Laboratory Maintenance of Mycobacterium marinum

Lian‐Yong Gao1, Joanna Manoranjan1

1 University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10B.1
DOI:  10.1002/9780471729259.mc10b01s00
Online Posting Date:  July, 2005
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Abstract

M. marinum naturally infects fish and amphibians and causes diseases in these animals with pathological features similar to the human disease caused by M. tuberculosis. At the genetic and biochemical levels, M. marinum is closely related to M. tuberculosis. Because of these and other properties of M. marinum (such as its fast growth rate and convenient laboratory handing on the benchtop), M. marinum has been increasingly used as a model for studying M. tuberculosis pathogenesis. The protocols in this unit describe the methods for laboratory culturing (in liquid and solid media) and maintenance (subculturing, short‐ and long‐term storage) of M. marinum and the methods for processing M. marinum for infection assays. Important parameters for culturing and maintaining M. marinum and its processing for infection assays are discussed in detail.

Keywords: Mycobacterium marinum; laboratory culture; storage; maintenance; biosafety; infection assay

     
 
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Table of Contents

  • Basic Protocol 1: Culturing M. marinum in Liquid Medium
  • Basic Protocol 2: Culturing M. marinum on Solid Medium
  • Basic Protocol 3: Processing M. marinum for Infection Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Culturing M. marinum in Liquid Medium

  Materials
  • 7H9 liquid culture medium (see recipe) or Sauton's liquid culture medium (see recipe)
  • M. marinum: ATCC #BAA‐535 (human isolate) or ATCC #927 (fish isolate)
  • M. marinum freezing medium (see recipe)
  • Liquid nitrogen tank with canes to accommodate cryovials
  • Culture containers: sterile 250‐ml flask or bottle (glass or plastic)
  • 30° to 32°C incubator with orbital shaker
  • Spectrophotometer capable of measurements at 600 nm
  • 50‐ml conical centrifuge tubes
  • Cryovials

Basic Protocol 2: Culturing M. marinum on Solid Medium

  Materials
  • M. marinum: ATCC #BAA‐535 (human isolate) or ATCC #927 (fish isolate) growing in liquid culture (see protocol 1)
  • 100‐mm 7H10 agar plates (see recipe)
  • Ethanol
  • 15‐ and 50‐ml conical centrifuge tubes
  • 10‐ml syringes with 26‐G needles
  • Tabletop centrifuge
  • Hemacytometer
  • Microscope with 40× objective
  • 30° to 32°C incubator
  • Spreader
  • Sealable plastic bags to accommodate petri dishes (Fisher)
  • Inoculating loop
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, )

Basic Protocol 3: Processing M. marinum for Infection Assays

  Materials
  • M. marinum: ATCC #BAA‐535 (human isolate) or ATCC #927 (fish isolate) growing in liquid culture (see protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 15‐ and 50‐ml conical centrifuge tubes
  • 10‐ml syringes with 26‐G needles
  • Tabletop centrifuge
  • Hemacytometer
  • Microscope with 40× objective
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Figures

Videos

Literature Cited

   Andersen P, Askgaard D, Ljungqvist L, Bennedsen J, Heron I. 1991. Proteins released from Mycobacterium tuberculosis during growth. Infect. Immun. 59:1905‐1910.
   Clark, H. F. and Shepard, C. C. 1963. Effect of environmental temperatures on infection with Mycobacterium marinum (Balnei) of mice and a number of poikilothermic species. J. Bacteriol. 86:1057‐1069.
   Gao, L. Y., Groger, R., Cox, J. S., Beverley, S. M., Lawson, E. H., and Brown, E. J. 2003. Transposon mutagenesis of Mycobacterium marinum identifies a locus linking pigmentation and intracellular survival. Infect. Immun. 71:922‐929.
   Gao, L. Y., Guo, S., McLaughlin, B., Morisaki, H., Engel, J. N., and Brown, E. J. 2004. A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT‐6 secretion. Mol. Microbiol. 53:1677‐1693.
   Lefevre, P., Braibant, M., de Wit, L., Kalai, M., Roeper, D., Grotzinger, J., Delville, J. P., Peirs, P., Ooms, J., Huygen, K., and Content, J. 1997. Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG. J. Bacteriol. 179:2900‐2906.
   Middlebrook, G. and Cohn, M. L. 1958. Bacteriology of tuberculosis: Laboratory methods. Am. J. Public Health 48:844‐853.
   Ramakrishnan, L, Valdivia, R. H., McKerrow, J.H, and Falkow, S. 1997. Mycobacterium marinum causes both long‐term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infect. Immun. 65:767‐773.
   Sauton, B. 1912. Sur la nutrition minerale du bacille tuberculeux. C. R. Hebd. Seances Acad. Sci. 92:85‐93.
   Strober, W. 2003. Monitoring cell growth. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, and W. Strober, eds.) pp. A.3A.1‐A.3A.2. John Wiley & Sons, Hoboken, N.J.
   Talaat, A. M., Reimschuessel, R. R., Wasserman, S. S., and Trucksis, M. 1998. Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infect. Immun. 66:2938‐2942.
   Tonjum, T., Welty, D. B., Jantzen, E., and Small, P. L. 1998. Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: Mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA‐DNA hybridization, and 16S rRNA gene sequence analysis. J. Clin. Microbiol. 36:918‐925.
Key References
   Clark and Shepard, 1963. See above.
  Demonstrates growth conditions, including temperature requirements, for M. marinum.
   Gao et al., 2003. See above.
  Demonstrates various culture methods and genetic manipulations in M. marinum.
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