Cultivation and Laboratory Maintenance of Chlamydia trachomatis

Marci A. Scidmore1

1 Cornell College of Veterinary Medicine, Ithaca, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 11A.1
DOI:  10.1002/9780471729259.mc11a01s00
Online Posting Date:  July, 2005
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Abstract

Chlamydia species are obligate intracellular bacteria that require growth inside mammalian cells for propagation and survival. As a result, Chlamydia cannot be grown on conventional bacteriological medium. This property makes Chlamydia difficult organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sac of the embryonated hen egg was the only way to isolate and propagate the organism. Since then, a tissue culture system has been available that allows easier laboratory culture of the Chlamydia species. However, with the exception of the LGV serovars, most C. trachomatis strains do not readily infect tissue culture cells. Chemical or mechanical assistance is used to increase their infectivity. Today, large numbers of infectious organisms can be purified through Renografin density gradient centrifugation of infected cell lysates. The ability to propagate C. trachomatis in the laboratory has greatly increased the understanding of the pathogenesis of C. trachomatis organisms.

Keywords: Chlamydia trachomatis; trachoma; lymphogranuloma venereum; tissue culture techniques; sexually transmitted diseases; renografin density gradient

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of C. trachomatis from Clinical Specimens
  • Basic Protocol 2: Chlamydial Infection of Monolayer Tissue Culture Cells Pretreated with DEAE‐Dextran
  • Alternate Protocol 1: Infection of Monolayer Tissue Culture Cells by Centrifugaton
  • Basic Protocol 3: Density Gradient Purification of C. trachomatis Elementary Bodies
  • Support Protocol 1: Determining the Asepsis of Elementary Body Preparations
  • Support Protocol 2: Titering Infectious Forming Units by Indirect Immunofluorescence
  • Basic Protocol 4: Purification of C. trachomatis Reticulate Bodies
  • Support Protocol 3: Recovery and Passage of Frozen Monolayer Tissue Culture Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of C. trachomatis from Clinical Specimens

  Materials
  • Freshly trypsinized McCoy cells (see protocol 8)
  • Clinical specimens stored in chlamydial transport buffer (see recipe for chlamydial transport buffer), 4°C
  • Complete medium supplemented with 10% FBS (see ) supplemented with 1 µg/ml cycloheximide (see recipe), 37°C
  • Sucrose/phosphate/glutamate buffer (SPG; see recipe)
  • 12‐mm shell vials
  • 48‐well tissue culture plates
  • Sonicator
  • 5‐ml sterile tubes
  • Ten Broek tissue homogenizers (for tissue samples only)
  • Low‐speed bench‐top centrifuge with tissue plate adaptors
  • Additional reagents and equipment for fixing and staining cells (see protocol 5)

Basic Protocol 2: Chlamydial Infection of Monolayer Tissue Culture Cells Pretreated with DEAE‐Dextran

  Materials
  • Freshly trypsinized monolayer tissue culture cells (see protocol 8)
  • Complete tissue culture medium (see ) supplemented with 10% FBS and 10 µg/ml gentamycin, 37°C
  • 10× DEAE‐dextran (see recipe)
  • HBSS ( appendix 2A)
  • Stock of titered C. trachomatis EBs, −80°C (see protocol 6)
  • SPG (see recipe)
  • Complete medium (see ) supplemented with 10% FBS, 10 µg/ml gentamycin, and 1 µg/ml cycloheximide
  • Platform rocker at 37°C
  • Inverted phase‐contrast microscope
NOTE: DEAE‐dextran pretreatment of host cells (step ) can be omitted with C. trachomatis LGV strains.

Alternate Protocol 1: Infection of Monolayer Tissue Culture Cells by Centrifugaton

  • Low speed bench top centrifuge with microplate adaptors

Basic Protocol 3: Density Gradient Purification of C. trachomatis Elementary Bodies

  Materials
  • C. trachomatis–infected monolayer tissue culture cells (20 infected 150‐cm2 flasks; see protocol 2)
  • HBSS ( appendix 2A)
  • 70% and 95% (v/v) ethanol
  • 95% (v/v) ethanol‐soaked gauze strips
  • 30%, 40%, 44%, and 54% (v/v) Renografin solutions (see recipe)
  • Disposable large cell scrapers, 3.0‐cm blade width
  • 10‐ml disposable pipets, sterile
  • 250‐ and 40‐ml centrifuge tubes with sealing caps
  • Sonicator (e.g., Labsonic U Ultrasonic homogenizer with needle probe 40T, Ultrasonic Power)
  • Low‐speed refrigerated centrifuge (Beckman JA‐14 and JA‐21 rotors or equivalent)
  • 18‐G metal cannulae
  • 10‐ml disposable syringes
  • 25 × 89–mm ultraclear ultracentrifuge tubes (Beckman cat. no. 344058)
  • Ultracentrifuge with swinging bucket SW28 rotor
  • 1.5‐ml screw‐cap microcentrifuge tubes
NOTE: For large‐scale preparations of density gradient purified EBs, use approximately twenty 150‐cm2 flasks. Although volumes are given per 150‐cm2 flask, this procedure may be scaled up or down accordingly, depending on the numbers of infectious EBs desired.NOTE: All solutions and centrifuge tubes should be pre‐chilled on ice prior to use and kept on ice during use.

Support Protocol 1: Determining the Asepsis of Elementary Body Preparations

  Materials
  • BHI blood agar plates (see recipe), 37°C
  • Purified chlamydial organisms

Support Protocol 2: Titering Infectious Forming Units by Indirect Immunofluorescence

  Materials
  • Freshly trypsinized monolayer tissue culture cells (see protocol 8)
  • Complete tissue culture medium (see ) supplemented with 10% FBS, 10 µg/ml gentamycin, and 1 µg/ml cycloheximide, 37°C
  • Frozen stocks of C. trachomatis EBs (see protocol 4) or infected cells (see protocol 2 or protocol 3)
  • HBSS ( appendix 2A)
  • 70% (v/v) ethanol
  • SPG (see recipe)
  • 100% methanol
  • PBS ( appendix 2A)
  • PBS supplemented with 3% (w/v) BSA (see recipe)
  • Primary antibody specific to C. trachomatis (e.g., Abcam or Chemicon)
  • Fluorophore‐conjugated secondary antibody
  • 24‐well tissue culture plates
  • Biohazard swinging‐bucket rotor adapted for microtiter plates (e.g., Sorvall biohazard microtiter rotor, cat. no. 75006‐444)
  • 12‐mm‐diameter coverslips (no. 1 thickness), optional
  • 37°C water bath
  • Inverted epi‐fluorescence microscope with appropriate filter sets
  • Hand‐held counter

Basic Protocol 4: Purification of C. trachomatis Reticulate Bodies

  Materials
  • Infected cells in 150‐cm2 flasks (see protocol 2)
  • 10× K‐36 buffer (see recipe)
  • Large cell scraper
  • 250‐ml centrifuge tube
  • 40‐ml Dounce homogenizer with a tight‐fitting pestle
  • Glass microscope slides and coverslips
  • Phase‐contrast microscope
  • Beckman JA‐14 rotor or equivalent
  • Additional reagents and equipment for density gradient purification (see protocol 4)

Support Protocol 3: Recovery and Passage of Frozen Monolayer Tissue Culture Cells

  Materials
  • Frozen immortalized tissue culture cell lines in liquid nitrogen: HeLa 229 (ATCC no. CCL‐2.1), McCoy (ATCC no. CRL‐1696), or L929 cell lines (ATCC no. CCL‐1)
  • 70% (v/v) ethanol
  • Complete medium (see ) supplemented with 10% FBS and 10 µg/ml gentamycin, 37°C
  • HBSS ( appendix 2A) without Ca2+ and Mg2+
  • Trypsin/EDTA solution (see recipe), 37°C
  • 37°C water bath
  • 15‐ml sterile centrifuge tubes
  • 75‐cm2 tissue culture flasks
  • Inverted phase‐contrast microscope
  • Hemacytometer slide with coverslip
  • Light microscope
  • Hand‐held cell counter
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Figures

Videos

Literature Cited

   Caldwell, H.D., Kromhout, J., and Schachter, J. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:1161‐1176.
   Moulder, J.W. 1991. Interaction of chlamydiae and host cells in vitro. Microbiol. Rev. 55:143‐190.
   Schachter, J. 1999. Infection and disease epidemiology. In Chlamydia: Intracellular Biology, Pathogenesis, and Immunity (R.S. Stephens, ed.) pp. 139‐169. American Society for Microbiology Press, Washington, D.C.
   Schachter, J. and Wyrick, P.B. 1994. Culture and isolation of Chlamydia trachomatis. Methods Enzymol. 236:377‐390.
   T'ang, F.F., Chang, H.L., Huang, Y.T., and Wang, K.C. 1957. Trachoma virus in chick embryo. Natl. Med. J. China 43:81.
   Thylefors, B., Negrel, A.D., Pararajasegaram, R., and Dadzie, K.Y. 1995. Global data on blindness. B. World Health Organ. 73:115‐121.
   World Health Organization. 1997. Chlamydia trachomatis genital tract infections—United States. Morb. Mortal. Weekly Rep. 46:193‐198.
Key References
   Phelan, M.C. 1998. Basic techniques for mammalian cell tissue culture. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) Unit 1.1. John Wiley and Sons, Inc., New York.
  This is an excellent resource for understanding the basic techniques of mammalian cell tissue culture.
   Schachter, J. and Wyrick, P.B. 1994. See above.
  Chapter provides detailed protocols for culturing, and purifying C. trachomatis using several different tissue culture systems.
   Richmond, J.Y. and McKinney, R.W. (eds.) 1999. Biosafety in Microbiological and Biomedical Laboratories, 4th ed. Government Printing Office, Washington, D.C.
  Book is essential when working with infectious organisms such as Chlamydia trachomatis as it describes the principles of biosafety, the biosafety levels, primary containment, proper handling, and the transport and transfer of infectious agents.
Internet Resources
   http://www.chlamydiae.com
  A comprehensive site that discusses all aspects of Chlamydia biology including, cell biology, molecular biology, pathogenesis, taxonomy, immunology, laboratory diagnosis, treatment, and research methods.
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