Laboratory Maintenance and Characterization of Yersinia pestis

Scott W. Bearden1, Robert D. Perry2

1 Centers for Disease Control and Prevention, Division of Vector‐Borne Infectious Diseases, Fort Collins, Colorado, 2 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5B.1
DOI:  10.1002/9780471729259.mc05b01s11
Online Posting Date:  November, 2008
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Abstract

This unit describes protocols for Yersinia pestis maintenance and growth in research and clinical laboratories, including some protocols for strain characterization. Strain‐dependent requirements for different Biosafety Level containments are also discussed. Curr. Protoc. Microbiol. 11:5B.1.1‐5B.1.13. © 2008 by John Wiley & Sons, Inc.

Keywords: plague; medium; strain storage; Yersinia selective medium; low‐calcium response; type III secretion; pigmentation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of Y. pestis in Liquid Media
  • Basic Protocol 2: Preservation of Laboratory Stocks by Freezing
  • Alternate Protocol 1: Preservation of Laboratory Stocks from Liquid Cultures or Plates by Freezing
  • Basic Protocol 3: Initiation of Growth on Solid Medium from Frozen Stocks
  • Basic Protocol 4: Preservation of Laboratory Stocks by Lyophilization
  • Basic Protocol 5: Initiation of Cultures from Lyophilized Stocks
  • Basic Protocol 6: Growth on CIN Agar
  • Basic Protocol 7: Colony Phenotypes on Congo Red Agar
  • Basic Protocol 8: Growth on TBA‐MgOX Plates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth of Y. pestis in Liquid Media

  Materials
  • Y. pestis strain on a CR plate (see recipe) or tryptose blood agar base (TBA; Difco) slant (prepare according to manufacturer's instructions, omitting blood)
  • HIB, BHI broth (Difco; see manufacturer's instructions) or other liquid medium
  • 33 mM potassium phosphate buffer (K‐PO 4; see recipe)
  • Inoculating loop (sterile, disposable, or metal loops with a Bunsen burner or Bacti‐cinerator; Oxford Labware)
  • Dilution tubes
  • Spectrophotometer
  • Shaking incubator

Basic Protocol 2: Preservation of Laboratory Stocks by Freezing

  Materials
  • Tryptose blood agar (TBA; Difco, prepare according to manufacturer's instructions)
  • Y. pestis strain grown on CR or TBA plates (see recipe for CR agar)
  • Sterile 33 mM potassium phosphate buffer (K‐PO 4), pH 7.0 (see recipe)
  • Sterile glycerol
  • 15‐ml conical centrifuge tubes, sterile
  • Inoculating loop, sterile
  • 30°C incubator
  • 5‐ml sterile capped tube
  • 2‐ml sterile freezer vials with screw‐caps
  • −70° to –80°C freezer

Alternate Protocol 1: Preservation of Laboratory Stocks from Liquid Cultures or Plates by Freezing

  Materials
  • Y. pestis strain
  • CR or 5% to 6% sheep blood agar (SBA) plates (see recipe)
  • BHI broth (Difco) with sterile glycerol added to 10% (v/v) final
  • 30° to 35°C incubator
  • Inoculating loop, sterile
  • 2‐ml sterile freezer vials with screw‐caps
  • Vortexer
  • Spectrophotometer or turbidity meter (e.g., Dade Microscan Turbidimeter)
  • −70°C to –80°C freezer

Basic Protocol 3: Initiation of Growth on Solid Medium from Frozen Stocks

  Materials
  • Frozen Y. pestis strain stock (see protocol 2 and protocol 3)
  • CR plates (see recipe for CR agar) or TBA slants (Difco, prepare according to manufacturer's instructions)
  • Benchtop cooler
  • Inoculating loop, sterile
  • 26° to 30°C incubator

Basic Protocol 4: Preservation of Laboratory Stocks by Lyophilization

  Materials
  • Y. pestis strain frozen or grown on an agar plate
  • SBA plates (see recipe) or TBA plates (Difco, prepare according to manufacturer's instructions)
  • Lyophilization medium (see recipe)
  • McFarland standards
  • BHI
  • 35° to 37°C incubator
  • 16 × 125–mm sterile, borosilicate glass tubes with screw caps (Fisher Scientific cat. no. 14‐959‐25C or equivalent)
  • Spectrophotometer or turbidity meter (e.g., Dade Microscan Turbidimeter)
  • 3‐ml sterile, glass serum tubing vials (e.g., Wheaton cat. no. 223684)
  • Inoculating loop, sterile
  • Vortex mixer
  • Automatic pipettor
  • Sterile, gray, butyl 2‐leg rubber stoppers (autoclaved separately; 7 × 13–mm i.d. × o.d.)
  • Aluminum foil
  • Lyophilizer equipped with an internal stoppering device
  • Aluminum crimp closures (13 mm)

Basic Protocol 5: Initiation of Cultures from Lyophilized Stocks

  Materials
  • Lyophilized Y. pestis stock (see protocol 5)
  • BHI or HIB (Difco), sterile
  • CR or 6% SBA plate (see reciperecipes)
  • TBA slant (Difco, prepare according to manufacturer's instructions)
  • Scissors, sterile
  • Inoculating loop, sterile
  • 26° to 30°C Incubator

Basic Protocol 6: Growth on CIN Agar

  Materials
  • Sample containing Y. pestis plus other bacteria
  • CIN agar plate (see recipe for CIN agar)
  • SBA, TBA, or CR plates (see reciperecipes)
  • 26° to 30°C incubator
  • Inoculating loop, sterile

Basic Protocol 7: Colony Phenotypes on Congo Red Agar

  Materials
  • Y. pestis strain (frozen or fresh culture)
  • CR plate (see recipe)
  • Inoculating loop, sterile
  • 26° to 30°C incubator

Basic Protocol 8: Growth on TBA‐MgOX Plates

  Materials
  • Y. pestis strain
  • HIB or BHI (Difco)
  • TBA plates (Difco, prepare according to manufacturer's instructions)
  • TBA‐MgOX plates (see recipe)
  • Inoculating loop, sterile
  • 26° to 30°C and 37°C incubators
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Figures

  •   FigureFigure 5.B0.1 Pgm+ (KIM6+; left) and Pgm (KIM6; right) colonies on a CR plate. Cells were streaked for isolated colonies and incubated 48 hr at 30°C. White spots on CR+ colonies are reflected light, not spontaneous Δ pgm mutants arising within the colony.

Videos

Literature Cited

Literature Cited
   Anisimov, A.P., Lindler, L.E., and Pier, G.B. 2004. Intraspecific diversity of Yersinia pestis. Clin. Microbiol. Rev. 17:434‐464.
   Beesley, E.D., Brubaker, R.R., Janssen, W.A., and Surgalla, M.J. 1967. Pesticins. III. Expression of coagulase and mechanism of fibrinolysis. J. Bacteriol. 94:19‐26.
   Ber, R., Mamroud, E., Aftalion, M., Tidhar, A., Gur, D., Flashner, Y., and Cohen, S. 2003. Development of an improved selective agar medium for isolation of Yersinia pestis. Appl. Environ. Microbiol. 69:5787‐5792.
   Brubaker, R.R. 1969. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 98:1404‐1406.
   Brubaker, R.R. 2005. Influence of Na+, dicarboxylic amino acids, and pH in modulating the low‐calcium response of Yersinia pestis. Infect. Immun. 73:4743‐4752.
   Chu, M.C. 2000. Laboratory Manual of Plague Diagnostic Tests. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, and World Health Organization, Geneva.
   Gage, K.L. and Kosoy, M.Y. 2005. Natural history of plague: Perspectives from more than a century of research. Annu. Rev. Entomol. 50:505‐528.
   Gong, S., Bearden, S.W., Geoffroy, V.A., Fetherston, J.D., and Perry, R.D. 2001. Characterization of the Yersinia pestis Yfu ABC iron transport system. Infect. Immun. 67:2829‐2837.
   Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Schoch‐Spana, M., and Tonat, K. 2000. Plague as a biological weapon: Medical and public health management. J. Amer. Med. Assoc. 283:2281‐2290.
   Israeli, E., Shaffer, B.T., and Lighthart, B. 1993. Protection of freeze‐dried Escherichia coli by trehalose upon exposure to environmental conditions. Cryobiology 30:519‐523.
   Jackson, S. and Burrows, T.W. 1956. The pigmentation of Pasteurella pestis on a defined medium containing haemin. Br. J. Exp. Pathol. 37:570‐576.
   Kirillina, O., Fetherston, J.D., Bobrov, A.G., Abney, J., and Perry, R.D. 2004. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms‐dependent biofilm formation in Yersinia pestis. Mol. Microbiol. 54:75‐88.
   Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H., and Crowe, L.M. 1995. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl. Environ. Microbiol. 61:3592‐3597.
   Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., and Pfaller, M.A. (Eds.) 2007. Manual of Clinical Microbiology, 9th Edition. ASM Press, Washington, D.C.
   Perry, R.D. and Fetherston, J.D. 1997. Yersinia pestis—Etiologic agent of plague. Clin. Microbiol. Rev. 10:35‐66.
   Schiemann, D.A. 1979. Synthesis of a selective agar medium for Yersinia enterocolitica. Can. J. Microbiol 25:1298‐1304.
   Staggs, T.M. and Perry, R.D. 1991. Identification and cloning of a fur regulatory gene in Yersinia pestis. J. Bacteriol. 173:417‐425.
   Straley, S.C. and Bowmer, W.S. 1986. Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect. Immun. 51:445‐454.
   Surgalla, M.J. and Beesley, E.D. 1969. Congo red‐agar plating medium for detecting pigmentation in Pasteurella pestis. Appl. Microbiol. 18:834‐837.
   Zahorchak, R.J. and Brubaker, R.R. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in Yersinia pestis. Infect. Immun. 38:953‐959.
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