Methods for Microbial Detection

Abstract

Methods for detecting microorganisms in a sample are provided, comprising detecting the confluence of the sample. Also provided are methods for assaying a test agent, comprising adding the test agent to a sample containing the microorganisms and measuring the confluence of the sample. In some embodiments, the detecting is automated. In some embodiments, the detecting is continuous. In some embodiments, the confluence is percent confluence.

Description

[Technical Field]

[0001] The general inventive concept relates to the field of microbial detection, and more particularly to a method for the automated detection of microorganisms by direct imaging.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 380,407, filed October 21, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Traditional microbiological testing is based on century-old technology using agar plates to enumerate microbial colony-forming units (CFUs) or visual detection of turbidity in liquid media, which indicates microbial growth. Traditional plate counts for microbial bioburden monitoring of in-process or finished product samples require 3-7 days of incubation for quantitative enumeration. Sterility testing is performed according to reference standards established by the United States Pharmacopeial Convention (USP). Non-sterile product bioburden testing is performed according to USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests and USP <62> Microbial counts are performed using either membrane filtration (0.45 μm), plate counts, or the most probable number method according to the USP. Total aerobic microbial counts (TAMC) are determined using soybean-casein digest agar after incubation at 30-35°C for 3-5 days. Total combined yeasts and molds counts (TYMC) are determined using Sabouraud dextrose agar after incubation at 20-25°C for 5-7 days. In contrast, USP <71> The presence / absence sterile product finished product sterility test according to USP requires a 14-day incubation period before visual confirmation of microbial growth. <71> In , sterile products are tested according to the minimum volume specified in the standard, using the minimum number of sample articles specified in the standard. To remove any potential antimicrobial properties, the product is filtered through a membrane filter (0.45 μm) to retain the microorganisms, washed with a diluent to remove the antimicrobial components, and then incubated in a medium to grow the microorganisms.The device is designed so that the solution to be tested can be introduced and filtered under sterile conditions, allowing the membrane to be transferred aseptically to the medium, or it is suitable for carrying out incubation after adding the medium to the device itself, as is done, for example, in the commercially available Steritest system. For sterile products without any antimicrobial properties, direct inoculation of the medium can be carried out. USP <71> specifies that sterility test media may be used, provided they meet the requirements for growth promotion testing of aerobic, anaerobic, and fungal bacteria. Official media found to be suitable for sterility testing are Fluid Thioglycollate Medium (FTM), which is primarily intended for the culture of anaerobic bacteria but also detects aerobic bacteria, and Soybean-Casein Digest Medium (Tryptic Soy Broth (TSB)), which is suitable for the culture of both fungi and aerobic bacteria. FTM and TSB samples are incubated at 30-35°C and 20-25°C, respectively, for 14 days and then visually inspected for the presence of turbidity, which indicates microbial growth. With the introduction of cell therapy products, USP informational chapter <1071> Rapid Sterility Testing of Short-Life Products: A Risk-Based Approach has recently been published.

[0004] For medical devices terminally sterilized with ionizing radiation, an incremental dose verification or sterilization dose demonstration is performed in accordance with ISO 11137. After exposure to the specified radiation dose, a sterility test is performed on the medical device or a sample item portion (SIP) of the medical device. The irradiated medical device sample is aseptically transferred to TSB, incubated at approximately 30°C for 7 days, and then visually inspected for turbidity, which indicates microbial growth.

[0005] Traditional microbiological methods are manual, provide slow retrospective results, are subject to subjective interpretation, have limited data traceability, and have potential data integrity issues. To address these limitations, recent advances have been made in the development of alternative and rapid microbiological methods (ARMMs), including, but not limited to, the development of CFR Part 11-compliant computer-based detection technologies, such as colorimetric CO2 detection, solid-phase laser scanning cytometry, flow cytometry, ATP bioluminescence, and automated detection of microcolony growth based on autofluorescence detection. However, all of these detection technologies require unique sample preparation, in some cases specialized reagents for rapid detection of microorganisms, and require costly and time-consuming revalidation efforts before use. In addition, surrogate detection of viable microorganisms without direct determination of microbial growth by some of these alternative detection technologies can be destructive tests that can produce false-positive results, which is unacceptable for certain industrial microbiology applications, such as sterility testing of finished products or in-process microbial screening. Summary of the Invention

[0006] There remains a need for methods for the automated detection of microorganisms by direct imaging.

[0007] A method for detecting a microorganism in a sample is provided, comprising detecting the confluence of the sample.

[0008] In some embodiments, confluence is detected using an image-based metric. In some embodiments, the image-based metric comprises direct imaging. In further embodiments, the direct imaging is direct cell imaging.

[0009] In some embodiments, the sample is in an incubator. In some embodiments, detecting comprises detecting using a microscope or an image sensor.

[0010] In some embodiments, the confluence of the sample is compared to the confluence of a control sample. In some embodiments, the confluence of the sample is monitored over a period of time. In some embodiments, the confluence of the sample is monitored continuously.

[0011] In some embodiments, the sample is contacted with an antibody to the microorganism.

[0012] In some embodiments, the sample is contacted with a fluorescent or bioluminescent agent.

[0013] In some embodiments, the system further comprises detecting fluorescence of the sample. In further embodiments, the means for detecting fluorescence is a fluorometer.

[0014] In some embodiments, the system further comprises detecting bioluminescence of the sample. In further embodiments, the means for detecting bioluminescence is a luminometer.

[0015] In some embodiments, the microorganism includes, but is not limited to, Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. In some embodiments, the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

[0016] In some embodiments, any genus or species of viable microorganism may be detected.

[0017] In some embodiments, detecting confluence is automated.

[0018] In some embodiments, the confluence is a percent confluence.

[0019] A method for assaying a test agent is provided, comprising adding the test agent to a sample containing a microorganism and detecting confluence of the sample.

[0020] In some embodiments, confluence is detected using an image-based metric.

[0021] In some embodiments, the image-based metric comprises direct imaging. In further embodiments, the direct imaging is direct cellular imaging.

[0022] In some embodiments, the sample is in an incubator. In some embodiments, detecting comprises detecting using a microscope or an image sensor.

[0023] In some embodiments, the confluence of the sample is compared to the confluence of a control sample. In some embodiments, the confluence of the sample is monitored over a period of time. In some embodiments, the confluence of the sample is monitored continuously.

[0024] In some embodiments, the test agent is a chemical compound, an adjuvant, an antibiotic, a bacteriophage, or a combination thereof.

[0025] In some embodiments, the method further comprises identifying the test agent as a prebiotic if the confluence of the sample is greater than the confluence of the control sample. In some embodiments, the method further comprises identifying the test agent as a prebiotic if the percent confluence of the sample is greater than the percent confluence of the control sample.

[0026] In some embodiments, the method further comprises identifying the test agent as a prebiotic if the confluence of the sample increases over a period of time.

[0027] In some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the confluence of the sample is less than the confluence of the control sample.

[0028] In some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the confluence of the sample decreases over a period of time.

[0029] In some embodiments, the sample is contacted with an antibody to the microorganism.

[0030] In some embodiments, the sample is contacted with a fluorescent or bioluminescent agent.

[0031] In some embodiments, the system further comprises detecting fluorescence of the sample. In further embodiments, the means for detecting fluorescence is a fluorometer.

[0032] In some embodiments, the system further comprises detecting bioluminescence of the sample. In further embodiments, the means for detecting bioluminescence is a luminometer.

[0033] In some embodiments, the microorganism includes, but is not limited to, Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. In some embodiments, the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

[0034] In some embodiments, any genus or species of viable microorganism may be detected.

[0035] In some embodiments, detecting confluence is automated.

[0036] In some embodiments, the confluence is a percent confluence. [Brief explanation of the drawings]

[0037] [Figure 1A] Illustrates the IncuCyte system. Illustrates a results table of time to detection of microorganisms with IncuCyte system automated direct imaging versus automated BacT / ALERT system CO2 colorimetric detection. [Figure 1B] The IncuCyte system is shown. The time to detection of Cutibacterium acnes on the IncuCyte is shown. The time course shows the change in % confluence over time. [Figure 1C] IncuCyte system. Time course of Cutibacterium acnes growth on IncuCyte. [Figure 2] IncuCyte CAR-T microbial screening. The graph shows a time course showing the change in confluence across different groups. CAR-T samples were spiked with approximately 5 or 55 CFU of Staphylococcus aureus (S. aureus, SA) in a 24-well microplate. Parallel TSB media-only samples spiked with approximately 5 or 55 CFU of SA were included in the 24-well microplate. Phase contrast imaging, 20x magnification, 36 images per well. [Figure 3] Figure 1 shows the % confluence detection of Bacillus subtilis spore spikes on CHO cells. The graph shows a time course demonstrating the detection of CHO cells contaminated with Bacillus subtilis. [Figure 4]Staphylococcus epidermidis (SE) prebiotic and antibacterial screening (1%) is shown. Component A (sterile filtered), SE prebiotic activity was detected by faster confluence % growth compared to the positive control. Component B (sterile filtered), SE antibacterial activity was detected by no change in confluence % growth over time. Component C (sterile filtered), SE prebiotic activity was detected by faster confluence % growth compared to the positive control. [Figure 5] Natural product extract Staphylococcus aureus (SA) antibacterial screening (0.1%). % confluence results map for a 96-well natural product extract microplate. Natural product extract SA antibacterial activity was detected by the lack of change in % confluence growth over time for sample wells A3, E6, B9, C11, and D11. DETAILED DESCRIPTION OF THE INVENTION

[0038] While the general inventive concept may be embodied in many forms, specific embodiments thereof are shown in the drawings and described in detail herein, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the general inventive concept. Accordingly, the general inventive concept is not intended to be limited to the specific embodiments shown herein.

[0039] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0040] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the object of the article. By way of example, "a cell" means one cell or more than one cell.

[0041] "About," as used herein, when referring to measurable values ​​such as amounts and temporal durations, is meant to encompass variations of ±5%, preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for practicing the disclosed methods.

[0042] "Confluence," as used herein, is based on the amount of surface area of ​​a culture vessel that appears to be covered by cells compared to the total surface area of ​​the culture vessel. The cells may be of any origin. In some embodiments, the cells are mammalian, insect, microbial, or a combination thereof. In some embodiments, confluence is percent confluence. In some embodiments, confluence may be measured on an image of the culture vessel. In some embodiments, the image is an image of a portion of the culture vessel. In some embodiments, confluence is based on the amount of surface area of ​​the image that appears to be covered by cells compared to the total surface area of ​​the image. Confluence may be measured or calculated, or may be determined by an algorithm. Confluence may be determined using an IncuCyte. Confluence may be measured over a period of time. Initial confluence is measured at time zero. Confluence may be measured continuously or at time intervals to determine confluence over time.

[0043] "Percent confluence" or "% confluence," as used herein, is based on the percentage of the surface area of ​​a culture vessel that appears to be covered by cells compared to the total surface area of ​​the culture vessel. The cells may be of any origin. In some embodiments, the cells are mammalian, insect, microbial, or a combination thereof. In some embodiments, the percent confluence may be measured on an image of the culture vessel. In some embodiments, the image is an image of a portion of the culture vessel. In some embodiments, the confluence is based on the amount of surface area of ​​the image that appears to be covered by cells compared to the total surface area of ​​the image. Percent confluence may be measured or calculated, or may be determined by an algorithm. Percent confluence may be determined using an IncuCyte. Percent confluence may be measured over a period of time. An initial percent confluence is measured at time zero. Percent confluence may be measured continuously or at time intervals to determine percent confluence over time.

[0044] The terms "antibody" and "antibodies," as used herein, are intended to have broad meanings and include immunoglobulin molecules, including polyclonal antibodies, monoclonal antibodies, including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, and single-chain antibodies.

[0045] Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0046] The term "antibody fragment" refers to a portion of an immunoglobulin molecule having a heavy chain and / or light chain antigen-binding site, for example, heavy chain complementarity determining regions (HCDRs) 1, 2, and 3, light chain complementarity determining regions (LCDRs) 1, 2, and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of one antibody arm; and a domain antibody (dAb) fragment consisting of the VH domain. Although VH and VL domains can be engineered and linked together via synthetic linkers to form a variety of single-chain antibody designs, the VH / VL domains pair intramolecularly, or, when the VH and VL domains are expressed as separate single-chain antibody constructs, pair intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or diabody. These are described, for example, in WO 1998 / 44001, WO 1988 / 01649, WO 1994 / 13804, and WO 1992 / 01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0047] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD38 is substantially free of antibodies that specifically bind to antigens other than human CD38). However, an isolated antibody that specifically binds CD38 may have cross-reactivity to other antigens, such as orthologs of human CD38, such as Macaca fascicularis (cynomolgus monkey) CD38. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0048] "Humanized antibody" refers to an antibody in which the antigen-binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Because humanized antibodies may contain substitutions within the framework regions, such frameworks may not be exact copies of expressed human immunoglobulin or germline gene sequences.

[0049] A "human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen-binding site are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody includes a heavy or light chain variable region "derived" from sequences of human origin when the variable region of the antibody is obtained from a system that uses human germline immunoglobulins or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage and transgenic non-human animals, such as mice, carrying human immunoglobulin loci described herein. A "human antibody" may contain amino acid differences when compared to human germline or rearranged immunoglobulin sequences, due, for example, to naturally occurring somatic mutations or the introduction of intentional substitutions in the framework or antigen-binding site. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to the amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0050] The isolated humanized antibody can be synthetic. Human antibodies are derived from human immunoglobulin sequences, but can be generated using systems such as phage display that incorporate synthetic CDRs and / or synthetic frameworks, or can undergo in vitro mutagenesis to improve the properties of the antibody, resulting in an antibody that does not naturally occur within the in vivo human antibody germline repertoire.

[0051] The term "recombinant antibody," as used herein, includes antibodies isolated from animals transgenic or transchromosomic for human immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody, antibodies isolated from recombinant combinatorial antibody libraries, as well as antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences, or antibodies generated in vitro using Fab arm exchange (e.g., bispecific antibodies).

[0052] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition, displaying a single binding specificity and affinity for a particular epitope, or, in the case of bispecific monoclonal antibodies, dual binding specificities for two distinct epitopes.

[0053] The term "epitope," as used herein, refers to the portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In discontinuous epitopes, amino acids in different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule.

[0054] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a synthetic or recombinant receptor comprising an antigen-specific domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises an extracellular hinge or spacer region, a transmembrane domain, or a combination thereof. In some embodiments, the antigen-specific domain is an scFv.

[0055] The term "chimeric antigen receptor T cell" or "CAR-T" as used herein refers to a T cell that expresses a CAR.

[0056] In some embodiments of any of the compositions or methods described herein, ranges are intended to include every integer or fraction or value within the range.

[0057] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" and / or "consisting essentially of" such features.

[0058] Currently, the use of automated direct microscopic imaging has not been proposed for microbial presence / absence screening of non-sterile product or in-process samples, presence / absence testing of finished product sterility test samples, bioprocess microbial contamination monitoring, antibiotic or bacteriophage susceptibility testing, or for prebiotic and antimicrobial product development applications. Provided herein are methods for automated, non-invasive, and non-destructive direct microscopic imaging of in-process and finished product non-sterile and sterility test samples prepared according to previously validated methods, while providing significantly faster time-to-detection than manual visual turbidity or colony-forming unit (CFU) formation on agar growth media. In some embodiments, the methods utilize a 21 CFR Part 11-compliant system for improved data traceability and data integrity. The methods described herein enable the use of direct microscopic imaging for bioprocess microbial contamination monitoring from aseptically collected bioreactor samples, antibiotic or bacteriophage susceptibility testing in clinical settings, and prebiotic and antimicrobial product development applications.

[0059] A method is provided for automated, non-destructive, and non-invasive detection of microorganisms by direct imaging in liquid media samples, improving data integrity and reducing time to detection compared to traditional manual visual detection based on media turbidity or CFU formation on agar growth media. In some embodiments, detection is continuous. Automated imaging systems, such as the Essen BioScience IncuCyte system, have a 10 μm detection limit for enumeration and are routinely used for mammalian cell imaging for enumeration and viability assessment applications. To overcome this 10 μm size limitation that prevents direct detection and enumeration of microorganisms in the 1 μm size range, the inventors have discovered that the % confluence detection metric of mammalian cell imaging systems can be used for rapid presence / absence determination for end-product sterility testing, presence / absence screening or in-process microbial screening of non-sterile products, bioprocess microbial contamination monitoring, antibiotic or bacteriophage susceptibility testing, and prebiotic ingredient and / or antimicrobial product development applications.

[0060] method A method for detecting a microorganism in a sample is provided, comprising detecting the confluence of the sample.

[0061] In some embodiments, the confluence is a percent confluence.

[0062] In some embodiments, confluence is detected using an image-based metric. In some embodiments, the image-based metric comprises direct imaging. In further embodiments, the direct imaging is direct cell imaging.

[0063] In some embodiments, the sample is in an incubator. In some embodiments, the sample is under controlled temperature incubation. In some embodiments, detecting comprises detecting using a microscope or an image sensor.

[0064] In some embodiments, the sample is at a temperature of about 20°C to about 40°C. In some embodiments, the temperature is about 20°C to about 37°C. In some embodiments, the temperature is 25°C to about 35°C. In further embodiments, the temperature is about 20°C to about 25°C. In still further embodiments, the temperature is 30°C to about 35°C.

[0065] In some embodiments, the sample is grown for about 0.1 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.25 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.25 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.5 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 1 day before measuring the confluence of the sample. In some embodiments, the sample is grown for about 2 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 3 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 4 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 5 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 5 days before measuring the confluence of the sample. In some embodiments, the samples are incubated for about 6 days before measuring the confluence of the samples. In some embodiments, the samples are incubated for about 7 days before measuring the confluence of the samples.

[0066] In some embodiments, the confluence of the sample is compared to the confluence of a control sample. In some embodiments, the confluence of the sample is monitored over a period of time.

[0067] In some embodiments, the sample is contacted with an antibody to the microorganism.

[0068] In some embodiments, the sample is contacted with a fluorescent or bioluminescent agent.

[0069] In some embodiments, the system further comprises detecting fluorescence of the sample. In further embodiments, the means for detecting fluorescence is a fluorometer.

[0070] In some embodiments, the system further comprises detecting bioluminescence of the sample. In further embodiments, the means for detecting bioluminescence is a luminometer.

[0071] In some embodiments, the microorganism includes, but is not limited to, Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. In some embodiments, the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

[0072] In some embodiments, any genus or species of viable microorganism may be detected.

[0073] In some embodiments, the sample confluence is monitored over a period of time. In further embodiments, the sample confluence is monitored at set intervals over a period of time. In still further embodiments, the sample confluence is monitored at random intervals over a period of time. In still further embodiments, the sample confluence is monitored continuously over a period of time. The period of time can be about 0.1 to about 1000 hours, about 0.1 to about 100 hours, about 0.1 to about 72 hours, about 0.1 to about 48 hours, about 0.1 to about 24 hours, about 0.1 to about 12 hours, and about 0.1 to about 6 hours.

[0074] In some embodiments, detecting confluence is automated.

[0075] cell In some embodiments, the sample comprises eukaryotic cells. In further embodiments, the eukaryotic cells produce a therapeutic product. The therapeutic product may be released into the cell culture medium, where it may be collected. The methods provided herein allow for the detection of contamination.

[0076] In some embodiments, the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.

[0077] In some embodiments, the eukaryotic cell includes, but is not limited to, a mouse cell, a CHO cell, a T cell, or a B cell. In some embodiments, the mouse cell is a mouse Sp2 / 0 cell. In some embodiments, the eukaryotic cell is a HEK293F cell. In some embodiments, the eukaryotic cell is a PER.C6 cell.

[0078] In some embodiments, the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).

[0079] In any of the methods described herein, confluence can be percent confluence.

[0080] Assay A method for assaying a test agent is provided, comprising adding the test agent to a sample containing a microorganism and detecting confluence of the sample.

[0081] In some embodiments, the confluence is a percent confluence.

[0082] In some embodiments, confluence is detected using an image-based metric.

[0083] In some embodiments, the image-based metric comprises direct imaging. In further embodiments, the direct imaging is direct cellular imaging.

[0084] In some embodiments, the sample is in an incubator. In some embodiments, the sample is under controlled temperature incubation. In some embodiments, detecting comprises detecting using a microscope or an image sensor.

[0085] In some embodiments, the sample is at a temperature of about 20°C to about 40°C. In some embodiments, the temperature is about 20°C to about 37°C. In some embodiments, the temperature is 25°C to about 35°C. In further embodiments, the temperature is about 20°C to about 25°C. In still further embodiments, the temperature is 30°C to about 35°C.

[0086] In some embodiments, the sample is grown for about 0.1 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.25 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.25 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 0.5 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 1 day before measuring the confluence of the sample. In some embodiments, the sample is grown for about 2 days before measuring the confluence of the sample. In some embodiments, the sample is grown for about 3 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 4 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 5 to about 7 days before measuring the confluence of the sample. In some embodiments, the sample is incubated for about 5 days before measuring the confluence of the sample. In some embodiments, the samples are incubated for about 6 days before measuring the confluence of the samples. In some embodiments, the samples are incubated for about 7 days before measuring the confluence of the samples.

[0087] In some embodiments, the confluence of the sample is compared to the confluence of a control sample. In some embodiments, the confluence of the sample is monitored over a period of time. In some embodiments, the percent confluence of the sample is compared to the percent confluence of a control sample. In some embodiments, the percent confluence of the sample is monitored over a period of time.

[0088] In some embodiments, the test agent is a chemical compound, an adjuvant, an antibiotic, a bacteriophage, or a combination thereof.

[0089] In some embodiments, the method further comprises identifying the test agent as a prebiotic if the confluence of the sample is greater than the confluence of the control sample. In some embodiments, the method further comprises identifying the test agent as a prebiotic if the percent confluence of the sample is greater than the percent confluence of the control sample.

[0090] In some embodiments, the method further comprises identifying the test agent as a prebiotic if the confluence of the sample increases over a period of time.

[0091] In some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the confluence of the sample is less than the confluence of the control sample, hi some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the percent confluence of the sample is less than the percent confluence of the control sample.

[0092] In some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the confluence of the sample decreases over a period of time. In some embodiments, the method further comprises identifying the test agent as an antimicrobial agent if the percent confluence of the sample decreases over a period of time.

[0093] In some embodiments, the sample is contacted with an antibody to the microorganism.

[0094] In some embodiments, the sample is contacted with a fluorescent or bioluminescent agent.

[0095] In some embodiments, the system further comprises detecting fluorescence of the sample. In further embodiments, the means for detecting fluorescence is a fluorometer.

[0096] In some embodiments, the system further comprises detecting bioluminescence of the sample. In further embodiments, the means for detecting bioluminescence is a luminometer.

[0097] In some embodiments, the microorganism includes, but is not limited to, Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. In some embodiments, the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

[0098] In some embodiments, any genus or species of viable microorganism may be detected.

[0099] In some embodiments, detecting confluence is automated.

[0100] cell In some embodiments, the sample comprises eukaryotic cells. In further embodiments, the eukaryotic cells produce a therapeutic product. The therapeutic product may be released into the cell culture medium, where it may be collected. The methods provided herein allow for the detection of contamination.

[0101] In some embodiments, the eukaryotic cell produces a protein, an antibody or fragment thereof, a duobody, a receptor, a chimeric antigen receptor, a glycoprotein, a viral vector, or a combination thereof.

[0102] In some embodiments, the eukaryotic cell includes, but is not limited to, a mouse cell, a CHO cell, a T cell, or a B cell. In some embodiments, the mouse cell is a mouse Sp2 / 0 cell. In some embodiments, the eukaryotic cell is a HEK293F cell. In some embodiments, the eukaryotic cell is a PER.C6 cell.

[0103] In some embodiments, the eukaryotic cell is a chimeric antigen receptor T cell (CAR-T cell).

[0104] In any of the assays described herein, confluence can be percent confluence. [Example]

[0105] Example 1: IncuCyte System Time Course Materials and Methods IncuCyte System Automated Direct Imaging vs. Automated BacT / Alert System BioBall™ samples (bioMerieux, USA) of the designated microorganisms were resuspended according to the manufacturer's instructions. Separate experiments for time to detection were performed by inoculating approximately 25 CFU (colony-forming units) into aerobic or anaerobic BacT / ALERT sample bottles, or into either tryptic soy broth (TSB) and liquid thioglycollate medium (FTM) for BacT / ALERT or IncuCyte system analysis, respectively. The majority of test microorganisms were incubated aerobically using TSB-based medium, while only Cutibacterium acnes was incubated anaerobically in anaerobic BacT / ALERT bottles containing TSB-based medium or FTM under anaerobic headspace conditions. Healthy T-cell donor CAR-T cells (1E4–1E6) were present in all microbial spiked samples except for the Cutibacterium acnes IncuCyte sample, which was FTM-only. Inoculum numbers for Cutibacterium acnes FTM alone were also less than 25 CFU for IncuCyte system experiments, as described below.

[0106] Plasma-treated 12-well cell culture plates containing approximately 6 mL of medium per well were used for all test microorganism IncuCyte experiments, except for Cutibacterium acnes, which was performed in plasma-treated 24-well plates containing approximately 3 mL of medium. The time to detection of positive microbial growth for the BacT / ALERT system was determined indirectly based on a colorimetric CO2 detection algorithm. In contrast, the time to detection of positive microbial growth for the IncuCyte system was determined by direct microscopic imaging based on the increase in the confluence % metric over time for all test microorganisms. Figure 1A shows the results of separate experiments for BacT / ALERT and IncuCyte time to detection.

[0107] Time to detection of Cutibacterium acnes in Incucyte A time course showing the change in % confluence over time was performed. A 200 mL bottle of FTM was spiked with Cutibacterium acnes at approximately 3 CFU / mL and then aseptically transferred (approximately 70 mL) to a 24-well microplate (approximately 3 mL / well). The sample plate was placed in the IncuCyte system in a 37°C incubator. Phase contrast imaging, 20x magnification, 36 images / well, was performed every 2 hours. Figure 1B shows the increase in the IncuCyte % confluence metric over time for Cutibacterium acnes growth in FTM.

[0108] Time lapse images of Cutibacterium acnes growth in IncuCyte As described above, the IncuCyte system was configured to capture 36 phase-contrast images per well of Cutibacterium acnes growth in a 24-well microplate every 2 hours. These time-resolved images of Cutibacterium acnes growth were saved and used by the system to calculate the % confluence metric of Cutibacterium acnes growth over time. Representative images of Cutibacterium acnes growth at the indicated time points are shown in Figure 1C.

[0109] result As shown in Figure 1A, automated direct microscopic imaging allows for a faster time to detection when compared with the BacT / ALERT system, an automated rapid sterility test previously validated for 7-day CAR-T final product sterility release. For media samples spiked with low levels of the designated test microorganism, the IncuCyte system's direct microscopic determination of % confluence increase was faster than the BacT / ALERT system's time to detection, which is based on indirect colorimetric detection of CO2 production in the sample media bottle. The slow-growing, aerotolerant microorganism Cutibacterium acnes was one of the challenging microorganisms that limited the BacT / ALERT system to a 7-day sterility release time. This was due to the time required to detect this microorganism in the BacT / ALERT anaerobic sample bottle after a low population spike and recovery validation study. As shown in Figures 1B and 1C, automated direct microscopic imaging enabled the detection of Cutibacterium acnes spiked into FTM at population counts of approximately 3 CFU / mL within 36 hours of incubation at 37°C. In a separate BacT / ALERT study, Cutibacterium acnes (formerly Propionibacterium acnes), spiked into anaerobic BacT / ALERT sample bottles at approximately 2 CFU / mL, demonstrated a time to detection of 94.3 hours based on indirect colorimetric detection of CO2 production in the sample media bottle, demonstrating the faster time to detection of direct microscopic imaging of this slow-growing microorganism. Based on the comparative ratio of faster times to detection, automated direct cell imaging could achieve a modest 3-day release time for sterility testing compared to the current 7-day BacT / ALERT sterility test, with the potential for release tests of less than 3 days to be demonstrated and implemented.

[0110] Additionally, presence / absence screening of non-sterile products can also be accomplished by performing a product dilution (i.e., 10x) and separating the contents into a bioburden sample and a 10 gram concentrated sample. The absence of growth in the 10x diluted bioburden sample demonstrates less than 10 CFU / g or mL in the original product sample. The IncuCyte system with multiple fluorescence channels is capable of detecting the presence of USP <62> It has the unique ability to develop applications for rapid presence / absence screening of specified organisms using fluorescently labeled primary or secondary antibodies for more rapid product release without the need for restreaking onto selective media to identify unwanted organisms as described in Microbiological Examination of Nonsterile Products: Tests For Specified Microorganisms, USP. <62> The capability of mammalian cell imaging systems for bioburden enumeration of non-sterile products according to the present invention can be realized by sample filtration onto a semi-transparent membrane, either freestanding or in a filter plate format, followed by incubation of the sample in medium until the 10-micron system size limit is reached. Applications are now available that can enable imaging of the entire semi-transparent sample membrane surface for quantification of microbial microcolonies. The fluorescent channel of the mammalian cell imaging system can also enable the development of multiplexed assays for viable cell count determination of mammalian cells and contamination screening for the presence / absence of microorganisms in the same bioreactor sample.

[0111] Example 2: IncuCyte's CAR-T Microbial Screening Materials and Methods Staphylococcus aureus (SA) Bioball™ (bioMerieux, USA) was resuspended according to the manufacturer's instructions. Two starting populations (approximately 5 and 55 CFU) were then inoculated into duplicate individual wells of a 24-well cell culture plate (plasma-treated) containing approximately 3 mL of TSB with and without approximately 10,000 healthy T-cell donor CAR-T cells. Approximately 10,000 CAR-T cells were aliquoted from 1 mL of CAR-T sample (LCAR + healthy human donor T cells, approximately 1E6 / mL). Phase-contrast imaging at 20x magnification, 36 images per well, was performed every hour. Time-resolved images captured by the IncuCyte system were then used to calculate the confluence % metric, and the time to detection was determined based on the increase in confluence %. The results are shown in Figure 2.

[0112] result Figure 2 shows IncuCyte CAR-T SA microbial screening results. The graph shows a time course showing the change in % confluence over time across different sample groups. Both 5 CFU and 55 CFU SA inocula spiked into 10,000 CAR-T cells were detected within 12 hours of incubation at 37°C based on the IncuCyte imaging software tracking the change in % confluence. As expected, sample wells containing CAR-T cells had higher starting % confluence values ​​for both SA inoculum numbers, with samples containing higher SA starting population numbers having a slightly faster time to detection based on the change in % confluence.

[0113] Example 3: CHO cell Bacillus subtilis spore spike confluence % detection Materials and Methods Bacillus subtilis spores were resuspended in Bioball™ (bioMerieux, USA) according to the manufacturer's instructions. 1 x 10 per well were plated in a 6-well cell culture plate (plasma-treated) containing approximately 8 mL of TSB in each well. 6Each well was inoculated with approximately 25 CFU of Bacillus subtilis spores, with the exception of one well, which served as a baseline control with only CHO cells. The plate was placed in an IncuCyte system in a 37°C incubator, and automated imaging was performed every 30 minutes using a 20x objective.

[0114] result Figure 3 shows the percent confluence detection of Bacillus subtilis spore spikes on CHO cells. The graph shows a time course demonstrating the detection of CHO cells contaminated with Bacillus subtilis spores. The presence of low levels of Bacillus subtilis spores (25 CFU / well) was detected within 8 hours based on changes in percent confluence. Results can be confirmed visually, and automated imaging is nondestructive, allowing for the identification of any detected contaminants. These results suggest that the IncuCyte system can be used in place of traditional plating for rapid in-process monitoring of mammalian cell bioreactors. The system can be deployed on the processing floor for at-line testing, and rapid results can be used to prevent the retention of contaminated bioreactors during the manufacturing process, resulting in significant cost savings.

[0115] Example 4: Staphylococcus epidermidis (SE) prebiotic and antibacterial screening Materials and Methods Stationary-phase Staphylococcus epidermidis (SE) cultures grown in TSB were diluted in TSB to obtain a population count of approximately 2,000 CFU per mL. A 10-microliter aliquot of this SE suspension (approximately 25 CFU) was then inoculated into each designated well of a 24-well cell culture plate (plasma-treated) containing a 1% soluble component suspension in approximately 1 mL of TSB. Each soluble component was filter-sterilized using a 0.22 μm filter membrane before creating a 1% test suspension. Designated wells containing only soluble components and only SE were included as negative and positive controls, respectively. The sample plate was then placed in an IncuCyte system contained within a 37°C incubator, and imaging was performed automatically every hour using a 20x objective.

[0116] result Figure 4 shows the Staphylococcus epidermidis (SE) prebiotic and antibacterial screening (1%). SE prebiotic activity of component A (sterile filtered) was detected based on faster confluence % growth compared to the positive control containing only SE in TSB. SE antibacterial activity of component B (sterile filtered) was detected by no change in confluence % growth over time. SE prebiotic activity of component C (sterile filtered) was detected by faster confluence % growth compared to the positive control. Of all components, only the negative control was negative for growth.

[0117] Example 5: Natural product extract Staphylococcus aureus (SA) antibacterial screening Materials and Methods A natural product extract library (Phytotitre, Caithness Biotechnologies, Leicester, United Kingdom) containing 50 μL of individual sample aliquots (10 mg / mL [starting concentration of 1% in DMSO]) in 96-well cell culture plates was screened for the presence of SA antimicrobials using the IncuCyte system. To generate SA microbial test suspensions, stationary-phase SA cultures grown in TSB were diluted in TSB to obtain a population count of approximately 3E6 CFU per mL. Ten μL of each designated natural product extract was then mixed with 90 μL of the SA TSB suspension in individual wells of a new, plasma-treated 96-well cell culture plate, resulting in a 0.1% natural product extract sample inoculated with approximately 20,000 CFU of SA per well. Three SA positive control wells containing only 90 μL of SA TSB suspension (approximately 2000 CFU / well) were included, in addition to three DMSO controls containing only 90 μL of SA TSB suspension (approximately 2000 CFU / well) and 10 μL of DMSO. The cell culture plate samples were then placed in an IncuCyte system at 37°C and automatically imaged every hour with a 20x objective.

[0118] result Figure 5 shows the natural product extract Staphylococcus aureus (SA) antibacterial screening (0.1%). 96-well natural product extract microplate % confluence results map. Natural product extract SA antibacterial activity was detected by the lack of change in % confluence growth over time for sample wells A3, E6, B9, C11, and D11. SA positive controls with and without DMSO contained representative positive growth, confirming that the presence of DMSO (10% concentration) in the sample wells did not inhibit SA microbial growth and that the antibacterial screening results were due to the natural product extract present in the test sample wells.

[0119] Embodiment The following exemplary embodiments further describe optional aspects of the technology of the present disclosure and are part of the detailed description. Although these exemplary embodiments are described in a format substantially similar to claims (each with a numerical designation followed by a capital letter), they are not technical claims of this application. The following exemplary embodiments are referenced to each other in a dependent relationship as "embodiments" instead of "claims." 1A. A method for detecting a microorganism in a sample, the method comprising detecting the confluence of the sample. 2A. The method of embodiment 1A, wherein confluence is detected using an image-based metric. 3A. The method of embodiment 2A, wherein the image-based metric comprises direct imaging. 4A. The method of embodiment 3A, wherein the direct imaging is direct cell imaging. 5A. The method of any one of embodiments 1A-4A, wherein the sample is in an incubator. 6A. The method of any one of embodiments 1A-5A, wherein detecting comprises detecting using a microscope or an image sensor. 7A. The method of any one of embodiments 1A-6A, wherein the confluence of the sample is compared to the confluence of a control sample. 8A. The method of any one of embodiments 1A-7A, wherein the confluence of the sample is monitored over a period of time. 9A. The method of any one of embodiments 1A-8A, wherein the sample is contacted with an antibody to the microorganism. 10A. The method of any one of embodiments 1A-9A, wherein the sample is contacted with a fluorescent or bioluminescent agent. 11A. The method of any one of embodiments 1A to 10A, further comprising detecting fluorescence or bioluminescence of the sample. 12A. The method of any one of embodiments 1A to 11A, wherein the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. 13A. The method of any one of embodiments 1A to 12A, wherein detecting confluence is automated. 14A. The method of any one of embodiments 1A-13A, wherein the confluence is percent confluence. 15A. A method for assaying a test agent, comprising adding the test agent to a sample containing a microorganism and detecting confluence of the sample. 16A. The method of embodiment 15A, wherein confluence is detected using an image-based metric. 17A. The method of embodiment 16A, wherein the image-based metric comprises direct imaging. 18A. The method of embodiment 17A, wherein said direct imaging is direct cell imaging. 19A. The method of any one of embodiments 15A to 18A, wherein the sample is in an incubator. 20A. The method of any one of embodiments 15A-19A, wherein detecting comprises detecting using a microscope or an image sensor. 21A. The method of any one of embodiments 15A to 20A, wherein the confluence of the sample is compared to the confluence of a control sample. 22A. The method of any one of embodiments 15A to 21A, wherein the confluence of the sample is monitored over a period of time. 23A. The method of any one of embodiments 15A-22A, wherein the test agent is a chemical compound, an adjuvant, an antibiotic, a bacteriophage, or a combination thereof. 24A. The method of any one of embodiments 15A-23A, further comprising identifying the test agent as a prebiotic if the confluence of the sample is greater than the confluence of the control sample. 25A. The method of any one of embodiments 15A-23A, further comprising identifying the test agent as a prebiotic if the confluence of the sample increases over a period of time. 26A. The method of any one of embodiments 15A-23A, further comprising identifying the test agent as an antibacterial agent if the confluence of the sample is less than the confluence of the control sample. 27A. The method of any one of embodiments 15A-24A, further comprising identifying the test agent as an antibacterial agent if the confluence of the sample decreases over a period of time. 28A. The method of any one of embodiments 15A to 27A, wherein the sample is contacted with an antibody to the microorganism. 29A. The method of any one of embodiments 15A to 28A, wherein the sample is contacted with a fluorescent or bioluminescent agent. 30A. The method of any one of embodiments 15A to 29A, further comprising detecting fluorescence or bioluminescence of the sample. 31A. The method of any one of embodiments 15A to 30A, wherein the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. 32A. The method of any one of embodiments 15A to 31A, wherein detecting confluence is automated. 33A. The method of any one of embodiments 15A to 32A, wherein the confluence is percent confluence.

[0120] References ASM Minireview, Sterility Testing for Cellular Therapies:What Is the Role of the Clinical Microbiology Laboratory.2020.Journal of Clinical Microbiology Vol.58 Issue 7 Pages1-14.

[0121] All publications and patents mentioned herein are incorporated herein by reference. Various modifications and variations of the subject matter described will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention that will be apparent to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. 1. A method for detecting a microorganism in a sample, comprising: detecting the confluence of said sample.

2. The method of claim 1 , wherein the confluence is detected using an image-based metric.

3. The method of claim 2 , wherein the image-based metric comprises direct imaging.

4. The method of claim 3 , wherein the direct imaging is direct cell imaging.

5. The method of any one of claims 1 to 4, wherein the sample is in an incubator.

6. The method of any one of claims 1 to 5, wherein said detecting comprises detecting using a microscope or an image sensor.

7. The method of any one of claims 1 to 6, wherein the confluence of the sample is compared to the confluence of a control sample.

8. The method of any one of claims 1 to 7, wherein the confluence of the sample is monitored over a period of time.

9. The method according to any one of claims 1 to 8, wherein the sample is contacted with an antibody against the microorganism.

10. The method of any one of claims 1 to 9, wherein the sample is contacted with a fluorescent or bioluminescent agent.

11. The method of any one of claims 1 to 10, further comprising detecting fluorescence or bioluminescence of the sample.

12. The method according to any one of claims 1 to 11, wherein the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

13. The method of any one of claims 1 to 12, wherein detecting confluence is automated.

14. The method of any one of claims 1 to 13, wherein the confluence is a percent confluence.

15. 1. A method for assaying a test agent, comprising: adding a test agent to a sample containing a microorganism; and detecting the confluence of the sample.

16. The method of claim 15 , wherein the confluence is detected using an image-based metric.

17. The method of claim 16 , wherein the image-based metric comprises direct imaging.

18. 18. The method of claim 17, wherein the direct imaging is direct cell imaging.

19. The method of any one of claims 15 to 18, wherein the sample is in an incubator.

20. 20. The method of any one of claims 15 to 19, wherein said detecting comprises detecting using a microscope or an image sensor.

21. The method of any one of claims 15 to 20, wherein the confluence of the sample is compared to the confluence of a control sample.

22. The method of any one of claims 15 to 21, wherein the confluence of the sample is monitored over a period of time.

23. The method of any one of claims 15 to 22, wherein the test agent is a chemical compound, an adjuvant, an antibiotic, a bacteriophage, or a combination thereof.

24. 24. The method of any one of claims 15 to 23, further comprising identifying the test agent as a prebiotic if the confluence of the sample is greater than the confluence of the control sample.

25. 24. The method of any one of claims 15 to 23, further comprising identifying the test agent as a prebiotic if the confluence of the sample increases over a period of time.

26. 24. The method of any one of claims 15 to 23, further comprising identifying the test agent as an antibacterial agent if the confluence of the sample is less than the confluence of the control sample.

27. 25. The method of any one of claims 15 to 24, further comprising identifying the test agent as an antibacterial agent if the confluence of the sample decreases over a period of time.

28. The method of any one of claims 15 to 27, wherein the sample is contacted with an antibody against the microorganism.

29. The method of any one of claims 15 to 28, wherein the sample is contacted with a fluorescent or bioluminescent agent.

30. 30. The method of any one of claims 15 to 29, further comprising detecting fluorescence or bioluminescence of the sample.

31. The method of any one of claims 15 to 30, wherein the microorganism is Cutibacterium acnes, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa.

32. The method of any one of claims 15 to 31, wherein detecting confluence is automated.

33. The method of any one of claims 15 to 32, wherein the confluence is a percent confluence.

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