Laboratory Maintenance of Treponema denticola

J. Christopher Fenno1

1 University of Michigan Schoolof Dentistry, Ann Arbor, Michigan
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12B.1
DOI:  10.1002/9780471729259.mc12b01s00
Online Posting Date:  October, 2005
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Abstract

This unit describes the methods, media, and equipment necessary for routine laboratory culture and handling of the anaerobic oral spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended media formulations, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. An additional protocol on isolation of T. denticola from clinical samples is included.

Keywords: spirochetes; anaerobes; complex medium; solid medium

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Routine Growth of T. denticola in Liquid Medium
  • Basic Protocol 2: Plating T. denticola on SemiSolid Media for Viability Counts
  • Isolation of T. denticola from Clinical Samples
  • Basic Protocol 3: Plating T. denticola in an Anaerobic Chamber
  • Basic Protocol 4: Limiting‐Dilution Isolation in 96‐Well Microtiter Plates Incubated in an Anaerobic Jar
  • Basic Protocol 5: Labeling of T. denticola with Radioisotopes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Routine Growth of T. denticola in Liquid Medium

  Materials
  • NOS, TYGVS, or OMIZ‐P4 broth medium (see recipes)
  • T. denticola stock culture (e.g., ATCC #35405)
  • 100% glycerol, sterile, at 37°C, prereduced
  • Dry ice/ethanol bath or liquid nitrogen
  • Preservation medium (see recipe)
  • 15‐ml glass or plastic (polystyrene or polypropylene) anaerobic culture tubes
  • Anaerobic chamber (see )
  • 2‐ml cryovials (Corning or equivalent)
  • Additional reagents and equipment for lyophilizing T. denticola (Cheng and Chan, ; optional) or storing in liquid nitrogen (Syed et al., ; optional)

Basic Protocol 2: Plating T. denticola on SemiSolid Media for Viability Counts

  Materials
  • NOS‐GN 1% agar semisolid medium (see recipe) or NOS 1.5% LMP agarose semi‐solid medium (see recipe)
  • T. denticola cell suspension (see protocol 1)
  • 37° to 39°C water bath
  • 25‐cm2 tissue culture flasks (Corning or equivalent)
  • 6‐well tissue culture plates (Corning or equivalent)
  • 100 × 25–mm extra‐deep petri dishes (Nalgene Nunc or equivalent)
  • Ice packs
  • Anaerobic chamber (see )

Basic Protocol 3: Plating T. denticola in an Anaerobic Chamber

  Materials
  • Subgingival plaque sample
  • Reduced transfer fluid (RTF; see recipe)
  • MTYGVS plates containing 5 µg/ml rifampicin (see recipe)
  • TYGVS medium (see recipe)
  • Anaerobic chamber (see ) containing 5 µg/ml rifampicin
  • Vortex mixer
  • Disposable cell spreaders (Fisher or equivalent)
  • Inoculating turntable
  • Semiautomatic spiral plater (Spiral Biotech Spiral Plater or equivalent; optional)
  • Pasteur pipets
  • 15‐ml glass or plastic (polystyrene or polypropylene) anaerobic culture tubes

Basic Protocol 4: Limiting‐Dilution Isolation in 96‐Well Microtiter Plates Incubated in an Anaerobic Jar

  Materials
  • Subgingival plaque sample
  • Reduced transfer fluid (RTF; see recipe)
  • OMIZ‐P4 medium with 5 µg/ml rifampicin and 100 µg/ml phosphomycin (see recipe; also see )
  • OMIZ‐P4 1.5% LMP agarose semisolid medium: prepare as described for NOS 1.5% LMP agarose semisolid medium (see recipe), substituting OMIZ‐P4 basal medium (see recipe) for NOS basal medium (see recipe)
  • 96‐well microtiter plates
  • Anaerobic jar (Becton Dickinson GasPak or equivalent; see )
  • 37°C incubator
  • Disposable cell spreaders, sterile (Fisher or equivalent)
  • Inoculating turntable

Basic Protocol 5: Labeling of T. denticola with Radioisotopes

  Materials
  • T. denticola broth culture (see protocol 1)
  • 3H‐, 14C‐, or 32P‐labeled substrate
  • Phosphate‐buffered saline (PBS; see appendix 2A)
  • 15‐ml polypropylene centrifuge tubes with caps (Corning or equivalent)
  • Anaerobic jar (Becton Dickinson GasPak or equivalent; see )
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Figures

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Literature Cited

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   Chan, E.C., Siboo, R., Keng, T., Psarra, N., Hurley, R., Cheng, S.L., and Iugovaz, I. 1993a. Treponema denticola (ex Brumpt 1925) sp. nov., nom. rev., and identification of new spirochete isolates from periodontal pockets. Int. J. Syst. Bacteriol. 43:196‐203.
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   Chan, E.C.S., DeCiccio, A., McLaughlin, R., Klitorinos, A., and Siboo, R. 1997. An inexpensive solid medium for obtaining colony‐forming units of oral spirochetes. Oral Microbiol. Immun. 12:372‐376.
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   Loesche, W.J., Hockett, R.N., and Syed, S.A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch. Oral Biol. 17:1311‐1325.
   Loesche, W.J., Syed, S.A., Laughon, B.E., and Stoll, J. 1982. The bacteriology of acute necrotizing ulcerative gingivitis. J. Periodontol. 53:223‐230.
   Loesche, W.J., Syed, S.A., Schmidt, E., and Morrison, E.C. 1985. Bacterial profiles of subgingival plaques in periodontitis. J. Periodontol. 56:447‐456.
   Miller, P.H., Wiggs, L.S., and Miller, J.M. 1995. Evaluation of AnaeroGen system for growth of anaerobic bacteria. J. Clin. Microbiol. 33:2388‐2391.
   Ohta, K., Mäkinen, K.K., and Loesche, W.J. 1986. Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect. Immun. 53:213‐220.
   Paster, B.J., Dewhirst, F.E., Weisburg, W.G., Tordoff, L.A., Fraser, G.J., Hespell, R.B., Stanton, T.B., Zablen, L., Mandelco, L., Woese, C.R. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173:6101‐6109.
   Paster, B.J., Boches, S.K., Galvin, J.L., Ericson, R.E., Lau, C.N., Levanos, V.A., Sahasrabudhe, A., and Dewhirst, F.E. 2001. Bacterial diversity in human subgingival plaque. J. Bacteriol. 183:3770‐3783.
   Rosebury, T. and Reynolds, J.B. 1964. Continuous anaerobiosis for cultivation of spirochetes. Proc. Soc. Exp. Biol. Med. 117:813‐815.
   Salvador, S.L., Syed, S.A., and Loesche, W.J. 1987. Comparison of three dispersion procedures for quantitative recovery of cultivable species of subgingival spirochetes. J. Clin. Microbiol. 25:2230‐2232.
   Schrank, K., Choi, B.K., Grund, S., Moter, A., Heuner, K., Nattermann, H., and Gobel, U.B. 1999. Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis. Int. J. Syst. Bacteriol. 49:43‐50.
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   Smibert, R.M. 1973. Spirochaetales: A review. CRC Crit. Rev. Microbiol. 2:491‐544.
   Smibert, R.M. 1976. Cultivation, composition, and physiology of avirulent treponemes. In The Biology of Parasitic Spirochetes. (R.C. Johnson, ed.) pp. 49‐56. Academic Press, New York.
   Socransky, S., MacDonald, J.B., and Sawyer, S. 1959. The cultivation of Treponema microdentium as surface colonies. Arch. Oral Biol. 1:171‐172.
   Socransky, S.S., Loesche, W.J., Hubersak, C., and Macdonald, J.B. 1964. Dependency of Treponema microdentium on other oral organisms for isobutyrate, polyamines, and a controlled oxidation‐reduction potential. J. Bacteriol. 88:200‐209.
   Socransky, S.S. and Hubersak, C. 1967. Replacement of ascitic fluid or rabbit serum requirement of Treponema dentium by alpha‐globulin. J. Bacteriol. 94:1795‐1796.
   Socransky, S.S. and Haffajee, A.D. 1991. Microbial mechanisms in the pathogenesis of destructive periodontal diseases: A critical assessment. J. Periodontal Res. 26:195‐212.
   Socransky, S.S. and Haffajee, A.D. 1992. The bacterial etiology of destructive periodontal disease: Current concepts. J. Periodontol. 63:322‐331.
   Socransky, S.S., Haffajee, A.D., Cugini, M.A., Smith, C., and Kent, R.L. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25:134‐144.
   Suzuki, M. and Loesche, W.J. 1989. Ceruloplasmin can substitute for rabbit serum in stimulating the growth of Treponema denticola. Infect. Immun. 57:643‐644.
   Syed, S.A., Makinen, K.K., Makinen, P.L., Chen, C.Y., and Muhammad, Z. 1993. Proteolytic and oxidoreductase activity of Treponema denticola ATCC 35405 grown in an aerobic and anaerobic gaseous environment. Res. Microbiol. 144:317‐326.
   Walker, R.L., Read, D.H., Loretz, K.J., and Nordhausen, R.W. 1995. Spirochetes isolated from dairy cattle with papillomatous digital dermatitis and interdigital dermatitis. Vet. Microbiol. 47:343‐355.
   Wyss, C. 1992. Growth of Porphyromonas gingivalis, Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii in a chemically defined medium. J. Clin. Microbiol. 30:2225‐2229.
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Key References
   Chan et al., 1993b. See above.
  These two articles describe efficient and repeatable methods of growing T. denticola as subsurface colonies.
   Chan et al., 1997. See above.
  This article documents oxygen sensitivity and other growth requirements of T. denticola.
   Cheng and Chan, 1983. See above.
  These two articles describe detailed methods for isolating Treponema species from clinical samples.
   Loesche, 1969. See above.
  These two classic reviews by an early investigator of anaerobic bacterial metabolism provide an excellent introduction into microbial physiology and the biochemistry of spirochetes. It is important to note that nomenclature of oral spirochetes was rather fluid before the advent of ribotyping. Most strains reported as T. microdentium, T. dentium, or T. oralis are currently classified as T. denticola.
   Salvador et al., 1987. See above.
  This article describes in great detail a defined medium capable of supporting growth T. denticola.
   Smibert, 1973. See above.
   Smibert, 1976. See above.
   Wyss, 1992. See above.
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