Research supported by New Hampshire-INBRE through an Institutional Development Award (IDeA), P20GM103506, from the National Institute of General Medical Sciences of the NIH.

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
<ol>
	<li>1x Phosphate Buffered Saline (PBS)
	<ol>
		<li>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 &#181;m 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.</li>
	</ol>
	</li>
	<li>Luria-Bertani (LB) Broth
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>AKI Medium Containing 0.03% (w/v) NaHCO3
	<ol>
		<li>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.</li>
		<li>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 &#181;m filter. NaHCO3 must be prepared immediately before use.</li>
		<li>Aseptically combine the two components creating a 1:10 ratio by filtering Component 2 into Component 1 prior to use.</li>
	</ol>
	</li>
	<li>Luria-Bertani (LB) Agar Plates
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>LB Agar Plates Supplemented with Polymyxin B
	<ol>
		<li>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.</li>
		<li>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/&#181;L and filter sterilize using a 0.22 &#181;m filter.</li>
		<li>Once Component 1 is cool to the touch, but not yet solidified, aseptically add 500 &#181;L of Component 2 to Component 1 creating a final concentration of 50 IU/&#181;L 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 &#176;C.</li>
	</ol>
	</li>
	<li>Minimal Citrate Medium Agar Plates
	<ol>
		<li>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.</li>
		<li>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 &#181;m filter. 10x VBMM can be stored at room temperature up to 6 months.</li>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>Milk Agar Plates
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#176;C. Prepare in 150 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes.</li>
	</ol>
	</li>
	<li>Motility Agar Plates
	<ol>
		<li>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.</li>
		<li>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 &#181;m filter. Store for up to 6 months at room temperature wrapped in foil to minimize light exposure.</li>
		<li>Add 2.5 mL of 1% (w/v) sterile TTC to autoclaved media.</li>
		<li>Pour media as thick as possible (&#8805;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 &#176;C. Prepare in 150 mm x 15 mm sterile Petri dishes by pouring mixed agar media into the Petri dishes.</li>
	</ol>
	</li>
	<li>Voges-Proskauer (VP)
	<ol>
		<li>For Component 1 (Methyl Red&#8211;Voges-Proskauer broth; MR&#8211;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.</li>
		<li>For Component 2 (5% (w/v) alpha (&#945;) naphthol): weigh 1.0 g &#945;-naphthol in a sterile vesicle and adjust volume to 20 mL with 95% ethanol. &#945;-naphthol can be stored at room temperature for up to 1 week wrapped in foil to minimize light exposure.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Maintenance and Growth of V. cholerae Strains
<ol>
	<li>Preparation and Use of Frozen Stock Cultures
	<ol>
		<li>Pellet 1.8 mL of liquid overnight culture for 2 min by centrifugation (&#8805;8,600 x g) in a sterile 2 mL microcentrifuge tube, remove supernatant, and re-suspend the cell pellet in 900 &#181;L of fresh LB broth by pipetting.</li>
		<li>Add 900 &#181;L of sterile 60% (v/v) glycerol to the culture and mix by vortexing.</li>
		<li>Transfer the mixture to a sterile 2 mL cryogenic tube and store indefinitely at -80 &#176;C.</li>
		<li>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 &#176;C after use to prevent cultures from completely thawing. Incubate plates lid-side down for 12 to 16 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Growth of Overnight Cultures in Liquid Medium
	<ol>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>Incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Growth Curve
	<ol>
		<li>Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.</li>
		<li>Make a 1:100 dilution of overnight culture by transferring 250 &#181;L of overnight culture to 25 mL of fresh LB broth in a sterile 250 mL Erlenmeyer flask.</li>
		<li>Measure the optical density of the liquid cultures at 600 nm (OD600) every hour beginning at the time of inoculation (T0).</li>
		<li>Incubate a 1:100 dilution of overnight culture with aeration in a shaker incubator at 225 rpm for up to 30 h at 37 &#176;C, or until culture reaches maximal density.</li>
		<li>Graph the OD600 vs. Time and use linear regression to determine the approximate doubling time for each strain.</li>
	</ol>
	</li>
	<li>El Tor Biotype Virulence Inducing Conditions
	<ol>
		<li>Streak for single colonies from frozen stock onto LB agar plates. Incubate plates lid-side down, for 12 to 16 h at 37 &#176;C.</li>
		<li>Inoculate 10 mL of AKI medium containing 0.03% (w/v) NaHCO3 with a single colony.</li>
		<li>Incubate culture without aeration at 37 &#176;C for 3.5 h.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Classical Biotype Virulence Inducing Conditions
	<ol>
		<li>Streak for single colonies from frozen stock onto LB agar plates. Incubate plates lid-side down for 12 to 16 h at 37 &#176;C.</li>
		<li>Inoculate 4 mL liquid LB broth (pH 6.5) with a single colony.</li>
		<li>Incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 30 &#176;C.</li>
	</ol>
	</li>
	<li>Washing Cell Pellet in 1x PBS
	<ol>
		<li>Pellet 1.8 mL of overnight culture for 2 min by centrifugation (&#8805;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.</li>
		<li>Repeat washing procedure three times followed by a final resuspension in 1.8 mL 1x PBS.</li>
	</ol>
	</li>
</ol>
3. Characterizing V. cholerae Biotypes
<ol>
	<li>PCR-based Genetic Screens using ctxB and tcpA 
	<ol>
		<li>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.</li>
		<li>Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.</li>
		<li>Isolate chromosomal DNA using a commercially available kit designed for Gram-negative bacteria. Purified chromosomal DNA can be stored indefinitely at -20 &#176;C. Chromosomal DNA isolation using commercially available kits generally take about 4 h.</li>
		<li>Use a micro-volume spectrophotometer to ensure the chromosomal DNA sample is of high quality (A260/280 &#62;1.8).</li>
		<li>For each chromosomal DNA isolate, prepare polymerase chain reaction(s) (PCR) on ice in sterile 200 &#956;L 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:
		<ol>
			<li>Initial denature at 95 &#176;C for 120 s.</li>
			<li>Denature at 95 &#176;C for 60 s.</li>
			<li>Anneal primers at 60 &#176;C for 45 s.</li>
			<li>Extend at 72 &#176;C for 90 s.</li>
			<li>Repeat steps 3.1.5.2 through 3.1.5.4 for 34 cycles.</li>
			<li>Final extension 72 &#176;C for 600 s.</li>
			<li>Infinite hold at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Dye 5 &#956;L of respective PCR product(s) using a standard 6x DNA gel loading dye, and load approximately 10 &#956;L of 1 kb ladder and 10 &#956;L of PCR product onto a 1% agarose gel in 1x Tris-Borate-EDTA (TBE) buffer.</li>
		<li>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.</li>
		<li>Upon verification of successful PCR amplification at the expected size, purify the remaining 45 &#956;L PCR product(s) using a commercially available DNA clean &#38; concentrator kit.</li>
		<li>Sequence cleaned PCR product(s) using the same primers used for PCR amplification, and prepare per local sequencing facility guidelines.</li>
		<li>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.</li>
		<li>ctxB: (classical) region between VC0395_A1059 to VC0395_A1060 (El Tor) VC_1456</li>
		<li>tcpA: (classical) VC0395_A0353 (El Tor) VC_0828</li>
	</ol>
	</li>
	<li>Phenotypic Assays for Biotype Classification via Spotting
	<ol>
		<li>Prepare overnight culture in liquid LB broth as previously stated in protocol 2.2.</li>
		<li>Wash the cell pellet(s) as specified in protocol 2.6.</li>
		<li>Use a pipette to spot 1 &#956;L of washed culture on respective medium. For medium selection and incubation specifications, refer to Table 2.</li>
	</ol>
	</li>
	<li>Motility Assay
	<ol>
		<li>Prepare overnight culture(s) in liquid LB broth as previously stated in protocol 2.2.</li>
		<li>Wash the cell pellet(s) as specified in protocol 2.6.</li>
		<li>Inoculate motility agar plates by inserting inoculating stab into the washed liquid culture and &#34;stab&#34; 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.</li>
		<li>Incubate plates lid-side up for 14 to 24 h at 37 &#176;C. After 14 h, monitor motility plates closely to prevent overgrowth of cultures.</li>
	</ol>
	</li>
	<li>Voges-Proskauer (VP) Assay
	<ol>
		<li>Prepare overnight culture(s) in liquid LB broth as previously stated in protocol 2.2.</li>
		<li>Inoculate 4 mL of methyl red Voges-Proskauer, or MR-VP broth, by pipetting 10&#160;&#956;L&#160;of previously prepared overnight culture into 4 mL MR-VP broth in a sterile culture tube&#160;and incubate with aeration in a shaker incubator at 225 rpm for 12 to 16 h at 37&#160;&#176;C.</li>
		<li>Add 150 &#956;L of 5% (w/v) &#945;-naphthol and 50 &#956;L 40% (w/v) KOH to 1 mL aliquots of MR-VP overnight culture in sterile culture tubes, respectively.</li>
		<li>Briefly vortex and let stand at room temperature for up to 4 h until color change develops.</li>
	</ol>
	</li>
</ol>

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<li>Iwanaga, M., Yamamoto, K., Higa, N., Ichinose, Y., Nakasone, N., Tanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+conditions+for+stimulating+cholera+toxin+production+by+Vibrio+cholerae+O1+El+Tor%5BTitle%5D)+AND+%22Microbiol.+Immunol%22%5BJournal%5D)">Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor.</a> Microbiol. Immunol. 30 (11), 1075-1083 (1986).</li>
<li>DiRita, V. J., Claude, P., Georg, J., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+cascade+controls+virulence+in+Vibrio+cholerae%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Regulatory cascade controls virulence in Vibrio cholerae.</a> Proc. Natl. Acad. Sci. USA. 88 (12), 5403-5407 (1991).</li>
<li>Kovacikova, G., Lin, W., Skorupski, K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15978075">Duel regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive Lys-R type regulator AlsR in Vibrio cholerae.</a> Mol. Microbiol. 57 (2), 420-433 (2005).</li>
<li>Vogel, H. J., Bonner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acetylornithinase+of+Escherechia+coli%3A+partial+purification+and+some+properties%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Acetylornithinase of Escherechia coli: partial purification and some properties.</a> J. Biol. Chem. 218 (1), 97-106 (1956).</li>
<li>Son, M. S., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+screens+and+biochemical+assays+to+characterize+Vibrio+cholerae+O1+biotypes%3A+classical+and+El+Tor%5BTitle%5D)+AND+%22Curr.+Protoc.+Microbiol%22%5BJournal%5D)">Genetic screens and biochemical assays to characterize Vibrio cholerae O1 biotypes: classical and El Tor.</a> Curr. Protoc. Microbiol. , 6A.2.1-6A.2.17 (2011).</li>
<li>Kovacikova, G., Skorupski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+virulence+gene+expression+in+Vibrio+cholerae+by+quorum+sensing%3A+HapR+functions+at+the+aphA+promoter%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter.</a> Mol. Microbiol. 46 (4), 1135-1147 (2002).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prevalence+of+functional+quorum-sensing+systems+in+recently+emerged+Vibrio+cholerae+toxigenic+strains%5BTitle%5D)+AND+%22Environ.+Microbiol.+Rep%22%5BJournal%5D)">The prevalence of functional quorum-sensing systems in recently emerged Vibrio cholerae toxigenic strains.</a> Environ. Microbiol. Rep. 3 (2), 218-222 (2011).</li>
<li>Sanders, E. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22617405">Aseptic laboratory techniques.</a> J. Vis. Exp. (63), e3064(2012).</li>
<li>Martinez, R. M., Megli, C. J., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+laboratory+maintenance+of+Vibrio+cholerae%5BTitle%5D)+AND+%22Curr.+Protoc%22%5BJournal%5D)">Growth and laboratory maintenance of Vibrio cholerae.</a> Curr. Protoc. , 6A.1.1-6A.1.6 (2010).</li>
</ol>

The authors have nothing to disclose.

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 variants13,17, have emerged that possess the El Tor biotype background, and harbor classical characteristics8,9,10,11</su...

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 potential1,2. Moreover, serogroup O139 has been primarily isolated to Southeastern Asia3,4, 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 environment5,6,7. Recently, strains have arisen which contain distinguishing characteristics of both classical and El Tor biotypes8,9,10,11,12,13,14,15,16,17and have since been termed El Tor variants13,17. 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 treatment8,9,18. 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 biotypes21,22,23. 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&#39;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 lost24,25. 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&#39;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&#39;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.

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

laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates

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&#39;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.

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) (...

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

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.

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

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

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10.9K Views.  Plymouth State University.  Dit manuscript beschrijft de juiste Vibrio cholerae onderhoudstechnieken naast een reeks biochemische analyses, collectief gebruikt voor snelle en betrouwbare differentiatie tussen klinische en milieuvriendelijke V. Cholerae biotypen in een laboratorium omgeving. 

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

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

I am Es.In MindUP, we study biology of human pathogens in non hosts environments. Many human pathogens after exiting their human host have to survive in the environment before infecting another host. So our goal is to better understand processes that control survival of human pathogens in the environment.And our goal is to then better understand transmission dynamics of infectious agents. So we are using Vibrio cholera, the cost of agent of diarrhea, disease, cholera as a model organism. Vibrio cholera is a facultative human pathogen and it has an aquatic lifecycle.Therefore, it provides us great opportunity to study the survival of human pathogen in a natural environment. There are many tools to study this organism. Its genome is completely sequenced and there are micro array representing every gene in the genome.And furthermore, there are many tools that we can manipulate the genome of this organism. My research program takes advantage of these tools and integrates them to study aquatic lifecycle of cholera. So in our experiments, we try to simulate the aquatic environments that organism lives in.And then we would like to know how does this organism so senses fluctuations in its environment with respect to nutrient load, temperature, salinity, or other biological stressors such as pages and tzo? So in aquatic environment, RIA cholera is found either in a free living form or in a surface attached form. And the surface attached forms are known as biofilms, which are simply composed of microorganisms and their extra polymeric substances, which can be exo polysaccharide and proteins and sometimes DNA.And this matrix acts as a glue and hold the organism together. And when grown in biofilm mode, these organisms are actually resistant to fluctuating parameters of their environments and they can be better protected from, for example, phage and proteasome probations. And these are factors that are controlling abundance of microorganisms in the environment.Some of the recent techno technological advance that helped us in our studies are the completion of the genome sequence of the organism and development of microray technology. Using these technologies, we can determine at the whole genome level how this organism responds to major fluctuations in its environment such as nutrient deprivation or changes in temperature. Or one of our major goal is to integrate our whole genome expression studies to a network analysis.So we would like to identify genetic regulatory network that controls response of to fluctuating environmental parameters. We would like to be able to model behavior of organism in the environment. And also we still do not know when in the environment with whom Vibra Cide interacts with.We do not know much about ecology of this organism, and we also do not know relevance of the genes and processes we identified to be important for survival of this organism in the laboratory to the real world. No are the genes that we identified express when Ria cholera is present in natural environment. We also would like to be able to identify and culture organisms that Vibrio Cora interacts with because in the environment, Vibrio Cora is a part of the complex microbial community.So I think understanding biology of Vibrio Cora in natural environment would be a, it's a challenging and it's critical for us to understand biology of the organism. Within the next five years, I hope that we would be able to better characterize op polysaccharide components of biofilms. For example, in Vibrio cholera, we still do not know structure of the op polysaccharide that is holding the cells together.We also do not know all the components of biofilm metrics. Eventually I think it be great to be able to quantify components of the biofilm metrics in natural biofilms of these organisms. I think another major challenge is to study, be OC in natural environment.And I hope that we would be able to take advantage of comparative genomic studies and combine whole genomic expression profiling comparative genomics to better understand evolution of IBR cholera as a pathogen.

Biology

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

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

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