HardyCHROM™ Vibrio

Cat. no. G319 HardyCHROM™ Vibrio, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics HardyCHROM™ Vibrio is recommended for use as a selective and differential growth medium for the cultivation, isolation, and differentiation of Vibrio spp. from food and environmental samples.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY AND PRINCIPLES

The Vibrio genus is comprised of Gram-negative, halophilic, non-spore forming rods that are straight or have a single, rigid curve. All Vibrio spp. are motile, and most species are oxidase- and catalase-positive. Several species in the genus are known human pathogens, the most notable of which are V. cholerae, V. parahaemolyticus, and V. vulnificus.(1-5) Most disease-causing strains are associated with gastroenteritis following ingestion of raw or uncooked shellfish or drinking contaminated water. Cases of open wound infection and septicemia have also been reported.(4,6)

V. cholerae is the most well known Vibrio species, as it is the causative agent of cholera. This organism is most often transmitted via contaminated drinking water in developing countries.(1,7) V. parahaemolyticus is the leading cause of bacterial diarrhea associated with seafood consumption. It is transmitted through ingestion of undercooked or mishandled seafood, or less commonly through open wounds exposed to seawater.(5,8) While not as common, V. vulnificus is the most fatal Vibrio species in the United States. Infection and death from this species occurs from consumption of Gulf Coast oysters and possibly wound infections acquired in or exposed to marine environments.(4,9)

HardyCHROM™ Vibrio Agar is a chromogenic medium, highly selective for isolation of V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus, as well as other Vibrio species. HardyCHROM™ Vibrio Agar has a high pH, which suppresses the growth of non-Vibrio species found in similar marine samples.(10) The media consists of animal proteins, sea salts, sodium citrate, sodium thiosulfate, sugars, and a chromogenic mix. Seas salts are incorporated into the medium to provide optimum growth and metabolic activity of halophilic Vibrio spp. Sodium thiosulfate provides a source of sulfur. The inclusion of chromogenic substrates allows for the differentiation of Vibrio species based on colony color. It is the only medium that will differentiate V. vulnificus from V. parahaemolyticus and V. cholerae.

FORMULA

Ingredients per liter of deionized water:*

Peptone 10.0g
Sea Salt Mixture 10.0g
Oxbile 10.0g
Sodium Thiosulfate 10.0g
Sucrose 10.0g
Yeast Extract 5.0g
Sodium Citrate 5.0g
Sodium Carbonate 1.5g
Lactose 2.0g
Sodium Pyruvate 0.5g
Chromogenic Mixture 1.25g
Agar 15.0g

Final pH 8.6 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample collection and preparation: Infectious material should be submitted directly to the laboratory within two to three hours of collection. Samples should be protected from excessive heat and cold. If there will be a delay in processing, the sample should be inoculated onto an appropriate transport medium and refrigerated until inoculation. Consult listed references for information on sample collection and preparation.(1-6)

Method of use: Plates should be warmed to room temperature and the agar surface should be dry prior to inoculating. Chapter Nine of the FDA Bacteriological Analytical Manual (BAM) has specific recommendations for the optimal recovery of Vibrio from food and water specimens.(6) When dilutions are necessary, Phosphate Buffered Saline (Cat. nos. K146, K161, or K163) serves as an appropriate diluent. Sample dilutions may be placed into Alkaline Peptone Water (Cat. no. K03) for 16-24 hours at 35°C as a generic enrichment for Vibrio , but the optimal enrichment broth, temperature and incubation time will vary depending upon the sample matrix and the target organism(s). Inoculate a 3mm loopful of enrichment broth onto HardyCHROM™ Vibrio and streak for isolation. Incubate aerobically at 35°C for 18-24 hours and examine for characteristic colonial morphology. If after 24 hours Vibrio spp. are not detected, plates should be discarded.

INTERPRETATION OF RESULTS

Examine plates for isolated colonies showing typical morphology and color.

Vibrio parahaemolyticus produces colonies that are torquoise in color.

Vibrio cholerae produces colonies that are magenta to purple in color and do not fluoresce under long-wave UV light (254mm).

Vibrio vulnificus produces colonies that are magenta in color and do fluoresce under long-wave UV light (254nm).

Vibrio alginolyticus and other Vibrio spp. produce colonies that are colorless to olive in color.

Enterococcus faecalis may grow on HardyCHROM™ Vibrio, but colonies appear sky blue.

LIMITATIONS

Color-blind individuals may encounter difficulty in distinguishing color differences on HardyCHROM™ Vibrio.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media or enrichment broths such as Phosphate Buffered Saline (Cat. nos. K146, K161, or K163) or Alkaline Peptone Water (Cat. no. K03), incinerators, and incubators, etc., as well as UV light and serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Vibrio parahaemolyticus
ATCC® 17802
B 24hr 35°C Aerobic Growth; torquoise colonies
Vibrio vulnificus
ATCC® 27562
B 24hr 35°C Aerobic Growth; magenta colonies, fluoresces under UV light
Vibrio alginolyticus
ATCC® 17749
B 24hr 35°C Aerobic Growth; colorless to olive colonies
Escherichia coli
ATCC® 25922
B 24hr 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL

PHYSICAL APPEARANCE

HardyCHROM™ Vibrio should appear opaque and off-white in color.

Vibrio vulnificus

Vibrio vulnificus (ATCC® 27562) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319) under UV. Incubated aerobically for 24 hours at 35ºC.

Vibrio Vulnificus

Vibrio vulnificus (ATCC® 27562) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319). Incubated aerobically for 24 hours at 35ºC.

Vibrio cholerae

Vibrio cholerae (ATCC® 9459) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319) under UV. Incubated aerobically for 24 hours at 35ºC.

Vibrio cholerae

Vibrio cholerae (ATCC® 9459) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319). Incubated aerobically for 24 hours at 35ºC.

Vibrio parahaemolyticus

Vibrio parahaemolyticus (ATCC® 17802) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319). Incubated aerobically for 24 hours at 35ºC.

Vibrio alginolyticus

Vibrio alginolyticus (ATCC® 17749) colonies growing on HardyCHROM™ Vibrio (Cat. no. G319). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Kaper, J. B., J. G. Morris Jr., and M. M. Levine. 1995. Cholera. Clin. Microbiol. Rev. 8(1):48-86.

2. McLaughlin, J.C. 1995. Vibrio, p 465-476. P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenevor,and R. H. Yolken (eds.), Manual of Clinical Microbiology, 6th ed. ASM Press, Washington, DC.

3. McPherson, V. L., J.A. Watts, L. M. Simpson, and J. D. Oliver. 1991. Physiological effects of the lipopolysaccharide of Vibrio vulnificus on mice and rats. Microbios. 67:141-149.

4. Oliver, J. D. 1989. Vibrio vulnificus, p 569-600. M.P. Doyle (ed.), Foodborne Bacterial Pathogens. Marcel Dekker, Inc., New York, New York.

5. Sakazaki, R., S. Iwanami, and H. Fukumi.1963. Studies on the enteropathogenic, facultatively halophilic bacteria, Vibrio parahaemolyticus. I. Morphological, cultural and biochemical properties and its taxonomic position. Jpn. J. Med. Sci. Biol. 16:161-188.

6. Rippey, S. R. 1994. Infectious diseases associated with molluscan shellfish consumption. Clin. Microbiol. Rev. 7:419-425.

7. Sack, D. A, R. B Sack, G. B. Nair, and A. K. Siddique. 2004. Cholera. Lancet. 363:223-233.

8. Klonts, K. C., L. Williams, L. M. Baldy, and M. Campos.1993. Raw oyster-associated Vibrio infections: Linking epidemiologic data with laboratory testing of oysters obtained from a retail outlet. J. Food Protect. 56:977-979.

9. Tacket, C. O., F. Brenner, and P. A. Blake. 1984. Clinical features and an epidemiological study of Vibrio vulnificus infections. J. Infect. Dis. 149:558-561.

10. U.S. Food and Drug Administration. Bacteriological Analytical Manual. Arlington, VA. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm

11. Ajello, et al. 1963. CDC Laboratory Manual for Medical Mycology, PHS Publication No. 994, U.S. Government Printing Office, Washington, D.C.

12. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

13. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology. C.V. Mosby Company, St. Louis, MO.

ATCC is a registered trademark of the American Type Culture Collection.

IFU-10469[A]