claim 7, wherein the in situ assembled microbial culture device is a Petri dish, and for the in situ assembly, execution of the computer-executable instructions further causes the system to perform the following actions: Remove the lid of a pre-loaded Petri dish having a base and a lid; introducing an appropriate amount of a sterile mixture containing one or more media and one or more solidifying polymers and / or gums; introducing an appropriate amount of said coded test sample, or extracts and / or dilutions thereof; placing the lid on the base of the Petri dish; and The system mixes the introduced sterile mixture with the sample, extract, or diluent thereof prior to gelation.

9. 9. The system of claim 8, wherein said introduction of said sterilization mixture and said introduction of said coded test sample, or extract and / or dilution thereof, are simultaneous.

10. 10. The system of claim 8, wherein the one or more solidifying polymers and / or gums in the introduced sterilization mixture do not solidify at room temperature in the absence of one or more cations, and prior to introducing the sterilization mixture and the sample, extract, or diluent thereof, execution of the computer-executable instructions further causes the system to: The system further comprises a base gel layer covering the base containing one or more cations, the one or more cations being diffusible from the base gel layer upon introduction of the sterilization mixture and the sample, extract thereof, or diluent.

11. 11. The system of claim 10, wherein the one or more solidifying polymers and / or gums in the introduced sterilization mixture comprise pectin and / or alginate, and the one or more cations in the base gel layer comprise divalent or trivalent cations.

12. The system of claim 10 , wherein the base gel layer comprises agar, gelatin, silica gel, or carrageenan.

13. The one or more cations are Ca 2+ The system of claim 10, comprising:

14. 9. The system of claim 8, wherein the sterilization mixture is introduced in liquid form, or the one or more solidifying polymers and / or gums in the introduced sterilization mixture are initially introduced as a coating in powder form, which absorbs and forms a gel over the base gel layer upon introduction of the culture medium and / or the coded test sample, or extract and / or diluent thereof, in liquid form.

15. 10. The system of claim 7, wherein the in situ assembled microbial culture device is a microfilm device having a backing card and a top cover, and for the in situ assembly, execution of the computer-executable instructions further causes the system to perform the following actions: applying a sterile medium to the surface of the backing card; introducing an appropriate amount of the coded test sample, or extract and / or dilution thereof, into contact with the medium to form a mixture; placing a top cover over the backing card; and The system stamps the positioned top cover to distribute the mixture over the desired test area of ​​the backing card.

16. 17. The system of claim 16, wherein the surface of the backing card defines a reservoir for containing the culture medium and the coded test sample, or extracts and / or dilutions thereof, the reservoir having a surface area that defines the desired test area.

17. 16. The system of claim 15, wherein execution of the computer-executable instructions further causes the system to perform the following actions before or after applying a sterile medium to the surface of the backing card: The system applies adhesive, gel, wax, or grease in a pattern that defines the desired area over which the mixture is to be distributed.

18. The system of claim 17 , wherein the adhesive comprises a pressure sensitive adhesive.

19. 16. The system of claim 15, wherein execution of the computer-executable instructions further causes the system to perform the following actions prior to placing the top cover: The system introduces a volume of enrichment medium that is diluted with the introduced coded test sample, or extract and / or diluent thereof.

20. 20. The system of claim 17, wherein the adhesive, gel, wax, or grease is applied before the sterilization medium.

21. 16. The system of claim 15, wherein applying the sterilization medium to the surface of the backing card comprises: printing the sterilization medium onto the surface of the backing card; and / or otherwise dispensing the sterilization medium onto the surface of the backing card and then drying.

22. 16. The system of claim 15, wherein the surface of the top cover is pre-coated with gelling agent powder, with or without dry medium or medium components.

23. 8. The system of claim 7, wherein the in situ assembled microbial culturing device is a Petri pouch having or configured to have an opening.

24. 24. The system of claim 23, wherein execution of the computer-executable instructions for the in-situ assembly further causes the system to perform the following actions: introducing an appropriate amount of a sterile mixture containing one or more culture media and one or more solidifying polymers and / or gums through said opening; introducing an appropriate amount of the coded test sample, or extracts and / or dilutions thereof, through the opening; mixing the introduced sterile mixture with the sample, extract, or dilution thereof in the pouch; rolling, pressing, or otherwise forming the pouch to distribute the mixed ingredients and achieve a desired gel thickness and surface area prior to gelling; and The system further comprising closing the opening before or after the mixing and / or the rolling.

25. 25. The system of claim 24, wherein the sterilization mixture is introduced in liquid form, or the one or more culture media and the one or more solidifying polymers and / or gums of the introduced sterilization mixture are initially introduced in powder form, which media and solidifying polymers and / or gums absorb and form a gel upon the introduction of the coded test sample, or extract and / or diluent thereof.

26. 25. The system of claim 24, wherein the one or more solidifying polymers and / or gums in the introduced sterilization mixture do not solidify at room temperature in the absence of one or more cations, and the inside of the Petri pouch has been pretreated with the one or more cations that are diffusible into the introduced sterilization mixture and the sample, extract thereof, or diluent.

27. 27. The system of claim 26, wherein prior to introducing the sterilization mixture and the sample, extract, or dilution thereof, execution of the computer-executable instructions further causes the system to perform the following actions: The system wherein the inside of the Petri pouch is pretreated with the one or more cations, which are diffusible into the introduced sterilization mixture and the sample, extract thereof, or diluent.

28. 28. The system of claim 27, wherein the one or more cations comprises a divalent or trivalent cation.

29. The one or more cations are Ca 2+ 30. The system of claim 28, comprising:

Images