Name: development of a more sensitive and specific chromogenic agar medium for the detection of vibrio parahaemolyticus and other vibrio species
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Description: Watch this Scientific Journal Video about Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species Video at JoVE.com

We thank M. Channey, E. Chau, and K. Tomas for their assistance on the project. Project supplies were partially funded by California Polytechnic State University.

1. Media and Culturing of Microbial Strains
NOTE: Use aseptic techniques in all experiments. Use sterile materials. Sterilize all containers, tools and reagent prior to use. Autoclave all waste materials prior to disposal because they are considered biohazardous. Autoclave temperature and time combination is &#8805;121 &#176;C x &#8805;15 min for all of the following procedures.
<ol>
	<li>To make ~1-L tryptic soy agar (TSA), first add 1 L deionized water in a 2-L Erlenmeyer flask containing ...

<ol>
	<li>Yeung, P. S., Boor, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+pathogenesis,+and+prevention+of+foodborne+Vibrio+parahaemolyticus+infections.">Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections.</a> Foodborne Pathog. Dis. 1 (2), 74-88 (2004).</li><li>Yeung, P. S. M., Boor, K. J., Faruque, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+molecular+biology,+and+detection+of+foodborne+Vibrio+parahaemolyticus+infections.">Epidemiology, molecular biology, and detection of foodborne Vibrio parahaemolyticus infections.</a> Foodborne and Waterborne Bacterial pathogens: Epidemiology, Evolution and Molecular Biology. , 153-184 (2012).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+Signs:+Incidence+and+Trends+of+Infection+with+Pathogens+Transmitted+Commonly+Through+Food+-+Foodborne+Diseases+Active+Surveillance+Network,+10+U.S.+Sites,+1996-2010.">Vital Signs: Incidence and Trends of Infection with Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 1996-2010.</a> Morbidity and Mortality Weekly Report. 10, 1996-2010 (2011).</li><li>. Summary of human Vibrio cases reported to CDC, 2008 Available from: <a target="_blank" href="https://stacks.cdc.gov/view/cdc/21591">https://stacks.cdc.gov/view/cdc/21591</a> (2008)</li><li>Scallan, E., 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=Foodborne+illness+acquired+in+the+United+States+-+major+pathogens.">Foodborne illness acquired in the United States - major pathogens.</a> Emerg. Infect. Dis. 17 (1), 7-15 (2011).</li><li>Baross, J., Liston, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+Vibrio+parahaemolyticus+and+related+hemolytic+vibrios+in+marine+environments+of+Washington+State.">Occurrence of Vibrio parahaemolyticus and related hemolytic vibrios in marine environments of Washington State.</a> Appl. Microbiol. 20 (2), 179-186 (1970).</li><li>Baross, J., Liston, J. <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+parahaemolyticus+from+the+Northwest+Pacific.">Isolation of Vibrio parahaemolyticus from the Northwest Pacific.</a> Nature. 217 (5135), 1263-1264 (1968).</li><li>Kueh, C. S., Chan, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+in+bivalve+shellfish+with+special+reference+to+the+oyster.">Bacteria in bivalve shellfish with special reference to the oyster.</a> J. Appl. Bacteriol. 59 (1), 41-47 (1985).</li><li>Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. , (2012).</li><li>Qadri, F., 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=Adaptive+and+inflammatory+immune+responses+in+patients+infected+with+strains+of+Vibrio+parahaemolyticus.">Adaptive and inflammatory immune responses in patients infected with strains of Vibrio parahaemolyticus.</a> J. Infect. Dis. 187 (7), 1085-1096 (2003).</li><li>MacFaddin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Media for isolation-cultivation-identification-maintenance of medical bacteria. 1, (1985).</li><li>Bottone, E. J., Robin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+parahaemolyticus:+suspicion+of+presence+based+on+aberrant+biochemical+and+morphological+features.">Vibrio parahaemolyticus: suspicion of presence based on aberrant biochemical and morphological features.</a> J. Clin. Microbiol. 8 (6), 760-763 (1978).</li><li>Lotz, M. J., Tamplin, M. L., Rodrick, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiosulfate-citrate-bile+salts-sucrose+agar+and+its+selectivity+for+clinical+and+marine+vibrio+organisms.">Thiosulfate-citrate-bile salts-sucrose agar and its selectivity for clinical and marine vibrio organisms.</a> Ann. Clin. Lab. Sci. 13 (1), 45-48 (1983).</li><li>Hara-Kudo, Y., Nishina, T., Nakagawa, H., Konuma, H., Hasegawa, J., Kumagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+detection+of+Vibrio+parahaemolyticus+in+seafood.">Improved method for detection of Vibrio parahaemolyticus in seafood.</a> Appl. Environ. Microbiol. 67 (12), 5819-5823 (2001).</li><li>Eddabra, R., Piemont, Y., Scheftel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+new+chromogenic+medium,+chromID+Vibrio,+for+the+isolation+and+presumptive+identification+of+Vibrio+choleare+and+Vibrio+parahaemolyticus+from+human+clinical+specimens.">Evaluation of a new chromogenic medium, chromID Vibrio, for the isolation and presumptive identification of Vibrio choleare and Vibrio parahaemolyticus from human clinical specimens.</a> Eur. J. Clin. Microbiol. Infect. Dis. 30 (6), 733-737 (2011).</li><li>Kodaka, H., Teramura, H., Mizuochi, S., Saito, M., Matsuoka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Compact+Dry+VP+method+for+screening+raw+seafood+for+total+Vibrio+parahaemolyticus.">Evaluation of the Compact Dry VP method for screening raw seafood for total Vibrio parahaemolyticus.</a> J. Food. Prot. 72 (1), 169-173 (2009).</li><li>Su, Y. C., Duan, J., Wu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectivity+and+specificity+of+a+chromogenic+medium+for+detecting+Vibrio+parahaemolyticus.">Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticus.</a> J. Food Prot. 68 (7), 1454-1456 (2005).</li><li>Bej, A. K., Patterson, D. P., Brasher, C. W., Vickery, M. C., Jones, D. D., Kaysner, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+total+and+hemolysin-producing+Vibrio+parahaemolyticus+in+shellfish+using+multiplex+PCR+amplification+of+tl,+tdh+and+trh.">Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.</a> J. Microbiol. Methods. 36 (3), 215-225 (1999).</li><li>Yeung, P. S. M., DePaola, A., Kaysner, C. A., Boor, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+assay+for+specific+detection+of+the+pandemic+Vibrio+parahaemolyticus+O3:K6+clone+from+shellfish.">A PCR assay for specific detection of the pandemic Vibrio parahaemolyticus O3:K6 clone from shellfish.</a> J. Food Sci. 68 (4), 1459-1466 (2003).</li><li>Yeung, P. S. M., Hayes, M. C., DePaola, A., Kaysner, C. A., Kornstein, L., Boor, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+phenotypic,+molecular,+and+virulence+characterization+of+Vibrio+parahaemolyticus+O3:K6+isolates.">Comparative phenotypic, molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates.</a> Appl. Environ. Microbiol. 68 (6), 2901-2909 (2002).</li><li>Duan, J., Su, Y. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+chromogenic+medium+with+thiosulfate-citrate-bile+salts-sucrose+agar+for+detecting+Vibrio+parahaemolyticus.">Comparison of a chromogenic medium with thiosulfate-citrate-bile salts-sucrose agar for detecting Vibrio parahaemolyticus.</a> J. Food Sci. 70, M125-M128 (2005).</li><li>Pinto, A. D., Terio, V., Novello, L., Tantillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+thiosulphate-citrate-bile+salt+sucrose+(TCBS)+agar+and+CHROMagar+Vibrio+for+isolating+Vibrio+parahaemolyticus.">Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus.</a> Food Control. 22 (1), 124-127 (2011).</li><li>Canizalez-Roman, A., Flores-Villase&#241;or, H., Zazueta-Beltran, J., Muro-Amador, S., Le&#242;n-Sicairos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+a+chromogenic+agar+medium-PCR+protocol+with+a+conventional+method+for+isolation+of+Vibrio+parahaemolyticus+strains+from+environmental+and+clinical+samples.">Comparative evaluation of a chromogenic agar medium-PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples.</a> Can J Microbiol. 57 (2), 136-142 (2011).</li><li>Kriem, M. R., 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=Prevalence+of+Vibrio+spp.+in+raw+shrimps+(Parapenaeus+longirostris)+and+performance+of+a+chromogenic+medium+for+the+isolation+of+Vibrio+strains.">Prevalence of Vibrio spp. in raw shrimps (Parapenaeus longirostris) and performance of a chromogenic medium for the isolation of Vibrio strains.</a> Lett Appl Microbiol. 61 (3), 224-230 (2015).</li><li>Food Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+guidance+on+reporting+results+from+studies+evaluating+diagnostic+tests.">Statistical guidance on reporting results from studies evaluating diagnostic tests.</a> http://www.fda.gov/RegulatoryInformation/Guidances/ucm071148.htm. , (2007).</li><li>Burd, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+laboratory-developed+molecular+assays+for+infectious+diseases.">Validation of laboratory-developed molecular assays for infectious diseases.</a> Clin Microbiol Rev. 23 (3), 550-576 (2010).</li></ol>

This study focuses on culture media development and evaluation. Conventionally, TCBS is the selective and differential medium used for isolating and detecting V. parahaemolyticus, V. cholerae and V. vulnificus12. However, limitations have been reported for this medium, such as the inability to differentiate V. cholerae from other Vibrio species. Sucrose and pH indicator are the differentiation agents of TCBS. Thus, acid production by sucrose fermenter causes color change of ...

As a member of the Vibrio genus, V. parahaemolyticus is a Gram-negative, non-spore-forming, curved, rod-shaped bacterium. It exhibits high motility in both liquid and semi-solid environments. Most V. parahaemolyticus strains are non-pathogenic to humans, yet the pathogenic subtypes have caused epidemics and pandemics, hence this species is considered to be an important foodborne pathogen in many countries1,2. The incidence of Vibrio infection in the US has shown an upward trend since 20003. Among Vibrio spp., V. parahaemolyticus is the most frequently reported species causing illnesses in the US4,5. Other clinically relevant species include V. alginolyticus, V. vulnificus, V. cholerae, etc. A small percentage of the illnesses is caused by multiple species simultaneously.
V. parahaemolyticus is a natural inhabitant of marine water and therefore widely distributed in marine waters throughout the world including the estuaries. The species was discovered in 1950 following an outbreak of food poisoning in Japan. In the US, the species was first isolated in seawater, sediments, and shellfish in the Puget Sound region6,7. Filter feeders in marine habitats, such as bivalve shellfish, can harbor V. parahaemolyticus as part of their natural flora8. As such, V. parahaemolyticus infections in human are often linked to the consumption of contaminated seafood, especially raw or undercooked shellfish. A less common route of entry occurs when open wound is exposed to seawater, leading to skin infection. Most V. parahaemolyticus strains do not cause human disease, yet certain subtypes harboring virulence factors such as thermostable direct hemolysin (TDH) are pathogenic. The most prevalent symptoms of foodborne V. parahaemolyticus infection are diarrhea and abdominal pain, followed by nausea, vomiting, and fever. Headache and chills are also reported. The median incubation period is 15 hr, but can be up to 96 hr after consumption of sufficient amount of pathogenic strains9. The illness lasts from two to three days. The gastroenteritis symptoms caused by V. parahaemolyticus are largely self-limiting and therefore special treatment is not necessary. Mild cases of gastroenteritis can be effectively treated by oral rehydration. More severe illnesses can be treated by antibiotics such as tetracycline or ciprofloxacin10. Mortality rate is about 2% for gastroenteritis cases, but may be as high as 29% for those who develop bloodstream infection or septicemia. Any person who consumes seafood or has open wound exposed to seawater is at risk of V. parahaemolyticus infection. The more severe form of illnesses, life-threatening septicemia, is more common in a subpopulation with underlying medical conditions11, which include alcoholism, liver disease, diabetes, renal disease, malignancy, and other conditions leading to a weakened immune response. Notably, this group of individuals is also at a higher risk for contracting severe illnesses caused by V. vulnificus, which can be found in natural habitats similar to V. parahaemolyticus.
V. parahaemolyticus is routinely isolated using thiosulfate-citrate-bile salts-sucrose (TCBS) agar as a selective and differential medium. Enrichment in alkaline peptone water may precede isolation on TCBS agar. Presumptive colonies on TCBS are then further tested in an array of biochemical tests and/or molecular assays targeting the presence of species-specific genes. PCR-based methods are often used to confirm the identities of V. parahaemolyticus by amplifying the thermolabile hemolysin gene, tlh12. 
Regardless of the choice of confirmation methods, it is important to have an effective medium to isolate and differentiate V. parahaemolyticus from other marine vibrios in the first place. TCBS has routinely been used to differentiate species within the Vibrio genus according to their abilities to ferment sucrose12. Positive fermentation reaction is accompanied by a color change of the pH indicator Bromothymol blue. V. parahaemolyticus colonies are fairly distinctive on TCBS, exhibiting blue to green color. However, this medium cannot easily differentiate V. alginolyticus and V. cholerae. Sucrose-fermenting Proteus species may produce yellow colonies resembling V. cholerae or V. alginolyticus13. On initial isolation on TCBS, V. parahaemolyticus may also be misidentified as Aeromonas hydrophila, Plesiomonas shigelloides, and Pseudomonas spp14. Strains with delayed sucrose fermentation may be confused with other sucrose nonfermenting Vibrio13, which include V. parahaemolyticus. TCBS was found to be not sensitive against Escherichia coli, Pseudomonas putrefaciens, among others. Several other species yield green to gray colonies which are potentially confused with V. parahaemolyticus or V. vulnificus15. As a result, it is desirable to develop alternative culture media with better sensitivity and specificity toward detecting and isolating V. parahaemolyticus and other closely related species.
Several media alternatives have been recently developed. In addition to the inclusion of selective agents, most incorporate chromogenic substrates to differentiate species based on their differential enzymatic activities. For example, indoxyl-&#946;-glucoside and indoxyl-&#946;-galactoside have been used as the chromogenic substrates to differentiate V. parahaemolyticus colonies (which appear bluish-green) from those of V. cholerae (purple) due to their differential abilities to produce &#946;-glucosidase and &#946;-galactosidase16. Different formulations of chromogenic agar developed by several groups have been evaluated and were reported to perform comparably to or better than TCBS17,18,19. An advantage of using a chromogenic medium is that the coloring of the surrounding medium is minimal thereby facilitating the isolation of particular colonies. In this study, we evaluated the ability of a newly formulated chromogenic medium to detect and isolate V. cholerae, V. parahaemolyticus, and V. vulnificus; with a special focus on its ability to differentiate V. parahaemolyticus from other species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent/Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-15-4</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >BHI powder</td>
			<td>Fisher Scientific</td>
			<td>DF0418-17-7</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Blender</td>
			<td>Any</td>
			<td></td>
			<td>to blend oyster meat</td>
		</tr>
		<tr >
			<td >CampyGen gas generator</td>
			<td>Hardy Diagnostics</td>
			<td>CN035A</td>
			<td>to provide a microaerophilic atmosphere; may use other brands</td>
		</tr>
		<tr >
			<td >Chocolate agar plates</td>
			<td>Hardy Diagnostics</td>
			<td>E14</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Common PCR reagents (dNTPs, MgCl2, Taq Polymerase)</td>
			<td>Any</td>
			<td></td>
			<td>or use PCR beads (Fisher Sci 46-001-014)</td>
		</tr>
		<tr >
			<td >Culture tubes</td>
			<td>Fisher Scientific</td>
			<td>S50712</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>S348903</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Gel doc</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HardyChrom Vibrio agar plates</td>
			<td>Hardy Diagnostics</td>
			<td>G319</td>
			<td>This study evaluates this medium</td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculating loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-606</td>
			<td>10 microliter-size was used in this study</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Oysters</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Fisher Scientific</td>
			<td>R23701</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Pipette and tips</td>
			<td>Any</td>
			<td></td>
			<td>Sterilized tips</td>
		</tr>
		<tr >
			<td >Primers for tlh</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Scale</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Spreader</td>
			<td>Fisher Scientific</td>
			<td>08-100-11</td>
			<td>Beads may be used instead</td>
		</tr>
		<tr >
			<td >Stomacher blender</td>
			<td>Stomacher</td>
			<td >400</td>
			<td>Samples were homogenized at 200 rpm for 30 sec.&#160; Other homogenizer can be used.</td>
		</tr>
		<tr >
			<td >Sterile filter bags for blenders</td>
			<td>Fisher Scientific</td>
			<td>01-812-5</td>
			<td></td>
		</tr>
		<tr >
			<td >TCBS powder</td>
			<td>Hardy Diagnostics</td>
			<td >265020</td>
			<td>This study evaluates this medium</td>
		</tr>
		<tr >
			<td >Thermocycler</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TSB powder</td>
			<td>Fisher Scientific</td>
			<td>DF0370-07-5</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >UV viewing cabinet</td>
			<td>Any</td>
			<td></td>
			<td>Emit long-wave UV light</td>
		</tr>
		<tr >
			<td >Water bath</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Name</td>
			<td>Sources</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr >
			<td >Bacterial species and strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Aeromonas hydrophila</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Candida albicans</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Campylobacter jejuni</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Escherichia coli</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Proteus mirabilis</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pseudomonas aeruginosa</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Staphylococcus aureus</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Salmonella Choleraesuis</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella boydii</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella flexneri</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella sonnei</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vibrio alginolyticus</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. cholerae (serotypes include O139, O1, non O1, El Tor biovars)</td>
			<td >FDA, ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. damsela</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. fisherii</td>
			<td >Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. fluvialis</td>
			<td >CDC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. furnissii</td>
			<td >CDC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. hollisae</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. metschnikovii</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. mimicus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. parahaemolyticus(serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc)</td>
			<td >ATCC, FDA, CDC, Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. proteolyticus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. vulnificus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this study, 54 microbial strains were assembled, which included 22 strains within the V. parahaemolyticus species, 19 other Vibrio species, and 13 non-Vibrio species (Table 1). Most V. parahaemolyticus strains were either received from FDA, CDC or other state health departments. They represent diverse serotypes and isolation sources. These strains were previously identified by the regulatory agencies. We further confirmed the identit...

Watch this Scientific Journal Video about Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species Video at JoVE.com

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species Video (Video) | JoVE

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.

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 ...

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