Laboratory Maintenance of Pathogenic Leptospira

Richard L. Zuerner1

1 National Animal Disease Center USDA, ARS, MWA, Ames, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12E.1
DOI:  10.1002/9780471729259.mc12e01s00
Online Posting Date:  October, 2005
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Abstract

Analysis of Leptospira requires use of specialized media for growth, maintenance, and storage of viable bacteria that can be used in experimental protocols in a predictable manner. However, pathogenic Leptospira are fastidious bacteria with unusual nutritional requirements. These problems make primary isolation, routine propagation, and storage of Leptospira difficult. Defined and complex media are available for routine growth of pathogenic Leptospira, and each medium has unique characteristics that favor the growth or isolation of some strains, while not supporting the growth of others. This unit will describe the preparation of Leptospira media and methods for isolation and storage of these bacteria.

Keywords: Leptospira; leptospirosis; spirochete; growth media; isolation

     
 
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Table of Contents

  • Strategic Planning
  • Growth of Pathogenic Leptospira
  • Basic Protocol 1: Growth in Semisolid Media
  • Basic Protocol 2: Growth on Solid Media
  • Basic Protocol 3: Growth in Liquid Media
  • Storage and Recovery of Pathogenic Leptospira
  • Basic Protocol 4: Storage of Pathogenic Leptospira
  • Basic Protocol 5: Recovery of Frozen Cultures
  • Isolation of Pathogenic Leptospira
  • Basic Protocol 6: Isolation of Pathogenic Leptospira from Urine
  • Basic Protocol 7: Isolation of Pathogenic Leptospira from Tissues
  • Basic Protocol 8: Fluorescent Antibody Staining of Leptospira
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth in Semisolid Media

  Materials
  • Starter cultures of Leptospira
  • Semisolid Leptospira medium (see recipe)
  • Liquid medium (see recipes for EMJH medium, modified Stuart medium, or T80/40/LH medium)
  • 29° to 30°C incubator
  • 50‐ml culture tubes with loosely fitting caps

Basic Protocol 2: Growth on Solid Media

  Materials
  • Fresh culture
  • Leptospira storage medium (see recipe)
  • Liquid nitrogen
  • 2‐ml cryogenic vials, sterile
  • Styrofoam rack

Basic Protocol 3: Growth in Liquid Media

  Materials
  • Frozen cultures in cryogenic vials
  • Semisolid Leptospira medium (see recipe) in 16 × 25–mm tubes
  • Capped glass specimen jar

Basic Protocol 4: Storage of Pathogenic Leptospira

  Materials
  • Urine sample
  • Leptospira storage medium (see recipe)
  • Semisolid Leptospira culture medium (see recipe)
  • 16 × 125–mm capped tubes
  • 29°C incubator
  • Dark‐field microscope

Basic Protocol 5: Recovery of Frozen Cultures

  Materials
  • Tissue sample
  • Leptospira storage medium (see recipe)
  • Culture medium
  • Whirlpak bag
  • Tissue homogenizer (e.g., Stomacher 400, Seward Medical)
  • 29°C incubator
  • Dark‐field microscope

Basic Protocol 6: Isolation of Pathogenic Leptospira from Urine

  Materials
  • Urine sample (see protocol 6) or tissue homogenate (see protocol 7)
  • PBS ( appendix 2A)
  • Acetone
  • Rabbit anti‐Leptospira antisera conjugated with fluorescent tag (available from Mark Wilson, NVSL, Ames, IA 50010, 515‐663‐7595)
  • Flazo Orange counter stain (diluted 1:10 to 1:20 with PBS)
  • Buffered glycerol
  • 2‐ml microcentrifuge tubes
  • Microscope slides
  • Moist chamber: petri dish with lid, containing a circle of filter paper moistened with water
  • 37°C incubator
  • Coverslips
  • Fluorescent microscope
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Figures

Videos

Literature Cited

   Baseman, J.B. and Cox, C.D. 1972. Intermediate energy metabolism of Leptospira. J. Bacteriol. 97:992‐1000.
   Brenner, D.J., Kaufmann, A.F., Sulzer, K.R., Steigerwalt, A.G., Rogers, F.C., and Weyant, R.S. 1999. Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int. J. Sys. Bacteriol. 49:839‐858.
   Ellinghausen, H.C. and McCullough, W.G. 1965. Nutrition of Leptospira pomona and growth of 13 other serotypes: Fractionation of oleic albumin complex and medium bovine albumin and polysorbate 80. Am. J. Vet. Res. 26:45‐51.
   Ellis, W.A. and Thiermann, A.B. 1986. Isolation of Leptospira interrogans serovar bratislava from sows in Iowa. Am. J. Vet. Res. 47:1458‐1460.
   Faine, S. 1994. Leptospira and Leptospirosis. CRC Press, Boca Raton, Florida.
   Faine, S., Adler, B., Bolin, C., and Perolat, P. 1999. Leptospira and Leptospirosis, 2nd ed. MediSci, Melbourne, Australia.
   Johnson, R.C. and Harris, V.G. 1967. Differentiation of pathogenic and saprophytic leptospires. I. Growth at low temperatures. J. Bacteriol. 94:27‐31.
   Johnson, R.C. and Rogers, P. 1964. 5‐Fluorouracil as a selective agent for the growth of Leptospirae. J. Bacteriol. 87:422‐426.
   Lawrence, J.J. 1951. The growth of Leptospirae in semisolid media. Aust. J. Exp. Biol. Med. Sci. 29:195‐199.
   Levett, P.N. 2001. Leptospirosis. Clin. Microbiol. Rev. 14:296‐326.
   Stuart, R.D. 1946. The preparation and use of a simple culture medium for Leptospirae. J. Pathol. 58:343‐349.
   World Health Organization. 1999. Leptospirosis worldwide, 1999. Weekly Epidemiological Report 74:237‐244.
Key References
   Faine, S. 1994. See above.
  These two books provide comprehensive coverage of the organism, metabolism, and the disease.
   Faine et al. 1999. See above.
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