Laboratory Maintenance of Mycobacterium smegmatis

Anil Kumar Singh1, Jean‐Marc Reyrat1

1 Inserm U570, Unité de Pathogénie des Infections Systémiques, Paris, France
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10C.1
DOI:  10.1002/9780471729259.mc10c01s14
Online Posting Date:  August, 2009
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Abstract

This unit gives background information on Mycobacterium smegmatis, a mycobacterial model system, and covers all the laboratory maintenance for this species including growth in liquid and on solid medium. It also contains recommendations concerning long‐term strain storage. Although M. smegmatis is a Biosafety Level 1 organism, some rare infections in humans have been reported, and, thus all of the required safety measures are discussed here. Curr. Protoc. Microbiol. 14:10C.1.1‐10C.1.12. © 2009 by John Wiley & Sons, Inc.

Keywords: medium; Middlebrook; 7H9; Sauton; LB Miller; strain storage

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of M. smegmatis in Middlebrook Liquid Medium
  • Basic Protocol 2: Growth of M. smegmatis in Sauton Liquid Medium
  • Basic Protocol 3: Growth of M. smegmatis on Solid Medium
  • Basic Protocol 4: Storage of M. smegmatis as Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of M. smegmatis in Middlebrook Liquid Medium

  Materials
  • Middlebrook 7H9 liquid medium (see recipe)
  • M. smegmatis growing on plate ( protocol 3)
  • 50‐ml (for small‐volume culture) or 500‐ml (for large‐volume culture) Erlenmeyer or conical flask (Borosil, cat no. 4980; http://www.borosil.com)
  • Disposable plastic inoculation loops, sterile
  • Porous plugs for flasks
  • Incubator containing platform shaker
  • Additional reagents and equipment for bacterial culture techniques including inoculation of liquid medium ( appendix 4A)

Basic Protocol 2: Growth of M. smegmatis in Sauton Liquid Medium

  Materials
  • Sauton medium (see recipe)
  • 175‐cm2 Corning Cell Culture Flasks (Sigma, cat no. 431080)
  • Additional reagents and equipment for growth of M. smegmatis ( protocol 1)

Basic Protocol 3: Growth of M. smegmatis on Solid Medium

  Materials
  • M. smegmatis (mc2155; ATCC no. 70084)
  • Middlebrook 7H10 or 7H11 agar plates (see recipe)
  • Sterile plastic inoculating loops
  • Sterile plastic plate spreaders
  • Heavy‐duty aluminum foil or sterile metal plate canisters
  • Additional reagents and equipments for bacterial culture techniques including spreading and streaking plates ( appendix 4A)

Basic Protocol 4: Storage of M. smegmatis as Frozen Stocks

  Materials
  • M. smegmatis culture ( protocol 1)
  • Storage medium (see recipe)
  • Sterile 2‐ml cryotubes
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Figures

Videos

Literature Cited

Literature Cited
   Alvarez, E. and Tavel, E. 1885. Recherches sur le bacille de Lustgarden. Arch. Physiol. Norm. Pathol. 6:303‐321.
   Bardarov, S., Kriakov, J., Carriere, C., Yu, S., Vaamonde, C., McAdam, R.A., Bloom, B.R., Hatfull, G.F., and Jacobs, W.R. Jr. 1997. Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 94:10961‐10966.
   Brown‐Elliott, B. and Wallace, R. 2002. Clinical and taxonomic status of pathogenic non‐pigmented or late‐pigmenting rapidly growing mycobacteria. Clin. Microbiol. Rev. 15:716‐746.
   Catanho, M., Mascarenhas, D., Degrave, W., and Miranda, A.B. 2006. GenoMycDB: A database for comparative analysis of mycobacterial genes and genomes. Genet. Mol. Res. 5:115‐126.
   Chatterjee, D. 1997. The mycobacterial cell wall: Structure, biosynthesis and sites of drug action. Curr. Opin. Chem. Biol. 1:579‐588.
   Cohn, M.L., Kovitz, C., Oda, U., and Middlebrook, G. 1954. Studies on isoniazid and tubercle bacilli. II. The growth requirements, catalase activities, and pathogenic properties of isoniazid‐resistant mutants. Am. Rev. Tuberc. 70:641‐664.
   Daffé, M. 2008. The global architecture of the mycobacterial cell envelope. In The Mycobacterial Cell Envelope (Daffé, M. and Reyrat, J.M., eds.) pp. 3‐12. ASM Press, Washington, D.C.
   Deshayes, C., Perrodou, E., Gallien, S., Euphrasie, D., Schaeffer, C., Van‐Dorsselaer, A., Poch, O., Lecompte, O., and Reyrat, J.M. 2007. Interrupted coding sequences in Mycobacterium smegmatis: Authentic mutations or sequencing errors? Genome Biol. 8:R20.
   Gallien, S., Perrodou, E., Carapito, C., Deshayes, C., Reyrat, J.M., Dorsselaer, A.V., Poch, O., Schaeffer, C., and Lecompte, O. 2008. Ortho‐proteogenomics: Multiple proteomes investigation through orthology and a new MS‐based protocol. Genome Res. 19:128‐135.
   Guilhot, C., Otal, I., Van Rompaey, I., Martin, C., and Gicquel, B. 1994. Efficient transposition in mycobacteria: Construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol. 176:535‐539.
   Jacobs, W.R. Jr., Kalpana, G.V., Cirillo, J.D., Pascopella, L., Snapper, S.B., Udani, R.A., Jones, W., Barletta, R.G., and Bloom, B.R. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204:537‐555.
   Kendall, S.L., Withers, M., Soffair, C.N., Moreland, N.J., Gurcha, S., Sidders, B., Frita, R., Ten Bokum, A., Besra, G.S., Lott, J.S., and Stoker, N.G. 2007. A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Mol. Microbiol. 65:684‐699.
   Kocíncová, D., Singh, A.K., Beretti, J.L., Ren, H., Euphrasie, D., Liu, J., Daffé, M., Etienne, G., and Reyrat, J.M. 2008. Spontaneous transposition of IS1096 or ISMsm3 leads to glycopeptidolipid overproduction and affects surface properties in Mycobacterium smegmatis. Tuberculosis 88:390‐398.
   Lehmann, K.B. and Neumann, R.O. 1899. Bakteriologische Diagnostik, 2nd ed., vol. II, pp. 408‐413. L.F. Lehmanns Verlag, Munich, Germany.
   Lustgarten, S. 1885. The bacillus of syphilis. Lancet i:609‐610.
   Martinez, A., Torello, S., and Kolter, R. 1999. Sliding motility in mycobacteria. J. Bacteriol. 181:7331‐7338.
   Niederweis, M. 2003. Mycobacterial porins‐new channel proteins in unique outer membranes. Mol. Microbiol. 49:1167‐1177.
   Ojha, A., Anand, M., Bhatt, A., Kremer, L., Jacobs, W.R., and Hatfull, G.F. 2005. GroEL1: A dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 123:861‐873.
   Ojha, A. and Hatfull, G.F. 2007. The role of iron in Mycobacterium smegmatis biofilm formation: The exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth. Mol. Microbiol. 66:468‐483.
   Pelicic, V., Reyrat, J.M., and Gicquel, B. 1996a. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J. Bacteriol. 178:1197‐1199.
   Pelicic, V., Reyrat, J.M., and Gicquel, B. 1996b. Generation of unmarked directed mutations in mycobacteria, using sucrose counter‐selectable suicide vectors. Mol. Microbiol. 20:919‐925.
   Pelicic, V., Jackson, M., Reyrat, J.M., Jacobs, W.R. Jr., Gicquel, B., and Guilhot, C. 1997. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 94:10955‐10960.
   Provvedi, R., Kocíncová, D., Donà, V., Euphrasie, D., Daffé, M., Etienne, G., Manganelli, R., and Reyrat, J.M. 2008. SigF controls carotenoid pigment production, affects transformation efficiency and hydrogen peroxide sensitivity in Mycobacterium smegmatis. J. Bacteriol. 190:7859‐7863.
   Recht, J., Martinez, A., Torello, S., and Kolter, R. 2000. Genetic analysis of sliding motility in Mycobacterium smegmatis. J. Bacteriol. 182:4348‐4351.
   Recht, J. and Kolter, R. 2001. Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis. J. Bacteriol. 183:5718‐5724.
   Reyrat, J.M. and Kahn, D. 2001. Mycobacterium smegmatis: An absurd model for tuberculosis? Trends Microbiol. 9:472‐474.
   Skerman, V.B.D., McGowan, V., and Sneath, P.H.A. 1980. Approved lists of bacterial names. Intl. J. Syst. Bacteriol. 30:225‐420.
   Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T., and Jacobs, W.R. Jr. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4:1911‐1919.
   Stinear, T.P., Seemann, T., Harrison, P.F., Jenkin, G.A., Davies, J.K., Johnson, P.D., Abdellah, Z., Arrowsmith, C., Chillingworth, T., Churcher, C., Clarke, K., Cronin, A., Davis, P., Goodhead, I., Holroyd, N., Jagels, K., Lord, A., Moule, S., Mungall, K., Norbertczak, H., Quail, M.A., Rabbinowitsch, E., Walker, D., White, B., Whitehead, S., Small, P.L., Brosch, R., Ramakrishnan, L., Fischbach, M.A., Parkhill, J., and Cole, S.T. 2008. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res. 18:729‐741.
   Stoker, N.G., Sander, P., and Reyrat, J.M. 2005. Gene Replacement Systems. ASM Press, Washington, D.C.
   Trevisan, V. 1889. I Generi e la Specie dell Batteriacee, p. 14. Zanaboni e Gabuzzi, Milan, Italy.
   Van Deun, A., Hossain, M.A., Gumusboga, M., and Rieder, H.L. 2008. Ziehl‐Neelsen staining: Theory and practice. Int. J. Tuberc. Lung Dis. 12:108‐110.
   van Kessel, J.C., and Hatfull, G.F. 2007. Recombineering in Mycobacterium tuberculosis. Nat. Methods 4:147‐152.
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