Laboratory Maintenance of Neisseria gonorrhoeae

Janice M. Spence1, Lori Wright1, Virginia L. Clark1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4A.1
DOI:  10.1002/9780471729259.mc04a01s8
Online Posting Date:  February, 2008
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Abstract

Neisseria gonorrhoeae is a human pathogen of mucosal surfaces, thus laboratory manipulations must include appropriate safety measures. The growth requirements and behavior of the gonococcus are significantly different from many bacteria, necessitating modifications of common laboratory techniques. A fastidious organism, N. gonorrhoeae requires enriched media in a CO2 atmosphere at 35° to 37°C for growth. In addition, N. gonorrhoeae expresses potent autolysins whose activity increases following glucose depletion during stationary phase, leading to cell death. Long believed to be an obligate aerobe, the gonococcus is capable of anaerobic growth when provided with a suitable electron acceptor. This unit provides information for both aerobic and anaerobic growth, basic long‐term and daily maintenance of gonococcal cultures, as well as safety considerations for laboratory studies. Curr. Protoc. Microbiol. 8:4A.1.1‐4A.1.26. © 2008 by John Wiley & Sons, Inc.

Keywords: Neisseria gonorrhoeae; bacterial growth; Neisseria media; anaerobic bacterial growth; frozen stock

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Aerobic Growth of N. gonorrhoeae on Streak Plates
  • Support Protocol 1: Oxidase Test
  • Support Protocol 2: Catalase Test
  • Basic Protocol 2: Establishing Broth Cultures for the Aerobic Growth of N. gonorrhoeae
  • Basic Protocol 3: Anaerobic Growth of N. gonorrhoeae on Solid Medium
  • Basic Protocol 4: Subculturing Anaerobic Cultures
  • Basic Protocol 5: Long‐Term Storage of N. gonorrhoeae
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Aerobic Growth of N. gonorrhoeae on Streak Plates

  Materials
  • Gonococci inoculum
  • GCMB plates (see recipe for GCMB solid medium), at room temperature
  • Microbiological loop or sterile cotton‐tipped applicator
  • 37°C incubator with 3% to 10% CO 2 flow or CO 2‐generating GasPak or candle jar (Wheaton cat. no. W216938) with candle
  • Sterile wooden applicators (opposite end of cotton‐tipped swab) or flat tooth‐picks, sterilized

Support Protocol 1: Oxidase Test

  Materials
  • Supplemented GCP (see recipe)
  • Overnight plate culture of N. gonorrhoeae (see protocol 1)
  • 3‐ to 10‐ml sterile tubes
  • Sterile culture flask (volume five times that of broth culture)
  • Sterile cotton‐tipped swab
  • Spectrophotometer capable of reading at 600 nm and cuvettes
  • 37°C shaking incubator

Support Protocol 2: Catalase Test

  Materials
  • GCMB solid medium (see recipe)
  • Fresh (1‐day‐old) N. gonorrhoeae plate culture
  • GCP broth (see recipe; non‐supplemented GCP is suitable for the temporary resuspension of GC)
  • 70% ethanol
  • 20% sodium nitrite solution, sterilized (see recipe)
  • 100‐mm plates
  • 3‐ to 10‐ml sterile tubes with caps
  • Sterile cotton‐tipped swabs
  • Sterilized glass spreaders ( appendix 4A)
  • Tweezers or forceps
  • Bunsen burner
  • Paper discs, ½‐in. diameter, sterilized (Schleicher and Schuell 740‐E)
  • Anaerobe chamber containing 37°C incubator (highly preferred) or 37°C incubator with H 2/CO 2‐generating GasPak (BBL) (BD cat. no. 260678)

Basic Protocol 2: Establishing Broth Cultures for the Aerobic Growth of N. gonorrhoeae

  Materials
  • 24‐ to 42‐hr anaerobic gonococcal culture
  • GCP stock (see recipe)
  • GCMB plates (see recipe for GCMB solid medium)
  • 20% sodium nitrite solution, sterilized (see recipe)
  • Squeeze bottle with 70% ethanol or 10% bleach
  • Anaerobe chamber containing 37°C incubator stocked with:
    • Pipets and sterile tips
    • Waste container lined with biohazard bag
    • Sterile swabs
    • Paper discs, ½‐in. diameter, sterilized (Schleicher and Schuell 740‐E)
    • Tweezers or forceps
    • Rack to hold tubes, tweezers, marker
    • Pipet aide
    • Container for holding inoculated plates
    • Lab‐wipes or paper towels
    • 3‐ to 5‐ml sterile tubes
    • Mechanical vortexer
    • Individually wrapped sterile glass spreaders or disposal sterile plastic Bacteriological loops, optional

Basic Protocol 3: Anaerobic Growth of N. gonorrhoeae on Solid Medium

  Materials
  • GCP stock (see recipe)
  • 18‐ to 24‐hr culture of N. gonorrhoeae
  • 50% glycerol (see recipe), autoclaved
  • Dry ice
  • 3‐ to 5‐ml sterile tubes
  • Sterile swab
  • Spectrophotometer and cuvettes
  • 2‐ml cryovials, sterilized
  • Ice bucket or other suitable container for holding dry ice
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Figures

Videos

Literature Cited

   Dillard, J.P. and Seifert, H.S. 2001. A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates. Mol. Microbiol. 41:263‐277.
   Hebeler, B. and Morse, S.A. 1976. Physiology and metabolism of pathogenic Neisseria: Tricarboxylic acid cycle activity in Neisseria gonorrhoeae. J. Bacteriol. 128:192‐201.
   Kellogg, D.S. Jr., Peacocky, W.L. Jr., Deacon, W.E., Brown, L., and Pirkle, C.I., 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274‐1279.
   Kellogg, D.S. Jr., Cohen, I.R., Norins, L.C., Schroeter, A.L., and Reising, G., 1968. Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro. J. Bacteriol. 96:596‐605.
   Knapp, J.S. and Clark, V.L. 1984. Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect. Immun. 46:171‐181.
   Melly, M.A., McGee, Z.A., and Rosenthal, R.S. 1984. Ability of monomeric peptidoglycan fragments from Neisseria gonorrhoeae to damage human fallopian‐tube mucosa. J. Infect. Dis. 149:378‐386.
   Morse, S.A. and Bartenstein, L. 1974. Factors affecting autolysis of Neisseria gonorrhoeae. Proc. Soc. Exp. Biol. Med. 145:1418‐1421.
   Morse, S.A. and Bartenstein, L. 1980. Purine metabolism in Neisseria gonorrhoeae: The requirement for hypoxanthine. Can. J. Microbiol. 26:13‐20.
   Morse, S.A., Stein, S., and Hines, J. 1974. Glucose metabolism in Neisseria gonorrhoeae. J. Bacteriol. 120:702‐714.
   Morse, S.A., Cacciapuoti, A.F., and Lysko, P.G. 1979. Physiology of Neisseria gonorrhoeae. Adv. Microb. Physiol. 20:251‐320.
   Schryvers, A.B. and Stojiljkovic, I. 1999. Iron acquisition systems in pathogenic Neisseria. Mol. Microbiol. 32:117‐123.
   Wegener, W.S., Hebeler, B.H., and Morse, S.A. 1977. Cell envelope of Neisseria gonorrhoeae: Relationship between autolysis in buffer and the hydrolysis of peptidoglycan. Infect. Immun. 18:210‐219.
Key References
   Morse, S.A. 1978. The biology of the gonococcus. CRC Crit. Rev. Microbiol. 7:93‐189.
  Stephen A. Morse played a major role in elucidating N. gonorrhoeae physiology.
   Perspectives on Pathogenic Neisseriae. 1989. Clin. Microbiol. Rev. 2: Supplement.
  This entire journal supplement is dedicated to pathogenic Neisseriae, with solicited review articles on major areas of Neisserial research.
   U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health. 2007. Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (BMBL). U.S. Government Printing Office, Washington.
  The document in its entirety is available online at http://www.cdc.gov/od/ohs
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