# US 2020/0399675 A1 · Devices and methods for examining drug effects on microorganisms

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- Number: US 2020/0399675 A1
- Document ID: US20200399675A1
- Status: Published application
- Kind: A1
- Application number: 16/640,254
- Filed: 2018-08-01
- Published: 2020-12-24
- Earliest claimed: 2017-08-01
- Assignees: Essenlix Corporation
- Applicants: Essenlix Corporation
- Inventors: Stephen Y. CHOU; Wei DING
- Web page: https://patentgrep.com/patent/US20200399675A1

## CPC

- C12Q 1/18: Testing for antimicrobial activity of a material
- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G06T 7/0016: {involving temporal comparison}
- G16H 10/40: for data related to laboratory analysis, e.g. patient specimen analysis
- G16H 30/40: for processing medical images, e.g. editing
- G16H 40/67: for remote operation
- G16H 80/00: ICT specially adapted for facilitating communication between medical practitioners or patients, e.g. for collaborative diagnosis, therapy or health monitoring
- G06T 2207/10016: Video; Image sequence
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- Y02A 90/10: Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides devices, systems, and methods, for performing biological and chemical assays.

## Claims

### Claim 1

A method for examining an effect of a test reagent on one or more microorganism individuals in a sample, the method comprising:
(a) putting the test reagent, the sample, and a detection agent between two plates, wherein the detection agent is a fluorescence stain dye that labels a microorganism individual, and wherein one or both plates have spacers;
(b) compressing the plates to form a uniform testing layer;
(c) using an imager to acquire, at at least two discrete time points, images of signals emanating from the microorganism individual in the uniform testing layer; and
(d) analyzing the images at the at least two discrete time points to determine a change of the microorganism individual between the at least two discrete time points;
wherein the two plates are movable relative to each other into different configurations, including an open configuration and a closed configuration;
wherein in the open configuration, the two plates are partially or entirely separated apart, the spacing between the plates is not regulated by the spacers, and the sample is deposited on one or both of the plates;
wherein in the closed configuration, which is configured after deposition of the sample in the open configuration: at least part of the sample is compressed by the two plates into the uniform testing layer having a highly uniform thickness, and the uniform thickness of the layer is confined by the sample contact surfaces of the plates and is regulated by the plates and the spacers;
wherein the imaging is in the closed configuration, and wherein the thickness of the testing layer at the closed configuration is configured such that there is substantially no overlap between any two microorganism individuals; and
wherein in the closed configuration, the thickness of the uniform testing layer is regulated by the two plates and the spacers within the sample area, and the height of the spacers is 250 microns or less.

### Claim 2

A method for examining an effect of a drug on one or more microorganism individuals in a sample, the method comprising:
(a) putting a drug and a sample containing one or more microorganism individuals between two plates to form a uniform testing layer;
(b) acquiring, using an imager, at least two images of the uniform testing layer at discrete time points;
(c) analyzing the images to determine a change of the one or more microorganism individuals between at least two of the discrete time points;
wherein the thickness of the uniform testing layer is regulated by the two plates and spacers inside of the sample and between the two plates;
wherein the thickness of the testing layer is configured such that there is substantially no overlap between two microorganism individuals;
wherein the two plates are movable relative to each other into different configurations, including an open configuration and a closed configuration;
wherein in the open configuration, the two plates are partially or entirely separated apart, the spacing between the plates is not regulated by the spacers, and the sample is deposited on one or both of the plates; and
wherein in the closed configuration, which is configured after deposition of the sample in the open configuration: at least part of the sample is compressed by the two plates into the uniform testing layer having a highly uniform thickness, and the uniform thickness of the layer is confined by the sample contact surfaces of the plates and is regulated by the plates and the spacers.

### Claim 3 (depends on 2)

The method of claim 2, further comprising a step of staining a microorganism using a cell viability dye that indicates whether a stained cell is alive or dead.

### Claim 4 (depends on 2)

The method of claim 2, further comprising an additional reagent that stains the microorganism individual.

### Claim 5 (depends on 4)

The method of claim 4, wherein the detection agent or the additional reagent or the test agent are coated on one or both of the plates before depositing the sample.

### Claim 6 (depends on 2)

The method of claim 2, wherein the one or more microorganisms are selected from the group consisting of bacteria, fungi, archaea, viruses, protists, micro-animals, myxozoa, arthropods, crustaceans, and microscopic nematodes.

### Claim 7 (depends on 2)

The method of claim 2, wherein the one or more microorganisms are selected from a genus, and the genus is selected from the group consisting of Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

### Claim 8 (depends on 2)

The method of claim 2, wherein the one or more microorganisms are selected from the group consisting of Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella Quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium, etani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella, neumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus, neumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis.

### Claim 9 (depends on 2)

The method of claim 2, wherein the test reagent is selected from the group consisting of an antibiotic, and an antifungal reagent.

### Claim 10 (depends on 9)

The method of claim 9, wherein the test reagent comprises an antibiotic, and wherein the antibiotic is selected from the group consisting of Actinomycin D, Actinonin, Aculeacin A, Acycloguanosine, Adenine 9-β-D-arabinofuranoside, Alamethicin, Alamethicin, L-Alanyl-L-1-aminoethylphosphonic acid, Albendazole, 17-(Allylamino)-17-demethoxygeldanamycin, Amastatin, Amikacin, Amikacin, 7-Aminoactinomycin D, 7-Aminocephalosporanic acid, N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide, (+)-6-Aminopenicillanic acid, Amoxicillin, Amphotericin B, Ampicillin, Anhydroerythromycin A, Anisomycin, Antimycin A, Antimycin A2, Antipain, Aphidicolin, Apicidin, Apoptolidin A, Apramycin, Artesunate, Ascochlorin, Ascomycin, 5-Azacytidine, Azaserine, Azithromycin, Azlocillin, Bacitracin, Bactenecin, Bafilomycin A1, Bafilomycin B1, Bestatin, Beta D-4 human recombinant, Beta D-1 (36 aa) human recombinant, Beta D-1 (47 aa) human recombinant, Bithionol VETRANAL™, Blasticidine S, Bleomycin, BM-Cyclin, Borrelidin, Brefeldin A, Caerulomycin A, Calcium ionophore III Selectophore™ Calcium Ionophore A23187, Calcium Ionophore A23187, Calcium Ionophore A23187, (S)-(+)-Camptothecin, Capreomycin, Carbadox, Carbenicillin, Carboplatin, Cecropin A, Cecropin B, Cecropin P1 Porcine, Cefaclor, Cefalexin VETRANAL™, Cefazoli, Cefixime, Cefmetazole, Cefoperazone, Cefotaxime, Cefsulodin, Ceftazidime, Ceftriaxone, Cephalexin, Cephalomannine, Cephalothin, Cephradine, Cercosporin, Cerulenin, Cetylpyridinium, Chloramphenicol, Chlorhexidine, Chloroquine, Chlortetracycline, Chromomycin A3, Chrysomycin A, Chrysomycin B, Cinnamycin, Cinoxacin, Ciprofloxacin, Clarithromycin, Clebopride, Clindamycin, Clofazimine, Clotrimazole, Cloxacillin, Colistin, Colominic acid, Compound 48/80, Concanamycin A, Cordycepin, Coumermycin A1, Cryptotanshinone, Crystal Violet, Cycloheximide, D-Cycloserine, Cyclosporin A, Cyclosporin C, Cytochalasin D, Cytochalasin B, Dacarbazine antineoplastic purine analog, Daunorubicin, 10-Deacetylbaccatin III, Decoyinine, Defensin HNP-1, Defensin HNP-2, Demeclocycline, 1-Deoxymannojirimycin, 1-Deoxynojirimycin, cis-Diamineplatinum(II), 6,9-Diamino-2-ethoxyacridine-DL-lactate, cis-Diammineplatinum(II), 6-Diazo-5-oxo-L-norleucine, 5,7-Dichloro-8-hydroxy-2-methylquinoline, Dichlorophene PESTANAL®, Dicloxacillin, Diethylcarbamazine, Diethylcarbamazine, Difloxacin, Dihydrostreptomycin, Dihydrostreptomycin, Diloxanide furoate, Dimetridazole, Dirithromycin, Doxorubicin, Doxycycline, Duramycin, Econazole, Elafin, Embelin, Emetine, Enrofloxacin, Erythromycin, Ethambutol, Etoposide, Fengycin, Filipin, Florfenicol, Flubendazol VETRANAL™, Fluconazole, Flumequine, Flumethasone, 5-Fluorocytosine nucleoside analog, Flurbiprofen cyclooxygenase inhibitor, Formycin A, Fumagillin, Fumitremorgin C, Furazolidone, Fusaric acid, G 418, Ganciclovir, Gatifloxacin, Geldanamycin, Gentamicin, Gentamicin, Gentian Violet, Gliotoxin, Gramicidins, Griseofulvin, Herbimycin A, Hexadecylpyridinium, Honokiol, Hydrocortisone 21, 8-Hydroxyquinoline, 4-Hydroxytamoxifen, (Z)-4-Hydroxytamoxifen, Hygromycin B, Ikarugamycin, Imipenem, Indomethacin, Indomethacin, Ionomycin, Irgasan, Itraconazole, Iturin A, Ivermectin, Josamycin, K-252a, K-252b, Kanamycins, Kasugamycin, Kendomycin, Ketoconazole, Kirromycin, L-(+)-Lactic acid, Lactoferricin B, Leptomycin A, Leptomycin B, Levamisol, Levofloxacin, Lincomycin, Listeria mono Selective Supplement I, LL-37, Lomefloxacin, Lysostaphin, Magainin I, Mebendazole, Meclocycline, Menadione, 2-Mercaptopyridine N-oxide, N-Methyl-1-deoxynojirimycin, 2-Methyl-4-isothiazolin-3-one hydrochloride, Metronidazole, Mevastatin, (±)-Miconazole, Minocycline, Mithramycin A, Mitomycin C, Monensin, Morantel, Moxalactam, Mupirocin, Mycosubtilin, Myxothiazol, Prothionamide, Nafcillin, Naftifine, Nalidixic acid, Narasin, Neocarzinostatin, Neomycin, Netilmicin, Netropsin, Niclosamide, Nigericin, Nikkomycin Z, Nisin, Nitrofurantoin, Nogalamycin, Nonactin, Norfloxacin, Nourseothricin, Novobiocin, NP-1, Nystatin, Ochratoxin A, Ofloxacin, Oligomycin, Oligomycin A, Oxacillin, Oxantel, Oxolinic acid quinolone, Oxytetracycline, Oxytetra, Paclitaxel, Paromomycin, Patulin, PD 404,182, Pediocin, Pefloxacin, D-Penicillamine, Penicillin G, Penicillin V, Pentamidine, PGLa, 1,10-Phenanthroline, Phenazine, Phenoxymethylpenicillinic acid, Phleomycin, Phosphomycin, Pimaricin, Pipemidic acid, Piperacillin, Pirarubicin, Platensimycin, Polymyxin B, Poly(vinylpyrrolidone)-Iodine complex, Potassium clavulanate: cellulose (1:1), Potassium Sorbate, Praziquantel, Puromycin, Pyrantel, Pyrazinecarboxamide, Pyronaridine, Pyrrolnitrin, Quinine, 8-Quinolinol, Radicicol, Ramoplanin, Rapamycin, Rebeccamycin, Reveromycin A, Ribavirin, Ribostamycin, Ricobendazole, Rifabutin, Rifampicin, Rifamycin SV, Rifapentine, Rifaximin, Ristomycin, Rolitetracycline, Roxithromycin, Salinomycin, Sangivamycin, Sinefungin, Sisomicin, Sorbic acid, Sordarin, Sparfloxacin, Spectinomycin, Spergualin, Spiramycin, Staurosporine, Streptolysin O, Streptomycin, Streptonigrin, Streptozocin, Succinylsulfathiazole, Sulconazole, Sulfabenzamide, Sulfachloropyridazine, Sulfadiazine, Sulfadimethoxine, Sulfadimidine, Sulfadoxin, Sulfaguanidine, Sulfameter, Sulfamethazine, Sulfamonomethoxine, Sulfanilamide, Sulfanitran, Sulfaquinoxaline, Sulfasalazine, Sulfathiazole, Sulochrin, Surfactin, Swainsonine, Syringomycin E, Tamoxifen, Tazobactam, Teicoplanin, Terbinafine, Terconazole, Tetracycline, Tetramisole, Thiabendazole, Thiamphenicol, Thimerosal, Thiolutin, Thiostrepton, Thio-TEPA, Thymol, Tiamulin, Ticarcillin, Tioconazole, Tobramycin, Aminoglycoside antibiotic, Tobramycin, Tolnaftate, Toyocamycin, Triacsin C, Trichlorfon, Trimethoprim, Tubercidin, Tunicamycin, Tunicamycin C2 homolog, Tylosin, Valacyclovir, Valinomycin, Vinblastine, Virginiamycin S1, Virginiamycin M1, an analog thereof, a salt thereof, and a derivative thereof.

### Claim 11 (depends on 9)

The method of claim 9, wherein the test reagent comprises an antifungal reagent, and wherein the antifungal regaent is selected from the group consisting of Polyene anti fungals (Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin), Imidazoles (e.g. Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole), Triazoles (Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propiconazole, Ravuconazole, Terconazole, Voriconazole), Thiazoles (e.g. Abafungin), Allylamines (e.g. Amorolfin, Butenafine, Naftifine, and Terbinafine), Echinocandins (e.g. Anidulafungin, Caspofungin, Micafungin), Aurones, Benzoic acid, Ciclopirox, Flucytosine and 5-fluorocytosine, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid, Crystal violet, Balsam of Peru, an analog thereof, a salt thereof, and a derivative thereof.

### Claim 12 (depends on 2)

The method of claim 2, wherein the imager comprises a camera.

### Claim 13 (depends on 2)

The method of claim 2, wherein the imager comprises a video camera.

### Claim 14 (depends on 2)

The device, system, or method of claim 2, wherein the uniform height of the spacers is equal to or less than an average dimension of the one or more microorganisms.

### Claim 15 (depends on 2)

The device, system, or method of claim 2, wherein the uniform height of the spacers is in the range of 75% to 125% of an average dimension of the one or more microorganisms.

### Claim 16 (depends on 2)

The method of claim 2, wherein at least one of the plates is transparent.

### Claim 17 (depends on 2)

The method of claim 2, wherein one or both of the plates comprise, on the respective sample contact area, a dye that, upon contacting the sample, is dissolved in the sample and stains the one or more microorganisms.

### Claim 18 (depends on 2)

The method of claim 2, wherein the sample comprises reagents that facilitate the survival and/or division of the one or more microorganisms.

### Claim 19 (depends on 2)

The method of claim 2, wherein one or both of the plates comprise, on the respective sample contact area, reagents that facilitate the survival and/or division of the microorganism.

### Claim 20 (depends on 2)

The method of claim 2, further comprising:
(a) a housing configured to hold the sample and to be mounted to the mobile communication device.

### Claim 21 (depends on 2)

The method of claim 2, wherein the imager is configured to acquire a series of images of the microorganism at discrete time points.

### Claim 22 (depends on 2)

The method of claim 2, wherein the further comprising a processor configured to receive and analyze the series of images, identify and count the microorganism in a first area of each of the images.

### Claim 23 (depends on 2)

The method of claim 2, wherein the imager is configured to acquire a video that comprises a series of images of the microorganisms over a first time period.

### Claim 24 (depends on 20)

The method of claim 20, wherein the housing comprises optics for facilitating the imaging and/or signal processing of the sample by a mobile communication device, and a mount configured to hold the optics on the mobile communication device.

### Claim 25 (depends on 24)

The method of claim 24, wherein the mobile communication device is configured to communicate test results to a medical professional, a medical facility or an insurance company.

### Claim 26 (depends on 25)

The method of claim 25, wherein the mobile communication device is further configured to communicate information on the subject with the medical professional, medical facility or insurance company.

### Claim 27 (depends on 26)

The method of claim 26, wherein the mobile communication device is configured to receive a prescription, diagnosis or a recommendation from a medical professional.

### Claim 28 (depends on 26)

The method of claim 26, wherein the mobile communication device communicates with the remote location via a wifi or cellular network.

### Claim 29 (depends on 24)

The method of claim 24, wherein the mobile communication device is a mobile phone.

### Claim 30 (depends on 2)

The method of claim 2, wherein the one or more microorganisms have an average dimension of less than 1 mm.

### Claim 31 (depends on 2)

The method of claim 2, wherein one or both of the plates comprise, on the respective sample contact area, a control zone and an experimental zone, wherein the experimental zone comprises one or more test reagents that, upon contacting the sample, are dissolved and diffuses in the sample, and wherein the control zone comprises no such test reagents.

### Claim 32 (depends on 2)

The method of claim 2, further comprising contacting the sample with an additional reagent, wherein the additional reagent is selected from the group consisting of staining dyes to label the microorganisms (e.g., cell viability dyes), life-supportive reagents to facilitate the survival of the microorganisms (e.g. nutrients, oxygen source, pH buffer), proliferative reagents to maintain or facilitate the proliferation of the microorganisms (e.g. hormones, siderophore).

### Claim 33 (depends on 32)

The method of claim 32, wherein the additional reagent comprising a staining dye, and wherein the staining dye comprises a fluorescent molecule selected from the group consisting of IRDye800CW, Alexa 790, Dylight 800, fluorescein, fluorescein isothiocyanate, succinimidyl esters of carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer of fluorescein dichlorotriazine, caged carboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine, Texas Red, propidium iodide, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethyl rhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester), tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, cyan-shifted green fluorescent protein, redshifted green fluorescent protein, yellow-shifted green fluorescent protein, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives, such as acridine, acridine isothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-c acid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate, erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF), 2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; ophthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of 5 sulforhodamine (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl hodamine isothiocyanate (TRITC); riboflavin; 5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CAL Fluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine, coumarins and related dyes, xanthene dyes such as rhodols, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol, and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, fluorescent europium and terbium complexes; combinations thereof, and the like. Suitable fluorescent proteins and chromogenic proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a GFP derived from Aequoria victoria or a derivative thereof, e.g., a “humanized” derivative such as Enhanced GFP; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi; “humanized” recombinant GFP (hrGFP); any of a variety of fluorescent and colored proteins from Anthozoan species; and any combination thereof.

### Claim 34 (depends on 2)

The method of claim 2, wherein it further comprising a life-supportive reagent that facilitates the survival of the microorganisms.