Name: laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates
Uploaded: 2017-05-30T13:00:00
Description: Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

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 ...

<ol>
	<li>Shimada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+serotyping+scheme+for+Vibrio+cholerae.">Extended serotyping scheme for Vibrio cholerae.</a> Curr. Microbiol. 28 (3), 175-178 (1994).</li><li>Yamai, S., Tadayuki, O., Toshio, S., Yasuji, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+serogroups+of+Vibrio+cholerae+non-O1+non-O139+with+specific+reference+to+their+ability+to+produce+cholera+toxin,+and+addition+of+novel+serogroups.">Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups.</a> Jpn. J. Infect. Dis. 71 (10), 1037-1045 (1997).</li><li>Karaolis, D. K., Lan, R., Reeves, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sixth+and+seventh+cholera+pandemics+are+due+to+independent+clones+separately+derived+from+environmental,+nontoxigenic,+non-O1+Vibrio+cholerae.">The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae.</a> J. Bacteriol. 177 (11), 3191-3198 (1995).</li><li>Albert, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+epidemic+of+cholera-like+disease+in+Bangladesh+caused+by+Vibrio+cholerae+O139+synonym+Bengal.">Large epidemic of cholera-like disease in Bangladesh caused by Vibrio cholerae O139 synonym Bengal.</a> Lancet. 342 (8868), 387 (1993).</li><li>Barua, D., Barua, D., Greenough III, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+Cholera.">History of Cholera.</a> Cholera. , 1-36 (1992).</li><li>Morales, R., Delgado, G., Cravioto, A., Faruque, S. M., Nair, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+Genetics+of+Vibrio+cholerae.">Population Genetics of Vibrio cholerae.</a> Vibrio cholerae-Genomics and Molecular Biology. , 29-47 (2008).</li><li>Samadi, A. R., Chowdhury, M. K., Huq, M. K., Khan, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonality+of+classical+and+El+Tor+cholera+in+Dhaka,+Bangladesh:+17-year+trends.">Seasonality of classical and El Tor cholera in Dhaka, Bangladesh: 17-year trends.</a> Trans. R. Soc. Trop. Med. Hyg. 77 (6), 853-856 (1983).</li><li>Son, M. S., Megli, C. J., Kovacikova, G., Qadri, F., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Vibrio+cholerae+O1+El+Tor+biotype+variant+clinical+isolates+from+Bangladesh+and+Haiti,+including+a+molecular+genetic+analysis+of+virulence+genes.">Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes.</a> J. Clin. Microbiol. 49 (11), 3739-3749 (2011).</li><li>Ghosh-Banerjee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+production+by+the+El+Tor+variant+of+Vibrio+cholerae+O1+compared+to+prototype+El+Tor+and+classical+biotypes.">Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes.</a> J. Clin. Microbiol. 48 (11), 4283-4286 (2010).</li><li>Ansaruzzaman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+in+Mozambique,+variant+of+Vibrio+cholerae.">Cholera in Mozambique, variant of Vibrio cholerae.</a> Emerg. Infect. Dis. 10 (11), 2057-2059 (2004).</li><li>Ansaruzzaman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+El+Tor+strains+of+Vibrio+cholerae+O1+with+hybrid+traits+isolated+from+Bangladesh+and+Mozambique.">Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique.</a> Int. J. Med. Microbiol. 297 (6), 443-449 (2007).</li><li>Lan, R., Reeves, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+spread+of+cholera:+genetic+diversity+and+relationships+within+the+seventh+pandemic+clone+of+Vibrio+cholerae+determined+by+amplified+fragment+length+polymorphism.">Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism.</a> J. Clin. Microbiol. 40 (1), 172-181 (2002).</li><li>Nair, G. B., Faruque, S. M., Bhuiyan, N. A., Kamruzzaman, M., Siddique, A. K., Sack, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+variants+of+Vibrio+cholerae+O1+biotype+El+Tor+with+attributes+of+the+classical+biotype+from+hospitalized+patients+with+acute+diarrhea+in+Bangladesh.">New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh.</a> J. Clin. Microbiol. 40 (9), 3296-3299 (2002).</li><li>Nair, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vibrio+cholerae+O1+strains+similar+to+pre-seventh+pandemic+El+Tor+strains+during+an+outbreak+of+gastrointestinal+disease+in+an+island+resort+in+Fiji.">Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji.</a> J. Med. Microbiol. 55 (11), 1559-1562 (2006).</li><li>Nair, G. B., Mukhopadhyay, A. K., Safa, A., Takeda, Y., Faruque, S. M., Nair, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+hybrid+variants+of+Vibrio+cholerae+O1.">Emerging hybrid variants of Vibrio cholerae O1.</a> Vibrio cholerae—Genomics and Molecular Biology. , 179-190 (2008).</li><li>Safa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+characteristics+of+Matlab+variants+of+Vibrio+cholerae+O1+that+are+hybrids+between+classical+and+El+Tor+biotypes.">Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.</a> J. Med. Microbiol. 55 (11), 1563-1569 (2006).</li><li>Nusrin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+CTX+phages+among+toxigenic+Vibrio+cholerae+O1+and+O139+strains+isolated+between+1994+and+2002+in+an+area+where+cholera+is+endemic.">Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic.</a> J. Clin. Microbiol. 42 (12), 5854-5856 (2004).</li><li>Carignan, B. M., Brumfield, K. D., Son, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphisms+in+regulator-encoding+genes+have+an+additive+effect+on+virulence+gene+expression+in+a+Vibrio+cholerae+clinical+isolate.">Single nucleotide polymorphisms in regulator-encoding genes have an additive effect on virulence gene expression in a Vibrio cholerae clinical isolate.</a> mSphere. 1 (5), e00253 (2016).</li><li>Iwanaga, M., Yamamoto, K., Higa, N., Ichinose, Y., Nakasone, N., Tanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+stimulating+cholera+toxin+production+by+Vibrio+cholerae+O1+El+Tor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+cascade+controls+virulence+in+Vibrio+cholerae.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylornithinase+of+Escherechia+coli:+partial+purification+and+some+properties.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+and+biochemical+assays+to+characterize+Vibrio+cholerae+O1+biotypes:+classical+and+El+Tor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+virulence+gene+expression+in+Vibrio+cholerae+by+quorum+sensing:+HapR+functions+at+the+aphA+promoter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+functional+quorum-sensing+systems+in+recently+emerged+Vibrio+cholerae+toxigenic+strains.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+laboratory+maintenance+of+Vibrio+cholerae.">Growth and laboratory maintenance of Vibrio cholerae.</a> Curr. Protoc. , 6A.1.1-6A.1.6 (2010).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1403">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1 kb DNA Ladder</td>
			<td style="width:183px;">New England Biolabs</td>
			<td style="width:171px;">N3232S</td>
			<td style="width:683px;">https://www.neb.com/products/n3232-1-kb-dna-ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Becto, Dickinson and Co.</td>
			<td>214030</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Peqlab</td>
			<td>732-2789</td>
			<td>https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&#38;_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&#38;_dynSessConf=1766917479792147141&#38;targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&#38;lastLanguage=en&#38;/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&#38;_D%3AcurrentLanguage=+&#38;currentLanguage=en&#38;_D%3AlastLanguage=+&#38;_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anhydrous K2HPO4</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&#38;searchTerm=P288500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Agar Plates</td>
			<td>Remel</td>
			<td>R01200</td>
			<td>Store at 4 &#176;C;&#160; http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=A73500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=B38810&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cirtric acid &#183;H2O</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 height="21">
			<td height="21" style="height:21px;">Deoxynucleotide (dNTP) Solution Kit</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/n0446-deoxynucleotide-solutionset</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=S311500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Clean &#38; Concentrator&#8482; -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 height="21">
			<td height="21" style="height:21px;">GelGreen Nucleic Acid Stain</td>
			<td>Biotium</td>
			<td>41005</td>
			<td>https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td>Corrosive;&#160; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&#38;searchTerm=A144212&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=P217500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</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&#38;searchTerm=P285500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=P250500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Loop</td>
			<td>Decon Labs Inc.</td>
			<td>MP190-25</td>
			<td>http://deconlabs.com/products/leloop/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Stab</td>
			<td>Decon Labs Inc.</td>
			<td>MP186-5</td>
			<td>http://deconlabs.com/products/lestab/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</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&#38;searchTerm=M63500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&#38;WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MR-VP Broth</td>
			<td>Difco</td>
			<td>216300</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</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&#38;searchTerm=S374500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=S27110&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</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&#38;searchTerm=S233500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaNH4HPO4&#183;4H2O</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&#38;searchTerm=S218500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Nonfat dry milk</td>
			<td>Nestle Carnation</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211677</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=FB0875714&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polymyxin B sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1004-10MU</td>
			<td>Store at 2-4 &#176;C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Cycler Bio-Rad C1000 Touch&#8482;&#160;</td>
			<td>Bio Rad Labs</td>
			<td>1840148</td>
			<td>http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&#38;_pageLabel=search_page&#38;sfMode=search&#38;sfStartNumber=1&#38;clearQR=true&#38;js=1&#38;searchString=1840148&#38;database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&#38;tabName=DIVISIONNAME</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triphenyltetrazolium chloride</td>
			<td>Alfa Aesar</td>
			<td>A10870</td>
			<td>https://www.alfa.com/en/catalog/A10870/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;searchTerm=BP1521&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8729;HCl</td>
			<td>Calbiochem</td>
			<td>9310</td>
			<td>http://www.emdmillipore.com/US/en/product/OmniPur-TRIS-Hydrochloride---CAS-1185-53-1---Calbiochem,EMD_BIO-9310-OP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211705</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becto, Dickinson and Co.</td>
			<td>212750</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-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) (...

Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

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 ...

The overall goal of this technique is to provide a collection of protocols that are simple to carry out and can help any investigator characterize a given clinical or environmental isolate of Vibrio cholerae. This method can help answer key questions in the bacteriology field such as the specific genotypic and phenotypic characteristics of any Vibrio cholerae isolate, especially those of clinical relevance. The main advantage of this technique is that it allows any investigator to use this collection of assays to reliably characterize and distinguish clinical or environmental Vibrio cholerae isolates.Together, these assays provide a systematic approach to be used as an alternative or in addition to costly labor intensive experiments and the characterization of Vibrio cholerae clinical and environmental isolates. Though this method can provide insight into the characterization of Vibrio cholerae biotypes, it can also be applied to characterize the biochemical and metabolic capabilities of other enteric bacteria. Generally, individuals new to this method will struggle with time management as the incubation times necessary to ensure optimal results differ between biochemical assays.Visual demonstrations of the spotting and stabbing protocols are critical as splatter can often lead to cross-contamination and maximal spacing is required to prevent merging between cultures. Begin the protocol by pelleting 1.8 mL of liquid overnight culture for two minutes by centrifugation at a minimum of 8, 600 xG in a sterile 2 mL microcentrifuge tube. After centrifugation, remove the supernatant and re-suspend the cell pellet in 900 mcL of fresh Luria-Bertani or LB broth by pipetting.Next, add 900 mcL of sterile 60%volume-to-volume glycerol to the culture and mix by vortexing. Transfer the mixture to a sterile 2 mL cryogenic tube and store the stock culture at 80 degrees Celsius. To use the stock, with a sterile inoculating loop, remove a small amount of frozen stock and streak the bacteria to obtain single colonies on LB agar plates.Immediately return the frozen stock to 80 degrees Celsius after use to prevent cultures from completely thawing. Incubate the plates lid-side down for 12-16 hours at 37 degrees Celsius. Obtain previously prepared plate.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 sterilized loop and transferring the colony into the liquid broth. Then, incubate the culture with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius. Prepare overnight culture in liquid LB broth.The next day, pellet the culture and wash the cells by re-suspending the cell pellet in 1.8 mL of 1x phosphate-buffered saline or PBS. Repeat the washing procedure three times. Follow the third wash with a final re-suspension in 1.8 mL of 1x PBS.Then, use a pipette tip to spot 1 mcL of washed culture on the respective medium. Wash the cell pellet obtained from overnight culture. Inoculate motility agar plates by inserting an inoculating stab into the washed liquid culture and stab vertically into the media.Ensure the inoculating stab does not bend while stabbing the agar. Between each inoculation, sterilize the wire stab using a Bunsen burner. Incubate the plates lid side up for 14-24 hours at 37 degrees Celsius.After 14 hours, monitor the motility plates closely to prevent the overgrowth of cultures. Inoculate 4 mL of Methyl Red Voges-Proskauer or MR-VP broth by pipetting 10 mcL of previously prepared overnight culture into 4 mL of MR-VP broth in a sterile culture tube and incubate with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius. Next, add 150 mcL of 5%weight per volume of Alpha-naphthol and 50 mcL of 40%weight per volume potassium hydroxide to 1 mL aliquots of the MR-VP overnight culture in sterile culture tubes respectively.Then, briefly vortex the tubes. Allow the tubes to stand at room temperature for up to four hours until color change develops. Biotype reference strains and representative El Tor variants were included in PCR base genetic screens and phenotypic assays to distinguish between biotype backgrounds of V.Cholerae clinical and environmental isolates.Wild-type classical O395 demonstrated classical ctxB and tcpA sequences. Conversely, wild-type El Tor strains N16961 and C6706 demonstrated El Tor ctxB and tcpA sequences. MQ1795 and BAA-2163 contains the classical biotype ctxB subunit comparable to 0395.Yet both El Tor variants contained the tcpA indicative of the El Tor biotype background. When grown on LB agar plates supplemented with polymyxin B, the wild-type classical biotype strain 0395 showed sensitivity to polymyxin B while wild-type El Tor biotype strain showed resistance. The representative El Tor variant strains demonstrated similar resistance to the antibiotic.After watching this video, you should have a good understanding of how to distinguish between Vibrio cholerae biotypes using the multiple assay identification system outlined in this manuscript. Don't forget that working with BSL-2 pathogens can be extremely hazardous and proper handling and disposal of all materials and waste products must be enforced according to institutional, local, state, and federal regulations. In addition to the protocols listed in this manuscript, further characterization of Vibrio cholerae isolates can be achieved through investigating other phenotypic properties such as biofilm formation and autoagglutination.

Research

JoVE Journal

Immunology and Infection

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Colonization of Euprymna scolopes Squid by Vibrio fischeri

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Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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 ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

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A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

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

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

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