Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

Summary

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Abstract

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000's clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Introduction

Cholera is a disease of the distal small intestine caused by the consumption of contaminated food or water containing the aquatic Gram-negative bacterium Vibrio cholerae. Symptoms of cholera include vomiting and uncontrollable watery diarrhea, leading to severe dehydration, which if not treated properly, will result in death. V. cholerae can be divided into over 200 serogroups based on the structure of the cell-surface lipopolysaccharide O-antigen. However, only 2 serogroups, O1 and O139, have shown epidemic or pandemic potential^1,2. Moreover, serogroup O139 has been primarily isolated to Southeastern Asia^3,4, while serogroup O1 is distributed worldwide. Furthermore, the O1 serogroup can be divided into 2 main biotypes: classical and El Tor. The classical biotype was responsible for the first 6 cholera pandemics between 1817 and 1923. The ongoing seventh pandemic is a result of the El Tor biotype, which has globally displaced the classical biotype in the environment^5,6,7. Recently, strains have arisen which contain distinguishing characteristics of both classical and El Tor biotypes^{8,9,10,11,12,13,14,15,16,17}and have since been termed El Tor variants^13,17. Some El Tor variants have demonstrated elevated virulence capabilities with more rapid and severe disease progression than previously observed, emphasizing the need for a more comprehensive approach to agent identification and disease prevention and treatment^8,9,18. While biotype identification does not immediately dictate treatment, further advancements in vaccine development and future therapeutic agents may benefit from biotype distinction.

The first series of protocols listed here will enable investigators to properly maintain V. cholerae strains in a laboratory setting. Consistency and subsequent analysis requires stock preparation and growth of isolates, which is not biotype-dependent. However, to optimally induce virulence gene expression, independent biotype specific culturing techniques are required¹⁹. Additionally, preparation for various genetic and biochemical assays are outlined in this manuscript.

Cholera toxin (CT) and the toxin co-regulated pilus (TCP) are two main virulence factors controlled by the master regulator ToxT in both biotypes of the V. cholerae O1 serogroup²⁰. CT is a bipartite toxin composed of five CtxB subunits surrounding a single CtxA subunit, and is responsible for the rapid electrolyte loss associated with cholera. TCP is a type IV pilus encoded by the tcp operon (tcpABQCRDSTEF), and is involved in attachment and colonization of the distal small intestine. tcpA is the first gene of the tcp operon which encodes the individual pilin subunits essential for construction of the pilus⁸. The gene sequence for ctxA is completely conserved between classical and El Tor biotypes, while ctxB and tcpA differ across the two biotypes but are conserved within each biotype⁸. ctxB is completely conserved between biotypes except at two base positions (115 and 203). In the El Tor biotype, thymine resides at base positions 115 and 203, while the classical biotype contains cytosine at these bases. tcpA is completely conserved within each biotype, yet differ at multiple bases between biotypes. These genetic distinctions serve as primary biotype identification markers, and after sequencing the polymerase chain reaction (PCR) amplification product including these sites, isolate sequences can be compared to wild-type (WT) classical O395 or WT El Tor N16961 to determine the biotype background of CT and TCP, respectively, in a given V. cholerae isolate.

Numerous protocols have been developed to characterize the phenotypic distinctions between the classical and El Tor biotypes^21,22,23. Polymyxin B is a peptide antibiotic that compromises the integrity of the outer cell membrane in Gram-negative bacteria, and polymyxin B resistance can be visualized through the polymyxin B resistance assay²¹. Citrate is a primary substrate of the Kreb's cycle, and the ability to metabolize citrate as a sole carbon source can be determined using the citrate metabolism assay²². hapR encodes a global regulator and the master quorum-sensing regulator in V. cholerae, HapR, which binds to various promoter regions and regulates gene and operon expression²⁴. Some pathogenic strains of V. cholerae have a naturally occurring frame-shift mutation in the hapR gene that has caused this density dependent regulation of virulence gene expression to be lost^24,25. Measuring HapR-regulated protease activity using milk agar media allows the researcher to identify whether a particular isolate contains a functional HapR²³. The hemolysis assay tests for a strain's ability to secrete hemolytic enzymes that lyse red blood cells; the degree of hemolysis can be visualized on blood agar plates²³. Motility is often associated with virulence in V. cholerae and can be analyzed using motility agar plates²³. The Voges-Proskauer assay tests for a strain's ability to ferment glucose as a sole carbon source and produce the byproduct acetoin²¹. With the emergence of El Tor variants, it is difficult to predict the results of any given phenotypic assay without extensive genotypic screening, and before deducing the biotype background of V. cholerae isolates, it is recommended to perform this assembly of assays²³ and compare the results to reference strains as in Table 2.

Herein, we have advanced a series of protocols, collectively utilizing the aforementioned genotypic and phenotypic assays for a more comprehensive approach to characterizing V. cholerae biotypes. Furthermore, we have described the genotypic and phenotypic distinctions of known V. cholerae El Tor variants (MQ1795 and BAA-2163), in comparison to commonly used biotype reference strains (WT classical O395, WT El Tor C6706, and WT El Tor N16961; Table 1). The emergence of El Tor variants has presented challenges to the reliability of previously employed single assay biotype characterization protocols; however, this multiple assay identification system will allow for more reliable characterization of clinical and environmental V. cholerae isolates.

Protocol

Note: Time considerations for each assay must be made as individual media preparations require different times. For example, solid agar plate media should be allowed to sufficiently cool and dry (1-2 days). Additional time considerations (i.e. single colony and overnight culture growth) are specified under each protocol and are found in Table 2.

1. Preparation of Media

1. 1x Phosphate Buffered Saline (PBS)
   1. Weigh 4.0 g NaCl, 0.1 g KCl, 0.72 g Na₂HPO₄, and 0.12 g KH₂PO₄. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 500 mL with deionized water, adjust pH to 7.2 using a pH meter and adding HCl dropwise to the solution, and autoclave or filter sterilize using a 0.22 µm filter. 1x PBS can be stored at room temperature indefinitely.
      CAUTION: HCl is a concentrated acid and should be handled according to institutional, local, state, and federal regulations. For appropriate handling guidelines refer to the safety data sheet provided by the manufacturer.
2. Luria-Bertani (LB) Broth
   1. Weigh 5.0 g tryptone, 2.5 g yeast extract, and 2.5 g NaCl. Combine the constituents in a 1 L bottle, adjust volume to 500 mL with deionized water, autoclave, and store at room temperature for up to 6 months. For classical biotype virulence inducing conditions, adjust pH to 6.5 using HCl prior to autoclaving.
3. AKI Medium Containing 0.03% (w/v) NaHCO₃
   1. For Component 1 (AKI medium): weigh 7.5 g peptone, 2.0 g yeast extract, and 2.5 g NaCl. Combine the constituents in a 1 L bottle, adjust volume to 450 mL with deionized water and autoclave. AKI medium can be stored at room temperature for up to 6 months.
   2. For Component 2 (NaHCO₃): weigh 1.5 g NaHCO₃, and adjust volume to 50 mL with deionized water in a sterile vesicle. Filter sterilize NaHCO₃ using a 0.22 µm filter. NaHCO₃ must be prepared immediately before use.
   3. Aseptically combine the two components creating a 1:10 ratio by filtering Component 2 into Component 1 prior to use.
4. Luria-Bertani (LB) Agar Plates
   1. Weigh 5.0 g tryptone, 2.5 g yeast extract, 2.5 g NaCl, and 7.5 g agar. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 500 mL with deionized water, autoclave, and prepare in standard sized 100 mm x 15 mm sterile Petri dishes. Plated media can be stored wrapped in plastic for up to 6 months, lid-side up at 4 °C.
5. LB Agar Plates Supplemented with Polymyxin B
   1. For Component 1 (LB agar): weigh 5.0 g tryptone, 2.5 g yeast extract, 2.5 g NaCl, and 7.5 g agar. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 500 mL with deionized water and autoclave.
   2. For Component 2 (polymyxin B): dissolve polymyxin B in deionized water in a sterile 2 mL microcentrifuge tube to a concentration of 50,000 IU/µL and filter sterilize using a 0.22 µm filter.
   3. Once Component 1 is cool to the touch, but not yet solidified, aseptically add 500 µL of Component 2 to Component 1 creating a final concentration of 50 IU/µL and mix thoroughly. Prepare in standard sized 100 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes. Plated media can be stored wrapped in plastic for up to 3 months, lid-side up at 4 °C.
6. Minimal Citrate Medium Agar Plates
   1. For Component 1 (agar with bromothymol blue): weigh 7.5 g agar and 0.02 g bromothymol blue. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 450 mL with deionized water and autoclave the mixture.
   2. For Component 2 (10x VBMM): weigh 1.0 g MgSO₄7H₂O, 10.0 g citric acidH₂O, 50.0 g anhydrous K₂HPO₄, and 17.5 g NaNH₄HPO₄4H₂O. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 500 mL with deionized water and autoclave or filter sterilize using a 0.22 µm filter. 10x VBMM can be stored at room temperature up to 6 months.
   3. Add 50 mL of Component 2 to Component 1 and prepare in standard sized 100 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes. Plated media can be stored wrapped in plastic for up to 6 months, lid-side up at 4 °C.
7. Milk Agar Plates
   1. For Component 1 (milk): weigh 8.0 g instant nonfat dry milk in a 1 L Erlenmeyer flask, adjust volume to 200 mL with deionized water and autoclave.
   2. For Component 2 (agar with brain-heart infusion): weigh 3.68 g brain-heart infusion and 6.0 g agar. Combine the constituents in a 500 mL Erlenmeyer flask, adjust volume to 200 mL with deionized water and autoclave.
   3. Combine the two components by pouring Component 2 into Component 1, after autoclaving and mix thoroughly. Prepare in 150 mm x 15 mm sterile Petri dishes. Milk plates can be stored for up to one week wrapped in plastic, lid-side down at 4 °C. Prepare in 150 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes.
8. Motility Agar Plates
   1. For Component 1 (LB agar): weigh 5.0 g tryptone, 2.5 g yeast extract, 2.5 g NaCl, and 2.0 g agar. Combine the constituents in a 1 L Erlenmeyer flask, adjust volume to 500 mL with deionized water and autoclave.
   2. For Component 2 (1% (w/v) triphenyltetrazolium chloride; TTC): weigh 0.25 g TTC. Adjust volume to 25 mL with deionized water in a sterile vesicle and filter sterilize using a 0.22 µm filter. Store for up to 6 months at room temperature wrapped in foil to minimize light exposure.
   3. Add 2.5 mL of 1% (w/v) sterile TTC to autoclaved media.
   4. Pour media as thick as possible (≥50 mL/plate) into large 150 mm x 15 mm sterile Petri dishes. Plated media can be stored wrapped in plastic for up to one week, lid-side up at 4 °C. Prepare in 150 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes.
9. Voges-Proskauer (VP)
   1. For Component 1 (Methyl Red–Voges-Proskauer broth; MR–VP): weigh 1.7 g MR-VP medium in a 500 mL Erlenmeyer flask, adjust volume to 100 mL with deionized water and autoclave. Sterile MR-VP broth can be stored at room temperature for up to 6 months.
   2. For Component 2 (5% (w/v) alpha (α) naphthol): weigh 1.0 g α-naphthol in a sterile vesicle and adjust volume to 20 mL with 95% ethanol. α-naphthol can be stored at room temperature for up to 1 week wrapped in foil to minimize light exposure.
   3. For Component 3 (40% (w/v) potassium hydroxide; KOH): weigh 20.0 g KOH in a sterile vesicle, adjust volume to 50 mL with sterile deionized water. KOH can be stored for up to 6 months at room temperature in a plastic vessel.

2. Maintenance and Growth of V. cholerae Strains

1. Preparation and Use of Frozen Stock Cultures
   1. Pellet 1.8 mL of liquid overnight culture for 2 min by centrifugation (≥8,600 x g) in a sterile 2 mL microcentrifuge tube, remove supernatant, and re-suspend the cell pellet in 900 µL of fresh LB broth by pipetting.
   2. Add 900 µL of sterile 60% (v/v) glycerol to the culture and mix by vortexing.
   3. Transfer the mixture to a sterile 2 mL cryogenic tube and store indefinitely at -80 °C.
   4. To use, remove a small amount of frozen stock using a sterile inoculating loop and streak for single colonies on LB agar plates. Immediately return frozen stock to -80 °C after use to prevent cultures from completely thawing. Incubate plates lid-side down for 12 to 16 h at 37 °C.
2. Growth of Overnight Cultures in Liquid Medium
   1. Streak for single colonies (as per section 2.1.4) from frozen stock onto LB agar plates. Incubate plates lid-side down for 12 to 16 h at 37 °C.
   2. Inoculate 4 mL of liquid LB broth in a sterile 10 mL culture tube with a single colony by touching the surface of the single colony with a sterile applicator stick and transferring the colony into the liquid broth.
   3. Incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 37 °C.
3. Growth Curve
   1. Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.
   2. Make a 1:100 dilution of overnight culture by transferring 250 µL of overnight culture to 25 mL of fresh LB broth in a sterile 250 mL Erlenmeyer flask.
   3. Measure the optical density of the liquid cultures at 600 nm (OD₆₀₀) every hour beginning at the time of inoculation (T₀).
   4. Incubate a 1:100 dilution of overnight culture with aeration in a shaker incubator at 225 rpm for up to 30 h at 37 °C, or until culture reaches maximal density.
   5. Graph the OD₆₀₀ vs. Time and use linear regression to determine the approximate doubling time for each strain.
4. El Tor Biotype Virulence Inducing Conditions
   1. Streak for single colonies from frozen stock onto LB agar plates. Incubate plates lid-side down, for 12 to 16 h at 37 °C.
   2. Inoculate 10 mL of AKI medium containing 0.03% (w/v) NaHCO₃ with a single colony.
   3. Incubate culture without aeration at 37 °C for 3.5 h.
   4. Remove 7 mL of culture and incubate the remaining 3 mL of culture with aeration in a shaker incubator at 225 rpm for an additional 4 h.
5. Classical Biotype Virulence Inducing Conditions
   1. Streak for single colonies from frozen stock onto LB agar plates. Incubate plates lid-side down for 12 to 16 h at 37 °C.
   2. Inoculate 4 mL liquid LB broth (pH 6.5) with a single colony.
   3. Incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 30 °C.
6. Washing Cell Pellet in 1x PBS
   1. Pellet 1.8 mL of overnight culture for 2 min by centrifugation (≥8,600 x g) in a sterile 2 mL microcentrifuge tube and remove the supernatant using a pipette. Re-suspend cell pellet in 1.8 mL 1x PBS by pipetting.
   2. Repeat washing procedure three times followed by a final resuspension in 1.8 mL 1x PBS.

3. Characterizing V. cholerae Biotypes

1. PCR-based Genetic Screens using ctxB and tcpA
   1. Design a set of primers, which anneal approximately 50-70 bp upstream and downstream of the translational start and stop sites of ctxB and tcpA, respectively.
   2. Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.
   3. Isolate chromosomal DNA using a commercially available kit designed for Gram-negative bacteria. Purified chromosomal DNA can be stored indefinitely at -20 °C. Chromosomal DNA isolation using commercially available kits generally take about 4 h.
   4. Use a micro-volume spectrophotometer to ensure the chromosomal DNA sample is of high quality (A_260/280 >1.8).
   5. For each chromosomal DNA isolate, prepare polymerase chain reaction(s) (PCR) on ice in sterile 200 μL PCR tube(s) and amplify regions of ctxB and tcpA using standard PCR components: Taq polymerase and buffer (or equivalents), dNTP solution, and forward/reverse primers. Use standard PCR parameters (~3 h). For example, use the following protocol:
      1. Initial denature at 95 °C for 120 s.
      2. Denature at 95 °C for 60 s.
      3. Anneal primers at 60 °C for 45 s.
      4. Extend at 72 °C for 90 s.
      5. Repeat steps 3.1.5.2 through 3.1.5.4 for 34 cycles.
      6. Final extension 72 °C for 600 s.
      7. Infinite hold at 4 °C.
   6. Dye 5 μL of respective PCR product(s) using a standard 6x DNA gel loading dye, and load approximately 10 μL of 1 kb ladder and 10 μL of PCR product onto a 1% agarose gel in 1x Tris-Borate-EDTA (TBE) buffer.
   7. Run gel electrophoresis at 130 V until dye front reaches the end, but not off, of the gel (approximately 90 min). Constant monitoring is recommended as voltage and running times can vary depending on the equipment.
   8. Upon verification of successful PCR amplification at the expected size, purify the remaining 45 μL PCR product(s) using a commercially available DNA clean & concentrator kit.
   9. Sequence cleaned PCR product(s) using the same primers used for PCR amplification, and prepare per local sequencing facility guidelines.
   10. Compare the FASTA format of gene sequences from each isolate to the published sequence available on the NCBI website (http://www.ncbi.nlm.nih.gov/gene/), by searching the following accession numbers in the search query box.
   11. ctxB: (classical) region between VC0395_A1059 to VC0395_A1060 (El Tor) VC_1456
   12. tcpA: (classical) VC0395_A0353 (El Tor) VC_0828
2. Phenotypic Assays for Biotype Classification via Spotting
   1. Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.
   2. Wash the cell pellet(s) as specified in protocol 2.6.
   3. Use a pipette to spot 1 μL of washed culture on respective medium. For medium selection and incubation specifications, refer to Table 2.
3. Motility Assay
   1. Prepare overnight culture(s) in liquid LB broth as previously stated in protocol 2.2.
   2. Wash the cell pellet(s) as specified in protocol 2.6.
   3. Inoculate motility agar plates by inserting inoculating stab into the washed liquid culture and "stab" vertically into the media. In between each inoculation, sterilize the wire stab using a Bunsen burner, and when stabbing agar, ensure the inoculating stab does not bend, as this can alter results.
   4. Incubate plates lid-side up for 14 to 24 h at 37 °C. After 14 h, monitor motility plates closely to prevent overgrowth of cultures.
4. Voges-Proskauer (VP) Assay
   1. Prepare overnight culture(s) in liquid LB broth as previously stated in protocol 2.2.
   2. Inoculate 4 mL of methyl red Voges-Proskauer, or MR-VP broth, by pipetting 10 μL of previously prepared overnight culture into 4 mL MR-VP broth in a sterile culture tube and incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 37 °C.
   3. Add 150 μL of 5% (w/v) α-naphthol and 50 μL 40% (w/v) KOH to 1 mL aliquots of MR-VP overnight culture in sterile culture tubes, respectively.
   4. Briefly vortex and let stand at room temperature for up to 4 h until color change develops.

Results

For proper maintenance and use of any bacterial strain, it is recommended to know the doubling time of the strain(s) of interest. Herein, the varying growth rates of commonly used V. cholerae strains were demonstrated through a growth curve, and approximate doubling times were calculated using linear regression. WT El Tor N16961 and El Tor variant MQ1795 demonstrated shorter doubling times (~1 h and ~1 h, respectively) than WT classical O395 (~2 h) (...

Discussion

Of the over 200 identified V. cholerae serogroups, only O1 and O139 have epidemic potential. The O1 serogroup can be divided into two biotypes: classical and El Tor. However, hybrid strains, termed El Tor variants^13,17, have emerged that possess the El Tor biotype background, and harbor classical characteristics^8,9,10,11

Materials

Name	Company	Catalog Number	Comments
1 kb DNA Ladder	New England Biolabs	N3232S	https://www.neb.com/products/n3232-1-kb-dna-ladder
60% Glycerol	Calbiochem	356352	http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352
Agar	Becto, Dickinson and Co.	214030	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch
Agar with brain-heart infusion	Becto, Dickinson and Co.	237500	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch
Agarose	Peqlab	732-2789	https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&_dynSessConf=1766917479792147141&targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&lastLanguage=en&/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&_D%3AcurrentLanguage=+&currentLanguage=en&_D%3AlastLanguage=+&_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+
Anhydrous K2HPO4	Fisher Scientific	P288-500	https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&searchTerm=P288500&searchType=RAPID
Blood Agar Plates	Remel	R01200	Store at 4 °C;  http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar
Boric Acid	Fisher Scientific	A73-500	https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&searchTerm=A73500&searchType=RAPID
Bromothymol Blue	Fisher Scientific	B388-10	https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&searchTerm=B38810&searchType=RAPID
Cirtric acid ·H2O	Fisher Scientific	S72836-3	https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363
Deoxynucleotide (dNTP) Solution Kit	New England Biolabs	N0446S	Store at -20 °C;  https://www.neb.com/products/n0446-deoxynucleotide-solutionset
Disodium EDTA	Fisher Scientific	S311-500	https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&searchTerm=S311500&searchType=RAPID
DNA Clean & Concentrator™ -25 Kit	Zymo Research	D4007	http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit
GelGreen Nucleic Acid Stain	Biotium	41005	https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10,000x-in-water/
Genesys 10SUV-VIS Spectrophotometer	Thermo Scientific	840-208100	https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100
Gentra Puregene Yeast/Bact. Kit	Qiagen	158567	https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation
HCl	Fisher Scientific	A144-212	Corrosive;  https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&searchTerm=A144212&searchType=RAPID
KCl	Fisher Scientific	P217-500	https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&searchTerm=P217500&searchType=RAPID
KH2PO4	Fisher Scientific	P285-500	https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&searchTerm=P285500&searchType=RAPID
KOH	Fisher Scientific	P250-500	https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&searchTerm=P250500&searchType=RAPID
Le Loop	Decon Labs Inc.	MP190-25	http://deconlabs.com/products/leloop/
Le Stab	Decon Labs Inc.	MP186-5	http://deconlabs.com/products/lestab/
MgSO4·7H2O	Fisher Scientific	M63-500	https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&searchTerm=M63500&searchType=RAPID
Mini-Sub Cell GT Horizontal Electrophoresis System	Bio Rad Labs	1704406	http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373
MR-VP Broth	Difco	216300	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch
Na2HPO4	Fisher Scientific	S374-500	https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&searchTerm=S374500&searchType=RAPID
NaCl	Fisher Scientific	S271-10	https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&searchTerm=S27110&searchType=RAPID
NaHCO3	Fisher Scientific	S233-500	https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&searchTerm=S233500&searchType=RAPID
NaNH4HPO4·4H2O	Fisher Scientific	S218-500	https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&searchTerm=S218500&searchType=RAPID
NanoDrop Lite Spectrophtometer	Thermo Scientific	ND-LITE-PR	https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR
Nonfat dry milk	Nestle Carnation	N/A	N/A
Peptone	Becto, Dickinson and Co.	211677	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch
Petri Dishes (100 mm x 15 mm)	Fisher Scientific	FB0875712	https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712
Petri Dishes (150 mm x 15 mm)	Fisher Scientific	FB0875714	https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&searchTerm=FB0875714&searchType=RAPID
Polymyxin B sulfate salt	Sigma-Aldrich	P1004-10MU	Store at 2-4 °C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&region=US
Taq DNA Polymerase	New England Biolabs	M0273S	Store at -20 °C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer
Taq Reaction Buffer	New England Biolabs	M0273S	Store at -20 °C;  https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer
Thermal Cycler Bio-Rad C1000 Touch™	Bio Rad Labs	1840148	http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&_pageLabel=search_page&sfMode=search&sfStartNumber=1&clearQR=true&js=1&searchString=1840148&database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&tabName=DIVISIONNAME
Triphenyltetrazolium chloride	Alfa Aesar	A10870	https://www.alfa.com/en/catalog/A10870/
Tris Base	Fisher Scientific	BP152-1	https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&searchTerm=BP1521&searchType=RAPID
Tryptone	Becto, Dickinson and Co.	211705	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch
Yeast Extract	Becto, Dickinson and Co.	212750	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch
α-napthol	MP Biomedicals	204189	http://www.mpbio.com/product.php?pid=05204189