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Assessing and Controlling Microbial Contamination in Cell Cultures

Rosalie Coté¹

¹Becton Dickinson Microbiology Systems, Sparks, Maryland

Publication Name:

Current Protocols in Cell Biology

Unit Number:

Unit 1.5

DOI:

10.1002/0471143030.cb0105s01

Online Posting Date:

May, 2001

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Abstract

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary.

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Unit Introduction
Basic Protocol 1: Testing for Bacterial and Fungal Contaminants
Basic Protocol 2: Testing for Mycoplasma Contamination by Direct Culture
Alternate Protocol 1: Indirect Testing for Mycoplasma by Staining for DNA
Alternate Protocol 2: Indirect Testing for Mycoplasma by PCR
Support Protocol: Agarose Gel Electrophoresis of PCR Products
Basic Protocol 3: Use of Antibiotics to Control Microbial Contamination
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol 1: Testing for Bacterial and Fungal Contaminants

Materials

Medium for bacterial detection: e.g., brain heart infusion (BBL, Difco), fluid thioglycollate medium (BBL, Difco), HTYE broth (see recipe), soybean/casein digest broth USP (e.g., trypticase soy broth, BBL; tryptic soy broth, Difco), or trypticase soy agar (BBL)
Medium for mycelial and yeast fungal detection: e.g., Sabouraud's dextrose agar (Emmon's modified; BBL, Difco), or YM agar (Difco)
Sterile, defibrinated sheep blood (e.g., Colorado Serum, Waltz Farm)
Cell culture test samples
Antibiotic-free culture medium (optional)

Conductivity meter (Corning model 162 or equivalent), if not integrated with the laboratory water purification system
50°C water bath
16 × 125–mm borosilicate screw-cap test tubes with rubber-lined caps
100 × 15–mm sterile plastic disposable petri dishes
Semiautomated repeat-volume filling unit to accurately dispense 5- to 24-ml aliquots (optional)
Incubators: 26°C, 35° to 37°C, and 37°C with 5% (v/v) CO₂

NOTE: To avoid inadvertent contamination of clean cell lines, bacterial and fungal testing should be segregated to a laboratory not used for general cell culture work.

Basic Protocol 2: Testing for Mycoplasma Contamination by Direct Culture

Materials

Cell line for testing
Mycoplasma broth medium (see recipe): 6 ml medium in 16 × 125–mm screw-cap test tubes
Mycoplasma agar plates (see recipe): 10 ml solidified medium in 60 × 15–mm petri dishes

37°C incubators: one without CO₂ and one humidified with 5% (v/v) CO₂
Inverted microscope with 100 to 300× magnification

NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.

Alternate Protocol 1: Indirect Testing for Mycoplasma by Staining for DNA

Materials

Complete EMEM-10: Eagle's minimum essential medium (EMEM) with Earle's salts (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) bovine calf serum (see unit 1.2 for media preparation methods)
Indicator cell line: e.g., African green monkey cell line Vero (ATCC #CCL81) or 3T6 murine cell line (ATCC #CCL96)
Cell culture for testing
Mycoplasma hyorhinis (ATCC #29052) or a known mycoplasma-infected cell line to use as a positive control, actively growing
Fixative: 3:1 (v/v) absolute methanol/glacial acetic acid
Hoechst stain (see recipe)
Mounting medium (see recipe)

60 × 15–mm culture dishes, sterile
No. 1 or no. 1 ½ coverslips, sterilized by autoclaving (unit 1.4)
37°C, 5% (v/v) CO₂/95% air incubator

NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.

Alternate Protocol 2: Indirect Testing for Mycoplasma by PCR

Materials

Cells for testing
10× PCR buffer (usually provided with Taq polymerase)
2.5 mM 4dNTP mix: 2.5 mM each dGTP, dCTP, dTTP, and dATP
25 mM MgCl₂
5 U/µl Taq DNA polymerase
Mineral oil (if needed for thermal cycler)
Mycoplasma detection kit (ATCC), containing first- and second-stage primer mixtures (total 7 primers), as well as two positive control mycoplasma DNAs (Mycoplasma pirum, Acholeplasms laidlawii)

Thin-wall microcentrifuge tubes
Aerosol-preventive micropipettor tips, sterile
Positive-displacement micropipettors
Picofuge
Thermal cycler

Additional reagents and equipment for agarose gel electrophoresis (see Support Protocol)

NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.

NOTE: To avoid amplification of contaminating DNA from laboratory workers, room contaminants, or previous mycoplasma DNA amplifications, all PCR should be performed using aseptic technique (also see special considerations for PCR experiments in appendix 2A).

Support Protocol: Agarose Gel Electrophoresis of PCR Products

Materials

Agarose (e.g., NuSieve, FMC Bioproducts)
1× TBE electrophoresis buffer (appendix 2A)
10 mg/ml ethidium bromide solution
Second-stage PCR products from test samples and controls (see Alternate Protocol 2)
6× electrophoresis sample buffer (see recipe)
Molecular weight marker (100-bp DNA ladder)

Electrophoresis apparatus with a 10 × 14–in. gel tray and a 1-mm, 10-tooth comb
Power supply
UV light box

Basic Protocol 3: Use of Antibiotics to Control Microbial Contamination

Materials

Contaminated cell culture
Sterile antibiotic stock solution(s) as appropriate (Table 1.5.1)

Additional reagents and equipment for identifying microbial contamination (see Basic Protocols 1 and 2; see Alternate Protocols 1 and 2)

Table 1.5.1 Antibiotics ^a

Organism	Antibiotic	Solvent	Stability (days at 37°C)	Working concentration

Bacteria (grem-positive only)	Ampicillin	Water	3	100 mg/liter
	Erythromycin	2 M HCI	3	100 mg/liter
	Gentamicin sulfate	Water	5	50 mg/liter
	Kanamycin sulfate	Water	5	100 mg/liter
	Neomycin sulfate	Water	5	50 mg/liter
	Penicillin-G, K⁺ salt	Water	3	10⁵ U/liter
	Streptomycin sulfate	Water	3	100 mg/liter
	Tetracycline HCI	Water	4	10 mg/liter
Fungi (molds and yeasts)	Amphotericin B	DMSO DMF^b	3	2.5 mg/liter
	Nystatin	DMF	3	2.5×10⁶ U/liter
Mycoplasma	Gentamicin sulfate	Water	5	50.0 mg/liter

^aAll antibiotics must be filter sterilized in the slovent noted. Antibiotics are meant only for short-term use; if contamination is not cleared after 14 days of treatment or two subcultures, discard culture.

^bAbbreviations: DMF; dimethylformamide; DMSO, dimethyl sulfoxide.

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Figures

Figure 1.5.1

Mycoplasma colonies at 230× magnification. Figure provided by W. Siegel, Bio-Whittaker, Inc.

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Literature Cited

Literature Cited
	Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Key References
	Barnhart, E.R. 1990. Physicians' Desk Reference. Medical Economics Company, Oradell, N.J.
Provides invaluable information on the mode of action of, for instance, antibiotics and antagonisms.
	Budavari, S., O'Neil, M.J., Smith, A., Heckelman, P.E., and Kinneary, J.F. (eds.) 1996. The Merck Index. Merck Research Laboratories Division of Merck & Co., Whitehouse Station, N.J.
A technical reference providing information on physical characteristics, solubilities, and stabilities of most chemicals (including antibiotics) used in a tissue culture laboratory.
	Freshney, R.I. 1994. Contamination. In Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., pp 243-252. Wiley-Liss, New York.
This chapter of the classic text on cell culture discusses various types of contamination and methods for detection and eradication.
	Hay, R.J., Caputo, J., and Macy, M.L. 1992. ATCC Quality Control Methods for Cell Lines, 2nded. American Type Culture Collection, Rockville, Md.
A manual of procedures used in this cell culture collection's quality control program.
	Sigma-Aldrich Co. 1998. Cell Culture Catalogue. Sigma-Aldrich Co., St. Louis, Mo.
Most specialty chemical companies feature a wealth of useful, technical information with their catalogues. This particular edition can be most helpful to the novice user of cell cultures.