Chapter 2 : Isolation, Enumeration, and Preservation of the

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One or a combination of the bacteriocidal agents is often present in most of the selective media commonly used for the selective isolation of vibrios. A scheme for the isolation of pathogenic vibrios often includes enrichment prior to plating on a selective agar, especially when vibrios are at low levels and competing with other bacteria that can outgrow them in general media. Several enrichment media have been used for the selection of one group or species of . Special effort has been devoted to enhance the presence of potential pathogenic species in contaminated food or in clinical samples. Thiosulfate citrate bile salt sucrose agar (TCBS) is an ideal medium for the selective isolation and purification of vibrios. Although this medium was originally designed for the isolation of and , most vibrios grow to healthy large colonies with many different colonial morphologies. Until now, TCBS was the best selective medium for isolation of vibrios, though some strains of , , , , , and may present slight growth on it. TCBS agar is also an excellent medium for the selective isolation of vibrios from environmental sources, but differences in media formulation between manufacturers might be reflected in the enumeration of vibrios. Enumeration molecular techniques include the use of polymerase chain reaction (PCR) analysis or DNA probes for colony hybridization. Often, vibrios recovered directly from cryopreservation require at least two passes to fully recuperate and longer incubation periods.

Citation: Gomez-Gil B, Roque A. 2006. Isolation, Enumeration, and Preservation of the , p 15-26. In Thompson F, Austin B, Swings J (ed), The Biology of Vibrios. ASM Press, Washington, DC. doi: 10.1128/9781555815714.ch2
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1. Adzhieva, A. A. 2000. A dried nutrient medium for the isolation of Vibrio cholerae. Zh. Mikrobiol. Epidemiol. Immunobiol. (Mar-Apr): 2931.
2. Alsina, M., and, A. R. Blanch. 1994. A set of keys for biochemical identification of environmental Vibrio species. J. Appl. Bacteriol. 76: 7985.
3. Alsina, M.,, J. Martinez-Picado,, J. Jofre, and, A. R. Blanch. 1994. A medium for presumptive identification of Vibrio anguillarum. Appl. Environ. Microbiol. 60: 16811683.
4. Alvarado-Aleman, F. J.,, J. Kumate-Rodríguez,, J. Sepulveda-Amor, and, C. Wong-Arámbula. 1994. Identification of Vibrio cholera O1 by flow cytometry. Rev. Latinoamer. Microbiol. 36: 283293.
5. Alvarez-Barrientos, A.,, J. Arroyo,, R. Canton,, C. Nombela, and, M. Sanchez-Perez. 2000. Applications of flow cytometry to clinical microbiology. Clin. Microbiol. Rev. 13: 167195.
6. Azam, F. 2001. Introduction, history, and overview: the methods to our madness. Methods Microbiol. 30: 112.
7. Baumann, P., and, R. H. W. Schubert. 1983. Vibrionaceae, p. 516550. In N. R. Krieg and, J. G. Holt (ed.), Bergey’s Manual of Systematic Bacteriology. Lippincott Williams & Wilkins, Baltimore, Md.
8. Brayton, P. R.,, P. A. West,, E. Russek, and, R. R. Colwell. 1983. New selective plating medium for isolation of Vibrio vulnificus biogroup 1. J. Clin. Microbiol. 17: 10391044.
9. Campbell, M. S., and, A. C. Wright. 2003. Real-time PCR analysis of Vibrio vulnificus from oysters. Appl. Environ. Microbiol. 69: 71377144.
10. Carlucci, A. F., and, D. Pramer. 1957. Factors influencing the plate method for determining abundance of bacteria in sea water. Proc. Soc. Exp. Biol. Med. 96: 392394.
11. Carruthers, M. M., and, W. J. Kabat. 1976. Isolation of Vibrio parahaemolyticus from fecal specimens on mannitol salt agar. J. Clin. Microbiol. 4: 175179.
12. Cerda-Cuellar, M.,, J. Jofre, and, A. R. Blanch. 2000. A selective medium and a specific probe for detection of Vibrio vulnificus. Appl. Environ. Microbiol. 66: 855859.
13. Cerda-Cuellar, M.,, L. Permin,, J. L. Larsen, and, A. R. Blanch. 2001. Comparison of selective media for the detection of Vibrio vulnificus in environmental samples. J. Appl. Microbiol. 91: 322327.
14. Chatterjee, B. D.,, P. K. De, and, T. Sen. 1977. Sucrose teepol tellu-rite agar: a new selective indicator medium for isolation of Vibrio species. J. Infect. Dis. 135: 654658.
15. Chun, D.,, J. K. Chung, and, S. Y. Seol. 1974. Enrichment of Vibrio parahaemolyticus in a simple medium. Appl. Microbiol. 27: 11241126.
16. Colodner, R.,, R. Raz,, I. Meir,, T. Lazarovich,, L. Lerner,, J. Kopelowitz,, Y. Keness,, W. Sakran,, S. Ken-Dror, and, N. Bisharat. 2004. Identification of the emerging pathogen Vibrio vulnificus biotype 3 by commercially available phenotypic methods. J. Clin. Microbiol. 42: 41374140.
17. De, S. P.,, D. Sen,, P. C. De,, A. Ghosh, and, S. C. Pal. 1977. A simple selective medium for isolation of vibrios with particular reference to Vibrio parahaemolyticus. Indian J. Med. Res. 66: 398399.
18. DePaola, A., and, C. A. Kaysner. 2004. Vibrio. [Online.] In Bacteriological Analytical Manual, Food and Drug Administration, Rockville, Md. http://www.cfsan.fda.gov/~ebam/bam-9.html.
19. DePaola, A.,, C. A. Kaysner,, J. Bowers, and, D. W. Cook. 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl. Environ. Microbiol. 66: 46494654.
20. DePaola, A.,, J. L. Nordstrom,, J. C. Bowers,, J. G. Wells, and, D. W. Cook. 2003. Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters. Appl. Environ. Microbiol. 69: 15211526.
21. Donovan, T. J., and, P. van Netten. 1995. Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples. Int. J. Food Microbiol. 26: 7791.
22. Eilers, H.,, J. Pernthaler,, F. O. Glockner, and, R. Amann. 2000. Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl. Environ. Microbiol. 66: 30443051.
23. Entis, P., and, P. Boleszczuk. 1983. Overnight enumeration of Vibrio parahaemolyticus in seafood by hydrophobic grid membrane filtration. J. Food Prot. 46: 783786.
24. Farmer, J. J.,, J. M. Janda, and, K. Birkhead. 2003. Vibrio, p. 706718. In P. R. Murray (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C.
25. Fischer, M.,, D. Hervio,, S. C. R. Loaec, and, M. Pommepuy. 2002. Detection of cytotoxin-hemolysin MRNA in non-culturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater. Appl. Environ. Microbiol. 68: 56415646.
26. Furniss, A. L.,, J. V. Lee, and, T. J. Donovan. 1978. The Vibrios. Monograph series. Her Majesty’s Stationery Office, London, England.
27. Gerhardt, P.,, R. G. E. Murray,, W. A. Wood, and, N. R. Krieg (ed.). 1994. Methods for General and Molecular Bacteriology. ASM Press, Washington, D.C.
28. Gherna, L. R. 1994. Culture preservation, p. 278292. In P. Gerhardt,, R. G. E. Murray,, W. A. Wood, and, N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. ASM Press, Washington, D.C.
29. Gilmour, A.,, M. C. Allan, and, M. F. McCallum. 1976. The un-suitability of high pH media for the selection of marine Vibrio species. Aquaculture 7: 8187.
30. Gomez-Gil, B.,, S. Soto-Rodriguez,, A. Garcia-Gasca,, A. Roque,, R. Vazquez-Juarez,, F. L. Thompson, and, J. Swings. 2004a. Molecular characterization of V. harveyi related isolates associated with diseased aquatic organisms. Microbiology 150: 17691777.
31. Gomez-Gil, B.,, F. L. Thompson,, C. C. Thompson,, A. Garcia-Gasca,, A. Roque, and, J. Swings. 2004b. Vibrio hispanicus sp. nov., isolated from Artemia sp. and sea water in Spain. Int. J. Syst. Evol. Microbiol. 54: 261265.
32. Gomez-Gil, B.,, L. Tron-Mayen,, A. Roque,, J. F. Turnbull,, V. Inglis, and, A. L. Guerra-Flores. 1998. Species of Vibrio isolated from hepatopancreas, haemolymph and digestive tract of a population of healthy juvenile Penaeus vannamei. Aquaculture 163: 19.
33. Hara-Kudo, Y.,, Y. Kasuga,, A. Kiuchi,, T. Horisaka,, T. Kawasumi, and, S. Kumagai. 2003. Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA. J. Food Prot. 66: 16751680.
34. Hara-Kudo, Y.,, T. Nishina,, H. Nakagawa,, H. Konuma,, J. Hase-gawa, and, S. Kumagai. 2001. Improved method for detection of Vibrio parahaemolyticus in seafood. Appl. Environ. Microbiol. 67: 58195823.
35. Harris, L.,, L. Owens, and, S. Smith. 1996. A selective and differential medium for Vibrio harveyi. Appl. Environ. Microbiol. 62: 35483550.
36. Harwood, V. J.,, J. P. Ghandi, and, A. C. Wright. 2004. Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review. J. Microbiol. Methods 62: 35483550.
37. Høi, L.,, I. Dalsgaard, and, A. Dalsgaard. 1998. Improved isolation of Vibrio vulnificus from seawater and sediment with cellobiose-colistin agar. Appl. Environ. Microbiol. 64: 17211724.
38. Holt, J. G., and, N. R. Krieg. 1994. Enrichment and isolation, p. 179215. In P. Gerhardt,, R. G. E. Murray,, W. A. Wood, and, N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. ASM Press, Washington, D.C.
39. Honda, T.,, C. Sornchai,, Y. Takeda, and, T. Miwatani. 1982. Immunological detection of the Kanagawa phenomenon of Vibrio parahaemolyticus on modified selective media. J. Clin. Microbiol. 16: 734736.
40. Hsu, W. Y.,, C. I. Wei, and, M. L. Tamplin. 1998. Enhanced broth media for selective growth of Vibrio vulnificus. Appl. Environ. Microbiol. 64: 27012704.
41. Jameson, J. E., and, N. W. Emberley. 1958. Teepol in substitution for bile salts. J. Gen. Microbiol. 18: 1238.
42. Jannasch, H. W., and, G. E. Jones. 1959. Bacterial populations in sea water as determined by different methods of enumeration. Limnol. Oceanog. 4: 128139.
43. Kauffman, G. E.,, G. M. Blackstone,, M. C. Vickery,, A. K. Bej,, J. Bowers,, M. D. Bowen, and, R. F. Meyer. 2004. Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix. J. Food Prot. 67: 24242429.
44. Kitaura, T.,, S. Doke,, I. Azuma,, M. Imaida,, K. Miyano,, K. Harada, and, E. Yabuuchi. 1983. Halo production by sulfatase activity of V. vulnificus and V. cholerae O1 on a new selective sodium do-decyl sulfate-containing agar medium: a screening marker in environmental surveillance. FEMS Microbiol. Lett. 17: 205209. [Online.] http://www.sciencedirect.com/.
45. Kobayashi, T.,, S. Enomoto,, R. Sakazaki, and, S. Kuwahara. 1963. A new selective isolation medium for the Vibrio group; on a modified Nakanishi’s medium (TCBS agar medium). Nippon Saikingaku Zasshi 18: 387392.
46. Kourany, M. 1983. Medium for isolation and differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus. Appl. Environ. Microbiol. 45: 310312.
47. Massad, G., and, J. D. Oliver. 1987. New selective and differential medium for Vibrio cholerae and Vibrio vulnificus. Appl. Environ. Microbiol. 53: 22622264.
48. Miceli, G. A.,, W. D. Watkins, and, S. R. Rippey. 1993. Direct plating procedure for enumerating Vibrio vulnificus in oysters ( Cras-sostrea virginica). Appl. Environ. Microbiol. 59: 35193524.
49. Miyamoto, Y.,, T. Kato,, Y. Obara,, S. Akiyama,, K. Takizawa, and, S. Yamai. 1969. In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bacteriol. 100: 11471149.
50. Monsur, K. A. 1961. A highly selective gelatin-taurocholate-tellu-rite medium for the isolation of Vibrio cholerae. Trans. R. Soc. Trop. Med. Hyg. 55: 440442.
51. Monsur, K. A. 1963. Bacteriological diagnosis of cholera under field conditions. WHO Bull. 28: 387389.
52. Muniesa-Perez, M.,, J. Jofre, and, A. R. Blanch. 1996. Identification of Vibrio proteolyticus with a differential medium and a specific probe. Appl. Environ. Microbiol. 62: 26732675.
53. Nakanishi, H., and, M. Murase. 1973. Enumeration of Vibrio parahaemolyticus in raw fish meat, p. 117121. In International Symposium on Vibrio parahaemolyticus. Tokyo, Japan.
54. Nakanishi, Y. 1963. An isolation agar medium for cholerae and enteropathogenic halophilic vibrios. Modern Media 9: 246.
55. Nealson, K. H., and, J. W. Hastings. 1979. Bacterial bioluminescence: its control and ecological significance. Microbiol. Rev. 43: 496518.
56. Nicholls, K. M.,, J. V. Lee, and, T. J. Donovan. 1976. An evaluation of commercial thiosulphate citrate bile salt sucrose agar (TCBS). J. Appl. Bacteriol. 41: 265269.
57. Nishibuchi, M.,, N. C. Roberts,, H. B. Bradford, Jr., and, R. J. Seidler. 1983. Broth medium for enrichment of Vibrio fluvialis from the environment. Appl. Environ. Microbiol. 46: 425429.
58. Nishibuchi, M., and, R. J. Seidler. 1985. Rapid microimmunodiffusion method with species-specific antiserum raised to purified antigen for identification of Vibrio vulnificus. J. Clin. Microbiol 21: 102107.
59. O’Brien, M., and, R. Colwell. 1985. Modified taurocholate-tellu-rite-gelatin agar for improved differentiation of Vibrio species. J. Clin. Microbiol. 22: 10111013.
60. Oevreas, L.,, D. Bourne,, R. A. Sandaa,, E. O. Casamayor,, S. Benlloch,, V. Goddard,, G. Smerdon,, M. Heldal, and, T. F. Thingstad. 2003. Response of bacterial and viral communities to nutrient manipulations in seawater mesocosms. Aquat. Microb. Ecol. 31: 109121.
61. Ozsan, K., and, F. Mercangoz. 1980. New media for the isolation of Vibrio cholerae. Zentbl. Bakteriol A 247: 7173.
62. Pal, S. C.,, G. V. Murty,, C. G. Pandit,, D. K. Murty, and, J. B. Shrivastav. 1967. A comparative study of enrichment media in the bacteriological diagnosis of cholera. Indian J. Med. Res. 55: 318324.
63. Panicker, G.,, M. L. Myers, and, A. K. Bej. 2004. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by realtime PCR. Appl. Environ. Microbiol. 70: 498507.
64. Pfeffer, C. S.,, M. F. Hite, and, J. D. Oliver. 2003. Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina. Appl. Environ. Microbiol. 69: 35263531.
65. Reichelt, J. L., and, P. Baumann. 1974. Effect of sodium chloride on growth of heterotrophic marine bacteria. Arch. Microbiol. 97: 329345.
66. Sakazaki, R. 1965. Vibrio parahaemolyticus, Isolation and Identification. Nihon Eiyo Kagaku Co. Press, Tokyo, Japan.
67. Sakazaki, R. 1972. BTB Teepol Agar in Media for Bacteriological Examination. Kindai Igaku Co., Tokyo, Japan.
68. Salles, C. A.,, S. Voros,, E. C. Marbell, and, L. Amenuvor. 1976. Colony morphology of Vibrio cholerae on SV medium. J. Appl. Bacteriol. 40: 213216.
69. Sanjuan, E., and, C. Amaro. 2004. Protocol for specific isolation of virulent strains of Vibrio vulnificus serovar E (biotype 2) from environmental samples. Appl. Environ. Microbiol. 70: 70247032.
70. Sawabe, T.,, Y. Oda,, Y. Shiomi, and, Y. Ezura. 1995. Alginate degradation by bacteria isolated from the gut of sea urchins and abalones. Microb. Ecol. 30: 192202.
71. Schneider, J., and, G. Rheinheimer. 1988. Isolation methods, p. 7394. In B. Austin (ed.), Methods in Aquatic Bacteriology. John Wiley & Sons, Chichester, England.
72. Scott, J., and, R. L. Thune. 1986. Bacterial flora of hemolymph from red swamp crawfish, Procambarus clarkii (Girard), from commercial ponds. Aquaculture 58: 161165.
73. Shimada, T.,, R. Sakazaki,, S. Fujimura,, K. Niwano,, M. Mishina, and, K. Takizawa. 1990. A new selective, differential agar medium for isolation of Vibrio cholerae O1: PMT (polymyxin-mannose-tellurite) agar. Jpn. J. Med. Sci. Biol. 43: 3741.
74. Sogaard, H. 1982. The pharmacodynamics of polymyxin antibiotics with special reference to drug resistance liability. J. Vet. Pharmacol. Ther. 5: 219231.
75. Spira, W. M. 1984. Tactics for detecting pathogenic vibrios in the environment, p. 251268. In R. R. Colwell (ed.), Vibrios in the Environment. John Wiley & Sons, New York, N.Y.
76. Takahashi, H.,, Y. Hara-Kudo,, J. Miyasaka,, S. Kumagai, and, H. Konuma. 2005. Development of a quantitative real-time poly-merase chain reaction targeted to the toxR for detection of Vibrio vulnificus. J. Microbiol. Methods 61: 7785.
77. Tamplin, M. L.,, A. L. Martin,, A. D. Ruple,, D. W. Cook, and, C. W. Kaspar. 1991. Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters. Appl. Environ. Microbiol. 57: 12351240.
78. Tamura, K.,, S. Shimada, and, L. M. Prescott. 1971. Vibrio agar: a new plating medium for isolation of Vibrio cholerae. Jpn. J. Med. Sci. Biol. 24: 125127.
79. Thompson, F. L.,, T. Iida, and, J. Swings. 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev. 68: 403431.
80. Vandenberghe, J.,, F. L. Thompson,, B. Gomez-Gil, and, J. Swings. 2003. Phenotypic diversity amongst Vibrio isolates from marine aquaculture systems. Aquaculture 219: 920.
81. Vandenberghe, J.,, L. Verdonck,, A. R. Robles,, G. Rivera,, A. Bolland,, M. Balladares,, B. Gomez-Gil,, J. Calderon,, P. Sorgeloos, and, J. Swings. 1999. Vibrios associated with Litopenaeus vannamei larvae, postlarvae, broodstock, and hatchery probionts. Appl. Environ. Microbiol. 65: 25922597.
82. Vanderzant, C., and, R. Nickelson. 1972. Procedure for isolation and enumeration of Vibrio parahaemolyticus. Appl. Microbiol. 23: 2633.
83. Vuddhakul, V.,, T. Nakai,, C. Matsumoto,, T. Oh,, T. Nishino,, C. H. Chen,, M. Nishibuchi, and, J. Okuda. 2000. Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification. Appl. Environ. Microbiol. 66: 35063514.
84. West, P. A., and, R. R. Colwell. 1984. Identification and classification of Vibrionaceae—an overview, p. 285363. In R. R. Colwell (ed.), Vibrios in the Environment. John Wiley & Sons, New York, N.Y.
85. West, P. A.,, E. Russek,, P. R. Brayton, and, R. R. Colwell. 1982. Statistical evaluation of a quality control method for isolation of pathogenic Vibrio species on selected thiosulfate-citrate-bile salts-sucrose agars. J. Clin. Microbiol. 16: 11101116.
86. Whitman, K. A. 2004. Finfish and Shellfish Bacteriology Manual. Techniques and Procedures. Iowa State Press, Blackwell Publishing Company, Ames, Iowa.
87. Wright, A. C.,, G. A. Miceli,, W. L. Landry,, J. B. Christy,, W. D. Watkins, and, J. G. Morris, Jr. 1993. Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe. Appl. Environ. Microbiol. 59: 541546.
88. Zobell, C. E. 1941. Studies on marine bacteria. 1. The cultural requirements of heterotrophic aerobes. J. Mar. Res. 4: 4275.


Generic image for table

Media used for growth of medically important vibrios (adapted from Donovan and van Netten, 1995)

Citation: Gomez-Gil B, Roque A. 2006. Isolation, Enumeration, and Preservation of the , p 15-26. In Thompson F, Austin B, Swings J (ed), The Biology of Vibrios. ASM Press, Washington, DC. doi: 10.1128/9781555815714.ch2

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