Laboratory Maintenance of Rickettsia rickettsii

Nicole C. Ammerman1, Magda Beier‐Sexton1, Abdu F. Azad1

1 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3A.5
DOI:  10.1002/9780471729259.mc03a05s11
Online Posting Date:  November, 2008
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Abstract

This unit includes protocols for the laboratory maintenance of the obligate intracellular bacterium Rickettsia rickettsii, including propagation in mammalian cell cultures, as well as isolation, counting, and storage procedures. Regulations for working with R. rickettsii in biosafety level 3 containment are also discussed. Curr. Protoc. Microbiol. 11:3A.5.1‐3A.5.21. © 2008 by John Wiley & Sons, Inc.

Keywords: Rickettsia; Rocky Mountain spotted fever; Vero cells; cell culture techniques

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Propagation of Rickettsia rickettsii in Cell Culture from Frozen Bacterial Stocks
  • Basic Protocol 2: Propagation of Rickettsia rickettsii–Infected Vero Cells
  • Alternate Protocol 1: Propagation of Rickettsia rickettsii in Other Cell Types
  • Basic Protocol 3: Monitoring Rickettsia rickettsii Infection in Vero Cells Using the Giménez Stain
  • Alternate Protocol 2: Monitoring Rickettsia rickettsii Infection in Vero Cells Using the Giemsa Stain
  • Basic Protocol 4: Partial Purification of Rickettsia rickettsii from Vero Cells by Sonication
  • Alternate Protocol 3: Partial Purification of Rickettsiae from Vero Cells Using Needle and Syringe
  • Basic Protocol 5: Purification of Rickettsia rickettsii by Isopycnic Density Gradient Centrifugation
  • Alternate Protocol 4: Purification of Rickettsia rickettsii Using a Renografin “Cushion”
  • Basic Protocol 6: Quantification of Rickettsia rickettsii by Plaque Assay
  • Basic Protocol 7: Preparation of Frozen Stocks of Rickettsia rickettsii‐Infected Vero Cells
  • Basic Protocol 8: Preparation of Frozen Stocks of Purified (Cell‐Free) Rickettsia rickettsii
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Propagation of Rickettsia rickettsii in Cell Culture from Frozen Bacterial Stocks

  Materials
  • R. rickettsii‐infected Vero cells ( protocol 11) or purified rickettsial seed ( protocol 12), frozen at −80°C; also see
  • Dulbecco's modification of Eagle medium (DMEM), supplemented with 5% heat‐inactivated fetal bovine serum (FBS) and 2 mM L‐glutamine, filter‐sterilized (see recipe)
  • Vero cells, 80% to 90% confluent monolayer in a 150‐cm2 flask (see appendix 4E)
  • 32°C water bath
  • 15‐ml conical tubes, sterile
  • 37°C shaking incubator (e.g., Labnet 211DS)
  • 34°C incubator with 5% CO 2
NOTE: All solutions should be warmed to room temperature or 37°C before contacting cells.

Basic Protocol 2: Propagation of Rickettsia rickettsii–Infected Vero Cells

  Materials
  • R. rickettsii‐infected Vero cells in a 150‐cm2 flask, maintained at 34°C with 5% CO 2 (see )
  • DMEM, supplemented with 5% heat‐inactivated FBS, filter sterilized (see recipe)
  • Vero cells (uninfected), 80% to 90% confluent in a 150‐cm2 flasks (see appendix 4E)
  • Cell scraper, sterile
  • 50‐ml conical tubes, sterile
  • 34°C incubator, 5% CO 2
NOTE: All solutions should be warmed to room temperature or 37°C before contacting cells.

Alternate Protocol 1: Propagation of Rickettsia rickettsii in Other Cell Types

  Materials
  • R. rickettsii‐infected Vero cells in 150‐cm2 flask, maintained at 34°C with 5% CO 2 (see )
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium or magnesium, filter sterilized (see appendix 2A)
  • Methanol, ice‐cold
  • DMEM supplemented with 5% heat‐inactivated FBS, filter sterilized (see recipe)
  • Carbol basic fuchsin stock, warmed to 37°C (see recipe)
  • 0.1 M sodium phosphate buffer (see appendix 2A)
  • 0.8% aqueous malachite green oxalate (see recipe)
  • Cell scrapers, sterile
  • Cytocentrifuge sample chambers (e.g., Cytopro sample chambers; Wescor)
  • Cytocentrifuge (e.g., Cytopro cytocentrifuge; Wescor)
  • Glass microscope slides
  • Light microscope

Basic Protocol 3: Monitoring Rickettsia rickettsii Infection in Vero Cells Using the Giménez Stain

  Materials
  • R. rickettsii‐infected Vero cells in 150‐cm2 flask, maintained at 34°C with 5% CO 2 (see )
  • DMEM supplemented with 5% heat‐inactivated FBS, filter‐sterilized (see recipe)
  • Sucrose‐phosphate‐glutamate (SPG; see recipe)
  • Cell scraper, sterile
  • 15‐ml and 50‐ml high‐speed conical tubes, sterile
  • Sonicator with hand‐held probe (e.g., Fisher Scientific, Sonic Dismembrator Model 100), must fit inside biosafety cabinet
  • Centrifuge
  • 0.22‐µm syringe‐driven membrane filter and 10‐ml syringe, sterile (optional)
  • 1.5‐ml microcentrifuge tubes, sterile
CAUTION: The sonication process creates aerosols, so all sonication must be done inside a class II biosafety cabinet within a BSL‐3 facility.

Alternate Protocol 2: Monitoring Rickettsia rickettsii Infection in Vero Cells Using the Giemsa Stain

  Materials
  • R. rickettsii‐infected Vero cells in 150‐cm2 flask, maintained at 34°C with 5% CO 2 (see )
  • DMEM supplemented with 5% heat‐inactivated FBS, filter‐sterilized (see recipe)
  • Cell scraper, sterile
  • 15‐ml and 50‐ml high‐speed conical tubes, sterile
  • 27‐G needles, sterile
  • 5‐ml or 10‐ml syringes, sterile

Basic Protocol 4: Partial Purification of Rickettsia rickettsii from Vero Cells by Sonication

  Materials
  • 32%, 36%, and 42% Renografin‐60 solutions (see recipe)
  • Partially purified R. rickettsii in SPG, on ice (see protocol 6; also see )
  • SPG, ice‐cold (see recipe)
  • Ultracentrifuge tubes (with at least 11‐ml capacity), sterile
  • Swinging‐bucket ultracentrifuge rotor (e.g., Beckman Coulter SW 41 Ti or equivalent)
  • Ultracentrifuge, inner chamber at 4°C
  • Pasteur pipets, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
NOTE: Renografin‐60 (Bracco Diagnostics) is a controlled substance. To use Renografin‐60 for research purposes, permission must be obtained from the United Stated Department of Justice Drug Enforcement Administration (DEA).

Alternate Protocol 3: Partial Purification of Rickettsiae from Vero Cells Using Needle and Syringe

  Materials
  • Partially purified R. rickettsii in SPG, on ice (see protocol 6; also see )
  • SPG, ice‐cold (see recipe)
  • 25% Renografin‐60 solution (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Centrifuge
  • 1‐ml disposable pipet or Pasteur pipets, sterile
NOTE: Renografin‐60 (Bracco Diagnostics) is a controlled substance. To use Renografin‐60 for research purposes, permission must be obtained from the United Stated Department of Justice Drug Enforcement Administration (DEA).

Basic Protocol 5: Purification of Rickettsia rickettsii by Isopycnic Density Gradient Centrifugation

  Materials
  • DMEM, supplemented with 10% FBS, filter‐sterilized, kept at 37°C (see recipe)
  • 1% agarose in water, autoclaved and maintained at 50°C
  • Purified (cell‐free) R. rickettsii stock ( protocol 8 or protocol 9; also see ), frozen
  • 6‐well cell culture plates with Vero cells, ∼90% confluent (see appendix 4E)
  • Neutral red solution, filter‐sterilized (see recipe)
  • 32°C water bath
  • Disposable pipet tips
  • 37°C incubator
  • 34°C incubator with 5% CO 2
  • Inverted microscope

Alternate Protocol 4: Purification of Rickettsia rickettsii Using a Renografin “Cushion”

  Materials
  • DMEM supplemented with 10% heat‐inactivated FBS, 2 mM L‐glutamine, and 2 mM sucrose, filter‐sterilized (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • R. rickettsii‐infected Vero cells in 150‐cm2 flask, maintained at 34°C with 5% CO 2 (see )
  • Cell scraper, sterile
  • Cryovials suitable for freezing at −80°C or in liquid nitrogen
CAUTION: DMSO is hazardous; see unit 1.3 for guidelines on handling, storage, and disposal.

Basic Protocol 6: Quantification of Rickettsia rickettsii by Plaque Assay

  Materials
  • Cell‐free R. rickettsii, purified as described in protocol 6 or protocol 8, resuspended in SPG, kept on ice
  • SPG, filter‐sterilized and ice‐cold (see recipe)
  • Cryovials suitable for freezing at −80°C or in liquid nitrogen, chilled on ice
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Figures

Videos

Literature Cited

   Bovarnick, M.R., Miller, J.C., and Snyder, J.C. 1950. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol. 59:509‐522.
   Dumler, J.S. and Walker, D.H. 2005. Rocky Mountain spotted fever—changing ecology and persisting virulence. N. Engl. J. Med. 353:551‐553.
   Ellison, D.W., Clark, T.R., Sturdevant, D.E., Virtaneva, K., Porcella, S.F., and Hackstadt, T. 2008. Genomic comparison of virulent Rickettsia rickettsii Sheila Smith and avirulent Rickettsia rickettsii Iowa. Infect. Immun. 76:542‐550.
   Giemsa, G. 1904. Eine Vereinfachung und Vervollkommnung meiner Methylenazur‐Methylenblau‐Eosin‐Farbemethode zur Erzielung der Romanowsky‐Nocht'schen Chromatinfarbung. Zentabl. Bakteriol. Parasitenkd. Infectkrankh. 37:308.
   Giménez, D.F. 1964. Staining rickettsiae in yolk‐sac cultures. Stain Technol. 39:135‐140.
   Hanson, B.A., Wisseman, C.L. Jr., Waddell, A., and Silverman, D.J. 1981. Some characteristics of heavy and light bands of Rickettsia prowazekii on Renografin gradients. Infect. Immun. 34:596‐604.
   Raoult, D., Torres, H., and Drancourt, M. 1991. Shell‐vial assay: Evaluation of a new technique for determining antibiotic susceptibility, tested in 13 isolates of Coxiella burnettii. Antimicrob. Agents Chemother. 1991:2070‐2077.
Key References
   Weiss, E., Coolbaugh, J.C., and Williams, J.C. 1975. Separation of viable Rickettsia typhi from yolk sac and L cell host components by Renografin density gradient centrifugation. Appl. Microbiol. 30:456‐463.
  This is the seminal article describing the use of a Renografin density gradient for the isolation of pure, viable rickettsiae.
   Kimman, T.G., Smit, E., and Klein, M.R. 2008. Evidence‐based biosafety: A review of the principles and effectiveness of microbiological containment measures. Clin. Microbiol. Rev. 21:403‐425.
  The review article discusses the principles and methods of biosafety regulations as determined by the NIH, the CDC, the WHO and the European Union.
Internet Resources
  http://www.cdc.gov/ncidod/dvrd/rmsf/index.htm
  The CDC Rocky Mountain spotted fever Web site. This Web site serves as a good general reference about R. rickettsii natural history, epidemiology, laboratory detection, treatment, prevention, and control.
  http://www.cdc.gov/od/sap
  The CDC Select Agent Program Web site. This Web site lists all select agents and provides information on public laws and regulations regarding research with select agents.
  http://www.selectagents.gov/securitydoc.html
  The National Select Agent Registry (NSAR) Web site. This Web site has posted guidelines for compliance with the security requirements of the select agent regulations.
  http://www.biosafety.be
  The Belgian Biosafety Server Web site. This Web site has posted guidelines for biosafety in Belgium and the European Union, as well as links to other international biosafety guidelines.
  http://internationalbiosafety.org
  The International Biosafety Working Group Web site. This Web site provides an international forum for the discussion of biosafety issues.
  http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/
  The World Health Organizations Epidemic and Pandemic Alert Response Laboratory Biosafety Manual (third edition) Web site. This Web site provides links for the WHO Laboratory Biosafety manual in English, French, Spanish, Portuguese, Chinese, Russian, Italian, Japanese, Serbian, and Vietnamese.
  http://www.cdc.gov/ncidod/dvrd/index.htm
  The CDC Division of Viral and Rickettsial Diseases (DVRD) Web site. This center is one of the WHO Collaborating Reference Centers that work with R. rickettsii. This center is a possible source for obtaining R. rickettsii.
  http://www.timone.univ‐mrs.fr/medecine/recherche/crecherche.html
  The Unité des Rickettsies at the Faculté de Médecine Web site. This center is also one of the WHO Collaborating Reference Centers that work with R. rickettsii. This center is also a possible source for obtaining R. rickettsii.
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