A New Chromogenic Medium for Rapid Detection of Vibrio Species

Abstract

Source: Yeung, M., et al. Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species. J. Vis. Exp. (2016)

This video demonstrates a culture-based method for identifying Vibrio species, highlighting chromogenic agar as a more accurate alternative to the commonly used thiosulfate-citrate-bile salts-sucrose or TCBS agar. Chromogenic agar provides superior differentiation of Vibrio strains by detecting species-specific enzymatic activities.

Protocol

Growth on Selective and Differential Media

1. Two to four days before the experiment, streak all microbial strains on nonselective medium (TSAS, BHI, or chocolate agar) for colony isolation. Incubate plates at 35-37 °C for 48 hr. Check the purity of the cultures by observing colony morphology after incubation. Pure cultures should yield colonies that exhibit similar colony morphology.
2. Transfer a few isolated colonies into 5 ml of broth. Incubate tubes at 35-37 °C for 16-24 hr.

NOTE: Use young colonies, which are less than four days old, to prepare overnight cultures in all experiments.

3. Streak a loopful of overnight cultures on selective and differential media (TCBS and chromogenic agar) for colony isolation. Incubate plates at 35-37 °C for up to 96 hr.
4. Record the overall growth of all strains by examining both the culture density on the plate and the size of isolated colonies. Record the color of colonies under ambient and/or UV light. Note other characteristics of the isolated colony, such as the elevation, margin, and form.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Equipment
Agar	Fisher Scientific	DF0140-15-4	may use other brands
BHI powder	Fisher Scientific	DF0418-17-7	may use other brands
CampyGen gas generator	Hardy Diagnostics	CN035A	to provide a microaerophilic atmosphere; may use other brands
Chocolate agar plates	Hardy Diagnostics	E14	may use other brands
Culture tubes	Fisher Scientific	S50712	may use other brands
HardyChrom Vibrio agar plates	Hardy Diagnostics	G319	This study evaluates this medium
Incubator	Any
Inoculating loops	Fisher Scientific	22-363-606	10 microliter-size was used in this study
Petri dish	Fisher Scientific	FB0875713	may use other brands
Pipette and tips	Any Sterilized tips
TCBS powder	Hardy Diagnostics	265020	This study evaluates this medium
TSB powder	Fisher Scientific	DF0370-07-5	may use other brands
UV viewing cabinet	Any		Emit long-wave UV light
Water bath	Any
Bacterial species and strains
Aeromonas hydrophila	ATCC
Candida albicans	ATCC
Campylobacter jejuni	ATCC
Escherichia coli	ATCC
Proteus mirabilis	ATCC
Pseudomonas aeruginosa	ATCC
Staphylococcus aureus	ATCC
Salmonella Choleraesuis	ATCC
Shigella boydii	ATCC
Shigella flexneri	ATCC
Shigella sonnei	ATCC
Vibrio alginolyticus	ATCC
V. cholerae (serotypes include O139, O1, non O1, El Tor biovars)	FDA, ATCC
V. damsela	FDA
V. fisheri	Environment
V. fluvialis	CDC
V. furnissii	CDC
V. hollisae	FDA
V. metschnikovii	ATCC
V. mimicus	FDA
V. parahaemolyticus (serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc.)	ATCC, FDA, CDC, Environment
V. proteolyticus	FDA
V. vulnificus	FDA