Laboratory Maintenance of Flavobacterium psychrophilum and Flavobacterium columnare

Kenneth D. Cain1, Benjamin R. LaFrentz1

1 University of Idaho, Moscow, Idaho
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 13B.1
DOI:  10.1002/9780471729259.mc13b01s6
Online Posting Date:  August, 2007
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Bacteria in the genus Flavobacterium are highly heterogeneous and are comprised of pathogenic and non‐pathogenic species. They are found in a wide variety of environments that include fresh and marine water, soil, and ocean sediments. This unit focuses on laboratory maintenance of two important fish pathogens within this genus. Flavobacterium psychrophilum and F. columnare are fastidious, slow‐growing organisms that require specialized medium for optimal growth in the laboratory. Methods are described for the preparation of media, broth and agar culture, cryopreservation, and enumeration of viable cells for these bacteria. In addition, the diseases caused by these pathogens are described along with methods of diagnosis and treatments. This unit serves as a general guide for the cultivation of F. psychrophilum and F. columnare and discusses some of the difficulties that have been encountered for each. Curr. Protoc. Microbiol. 6:13B.1.1‐13B.1.12. © 2007 by John Wiley & Sons, Inc.

Keywords: Flavobacterium; bacterial culture; pathogen; soil bacteria; aquatic bacteria

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Broth Culture of Flavobacterium psychrophilum and Flavobacterium columnare
  • Basic Protocol 2: Cryopreservation of Flavobacterium
  • Basic Protocol 3: Enumeration of Viable Flavobacterium Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Broth Culture of Flavobacterium psychrophilum and Flavobacterium columnare

  • Lyophilized or frozen stock of F. psychrophilum or F. columnare (see protocol 2)
  • Sterile KPBS ( appendix 2A)
  • TYES agar plates and broth (or alternative medium broth and plates; see recipe for TYES agar and broth)
  • Sterile inoculation loops
  • Sterile 1‐ml syringes and needles
  • Parafilm
  • Low‐temperature incubator (15° to 25°C)
  • 15‐ml tubes
  • Additional reagents and equipment for Gram staining ( appendix 3C) and light microscopy (unit 2.1)

Basic Protocol 2: Cryopreservation of Flavobacterium

  • Sterile glycerol stock (see recipe)
  • Log‐phase growth of Flavobacterium (see protocol 1)
  • Sterile 15‐ml polypropylene tubes
  • 1.0‐ml cryovials
  • Vortex
  • Cryovial storage box

Basic Protocol 3: Enumeration of Viable Flavobacterium Cells

  • Petri plates with TYES or alternative medium agar (see recipe)
  • Sterile KPBS ( appendix 2A)
  • Flavobacterium culture (see protocol 1)
  • 95% ethanol
  • Sterile culture tubes (13 × 100–mm)
  • Cell spreader
  • Parafilm
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Literature Cited

   AFS‐FHS (American Fisheries Society‐Fish Health Section). 2006. FHS Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens, 2006 Edition. AFS‐FHS, Bethesda, Maryland.
   Anacker, R.L. and Ordal, E.J. 1955. Study of a bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 70:738‐741.
   Bernardet, J.‐F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K., and Vandamme, P. 1996. Cutting a gordian knot: Emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. Nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978). Int. J. Syst. Evol. Microbiol. 46:128‐148.
   Bullock, G.L., Hsu, T.C., and Shotts, E.B. 1986. Columnaris disease of fishes. U.S. Fish and Wildlife Service, Fish Disease Leaflet 72. National Fisheries Research Center‐Leetown, National Fish Health Research Laboratory, Kearneysville, West Virginia.
   Cepeda, C., García‐Márquez, S., and Santos, Y. 2004. Improved growth of Flavobacterium psychrophilum using a new culture medium. Aquaculture 238:75‐82.
   Crump, E.M., Perry, M.B., Clouthier, S.C., and Kay, W.W. 2001. Antigenic characterization of the fish pathogen Flavobacterium psychrophilum. Appl. Environ. Microbiol. 67:750‐759.
   Daskalov, H., Austin, D.A., and Austin, B. 1999. An improved growth medium for Flavobacterium psychrophilum. Lett. Appl. Microbiol. 28:297‐299.
   Desolme, B. and Bernardet, J.‐F. 1996. Freeze‐drying of Flavobacterium columnare, Flavobacterium psychrophilum and Flexibacter maritimus. Dis. Aquat. Org. 27:77‐80.
   Evenson, O. and Lorenzen, E. 1996. An immunohistochemical study of Flexibacter psychrophilus infection in experimentally and naturally infected rainbow trout (Oncorhynchus mykiss) fry. Dis. Aquat. Org. 25:53‐61.
   Holt, R.A., Rohovec, J.S., and Fryer, J.L. 1993. Bacterial coldwater disease. In Bacterial diseases of fish (V. Inglis, R.J. Roberts, and N.R. Bromage, eds.) pp. 3‐23. Blackwell Scientific Publications, Oxford.
   Jooste, P.J. and Hugo, C.J. 1999. The taxonomy, ecology and cultivation of bacterial genera belonging to the family Flavobacteriaceae. Int. J. Food Microbiol. 53:81‐94.
   Lorenzen, E. 1993. The importance of the brand of the beef extract in relation to the growth of Flexibacter psychrophilus in Anacker & Ordal medium. Bull. Eur. Assoc. Fish Pathol. 13:64‐65.
   Michel, C., Antonio, D., and Hedrick, R.P. 1999. Production of viable cultures of Flavobacterium psychrophilum: Approach and control. Res. Microbiol. 150:351‐358.
   Michel, C. and Garcia, C. 2003. Virulence stability in Flavobacterium psychrophilum after storage and preservation according to different procedures. Vet. Res. 34:127‐132.
   Plumb, J.A. 1999. Health Maintenance and Principal Microbial Diseases of Cultured Fishes. Iowa State University Press, Ames, Iowa.
   Schmidtke, L.M. and Carson, J. 1995. Characteristics of Flexibacter psychrophilus isolated from Atlantic salmon in Australia. Dis. Aquat. Org. 21:157‐161.
   Shieh, H.S. 1980. Studies on the nutrition of a fish pathogen, Flexibacter columnaris. Microbios Lett. 13:129‐133.
   Song, Y.L., Fryer, J.L., and Rohovec, J.S. 1988. Comparison of six media for the cultivation of Flexibacter columnaris. Fish Pathol. 23:91‐94.
   Taylor, P.W. and Winton, J.R. 2002. Optimization of nested polymerase chain reaction assays for identification of Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum. J. Aquat. Anim. Health 14:216‐224.
   Thune, R.A. 1993. Pathogenesis of Gram‐negative bacterial infections in warmwater fish. In Annual Reviews of Fish Diseases (M. Fasial and F.M. Hetrick, eds.) pp. 37‐68. Elsevier Science, New York.
   Vastos, I.N., Thompson, K.D., and Adams, A. 2003. Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water. Dis. Aquat. Org. 56:115‐126.
   Wood, E.M. and Yasutake, W.T. 1956. Histopathology of fish III. Peduncle (‘coldwater’) disease. Prog. Fish Cult. 18:58‐61.
Internet Resources
  Website link to an extension bulletin of the Southern Regional Aquaculture Center (SRAC) on columnaris disease.
  Website link to an extension bulletin of the Western Regional Aquaculture Center (WRAC) on coldwater disease.
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