Cultural Revolution: Mycoplasma Testing Kits and Services

Date: October 11, 1999Mycoplasma Detection Kits Mycoplasma Testing Services Most scientists who engage in cell culture find it such a chore to split cells on a regular basis that conscientious habits like routine testing for mycoplasma remain an unrealized ideal. Although mycoplasma contamination can potentially foil the most crucial of experiments, one may argue that mycoplasma testing is expensive, time consuming, or complicated. Fortunately, times are changing. Several companies have developed commercial kits that make it quick and easy to add mycoplasma testing to your list of regular laboratory procedures. And if you don't want to take the time to do the testing yourself, a number of companies provide mycoplasma testing services. This article reveals the nature of mycoplasma that may be lurking in your cultures, examines the different types of products and services available for mycoplasma testing, and discusses the available options for eliminating mycoplasma from contaminated cultures. PCR detection with Stratagene's Mycoplasma Plus Primer Set Several separate studies have shown that at least 10 percent of cells may be contaminated with mycoplasma.1 This should be a concern for anyone doing cell culture. Mycoplasma can evade the typical precautions used in cell culture facilities, and contamination can affect cell metabolism globally, producing artifactual results in experimental systems. Mycoplasma are the simplest prokaryotes, devoid of a cell wall and consisting of only a plasma membrane, ribosomes, and a double-stranded genome of approximately 580 kb.2 Mycoplasma can form colonies as large as 600 µm in diameter, but can also exist during their life cycle as single cells as small as 0.15 µm in diameter.3 These parasites are present in numerous tissues of man and animals, with over 100 species identified to date. Due to its small size, a single mycoplasma can pass through the 0.22 or 0.45 µm filters typically used to sterilize tissue culture reagents and is not visible during routine examination of cultures by microscopy. To make matters worse, penicillin and streptomycin, two antibiotics that are routinely used to prevent microbial contamination, act on bacterial cell wall components and thus have no effect on mycoplasma. Mycoplasma can be introduced into a particular culture through many different routes: tissue culture reagents, laboratory personnel, the tissue used to establish primary cultures, and, most commonly, from another culture.4 Mouth pipetting was a common source of mycoplasma contamination in the past.3 Today, routine reliance on antibiotics in culture media has deemphasized the importance of good aseptic technique, which has led to the introduction of many organisms, including mycoplasma.5 Mycoplasma are often slow-growing contaminants and will not usually outnumber the cells of interest, but can still cause experimental artifacts. Mycoplasma contamination has been shown to alter cellular growth rates, morphology, and viability; affect metabolism of amino and nucleic acids; induce chromosomal and membrane aberrations; affect cell antigenicity; mimic viral actions; and interfere with viral replication.5,6,7 To avoid gathering artifactual data from unknowingly contaminated cultures, scientists should routinely test for the presence of mycoplasma. If detected, it may be possible to eliminate the contamination and avoid the need to discard contaminated cultures. Both direct culture techniques and indirect assay methods can be used for mycoplasma detection. Indirect methods include DNA staining, biochemical detection, nucleic acid hybridization, immunoassays, and polymerase chain reaction (PCR) DNA amplification. Indirect methods have the advantage of detecting "noncultivatable" mycoplasma species. Assay methods can typically detect several, if not all, of the most common cell culture contaminants (Mycoplasma hyorhinis, M. arginini, M. orale, M. fermentans, M. salivarium, and Acholesplasma laidlawii) as well as some of the more obscure species. Identification of the contaminating species, also called speciation, is useful in determining the source of the contamination.8 For example, identification of the human isolate M. orale in a mouse hybridoma line would suggest that laboratory personnel were the source of contamination. Whether testing is done on premises or farmed out, the advantages and disadvantages of each type of detection method should be considered, and often the use of two different methods is recommended to offset the limitations of any one method.1,5,6 Before one begins testing, it is essential to handle and prepare the cells properly. Enzymes (such as trypsin), pH extremes, and slow freezing of cells can degrade the plasma membrane of mycoplasma, releasing proteases and nucleases that could interfere with certain assays.1 Because both cryopreservants and antibiotics interfere with testing protocols, cryopreserved stocks and antibiotic-treated cells should be passaged at least twice in antibiotic-free media prior to testing.6 Direct culturing of cells or culture reagents to screen for mycoplasma is perhaps the most sensitive assay for mycoplasma contamination, theoretically capable of detecting a single viable mycoplasma cell.1 Cells or reagents are used to inoculate liquid broth or agar plates and both anaerobic and aerobic cultures maintained for up to four weeks. Cultures are then examined by light microscopy for the presence of mycoplasma colonies. True colonies can be distinguished from other types of cellular growth with a Dienes stain. While protocols exist for preparing the mycoplasma growth media and performing a direct culture assay, commercially available products can streamline the process.6 Life Technologies sells agar plates with mycoplasma broth for direct culture assays. The MYCOTRIM™ TC kit (Irvine Scientific) uses a triphasic (broth, air/agar, and agar) flask culture system that yields results in only four to seven days. Another option for direct culture testing is to use a testing service, eliminating the need to culture mycoplasma as a positive control and risk further contamination. Mycoplasma-contaminated cells detected by ICN Biomedicals' Hoechst stain The presence of mycoplasma in cell cultures can be assayed by DNA fluorochrome staining. Although other fluorochromes such as DAPI can be used, Hoechst stain is considered the method of choice. Fixed cells are stained with Hoechst 33258 (bisbenzamide) and examined under UV epifluorescence. If mycoplasma are associated with the cytoplasm of cells, stained cell nuclei will be surrounded by fluorescing structures in the cytoplasm. To increase sensitivity or to test suspension cell lines or culture reagents, cells or reagents can be inoculated onto an indicator line of Vero (African Green monkey kidney) cells. The large cytoplasm of these cells facilitates the interpretation of results. Accurate interpretation of results from DNA staining for mycoplasma detection requires an experienced eye, as all prokaryotes infecting the cytoplasm will fluoresce. Commercial Hoechst mycoplasma staining kits (Sigma-Aldrich and ICN Biomedicals) offer the advantage of fixed, mycoplasma-positive control slides. These allow a direct comparison of staining patterns without the necessity of growing mycoplasma cultures in your laboratory as a positive control. Life Technologies has developed the MYCOTECT™ Kit based on the unique biochemistry of mycoplasma. Mycoplasma contain significant levels of adenosine phosphorylase, which can convert 6-methylpurine deoxyriboside (6-MPDR) into two toxic products (6-methylpurine and 6-methylpurine riboside). In the MYCOTECT assay, an indicator cell line grown in a 24-well plate is inoculated with the cell line to be tested for mycoplasma. The 6-MPDR substrate is added, and after two days of additional growth, a crystal violet stain is added to check for cell viability. Mycoplasma-positive cultures produce toxic agents, causing the death of the indicator cell line; the cells in these test wells do not retain the crystal violet stain. Easily interpretable results and the use of adenosine phosphorylase as a positive control are two advantages of this system. The Gen-Probe Mycoplasma Tissue Culture NI (MTC-NI) Rapid Detection System (distributed through Fisher Scientific) is a commercially available kit using hybridization technology for detection of mycoplasma in cultured cells. The system uses a chemiluminescent, single-stranded DNA probe to detect levels of mycoplasma rRNA in cell extracts. The target rRNA is detected using Gen-Probe's Hybridization Protection Assay technology. Immunofluorescence and enzyme-linked immunosorbent assays (ELISAs) can be used to detect mycoplasma contamination. In the immunofluorescence assay (sold as a kit by both Accurate Chemical & Scientific and ICN Biomedicals), fixed cells are stained with a fluorescently conjugated monoclonal antibody that recognizes several mycoplasma species; the cells are then examined on a fluorescence microscope. If additional sensitivity is required, a fluorescently conjugated secondary antibody supplied with the kit can be used to enhance the signal. ELISA protocols use an antibody sandwich technique, with mycoplasma antigens captured in polyclonal antimycoplasma antibody-coated wells. Captured antigens are then bound by biotinylated antimycoplasma antibodies. After incubation with an avidin-enzyme conjugate, an enzymatic reaction produces a colored substrate that can be quantified. Roche Molecular Biochemicals' Mycoplasma Detection Kit includes four separate polyclonal antibodies for the user to coat plates with and allows identification of the infecting mycoplasma species. Stratagene's Mycoplasma EIA Detection Kit includes a 96-well plate precoated with an antibody that can recognize any of six different mycoplasma species and therefore does not pinpoint the offending mycoplasma species. Mycoplasma detection by PCR can be performed using a single set of primers or a nested PCR technique. The primers used in mycoplasma PCR kits anneal to conserved regions of the mycoplasma genome, allowing the detection of several species of mycoplasma. PCR detection kits from Stratagene, Panvera, and ATCC require that the amplified products be analyzed by agarose gel electrophoresis. Primer sets in commercial kits can produce either a single band as a plus-minus indicator of mycoplasma infection or multiple product banding patterns that must be interpreted to confirm the presence of mycoplasma. As an example, the Mycoplasma Plus™ PCR Primer Set from Stratagene amplifies a single 874 bp product if mycoplasmal DNA is present in an extract from the cultured cells. To determine the species responsible for the contamination, the PCR product can be digested with Sau 3A and the resulting fragments examined by gel electrophoresis. The banding pattern will represent a fingerprint that is unique to a particular mycoplasma species. The Mycoplasma PCR ELISA from Roche Molecular Biochemicals relies on a different principle for detecting mycoplasma and cannot determine the species contaminating a culture. This kit includes digoxigenin-dUTP in the nucleotide cocktail used for the PCR reaction. The PCR product is then captured in microtiter plate wells and incubated with an anti-digoxigenin-peroxidase conjugate. An enzymatic reaction with TMB substrate produces a colored product that can be quantified using a plate reader. Once a lab commits to routine (monthly or quarterly) mycoplasma testing, the next task is to decide what method will be used and whether the lab will do its own testing or engage a testing service. Scientists should consider the following variables when choosing a testing method: equipment available, cost, time required, type and number of samples to be tested, and if the work is subject to regulation by a governing body, such as the Food and Drug Administration (FDA).4 The gold standard for assaying mycoplasma contamination is to perform both a direct culture test and a DNA staining method using an indicator cell line.1 For research laboratories, the disadvantages of performing these types of assays on site may lead individual investigators to choose PCR or immunoassays if they want to do the test themselves. Numerous companies provide mycoplasma screening services, running the gamut from companies that do an occasional test by PCR on a custom basis to those that provide FDA-specified testing of cell lines that will be used to produce biologicals such as vaccines. FDA standards mandate the use of well-defined multiple direct culture protocols and an indirect DNA staining protocol. If a cell line is to be used for research purposes only, less stringent direct culture testing is available from several companies, including American Type Culture Collection, Anmed/Biosafe, Bionique Testing Laboratories, and ViroMed Laboratories. Bionique Testing Services, (Saranac Lake, N.Y.), has been in the mycoplasma detection services field for almost 20 years and is the only company in the world solely devoted to testing of cell cultures and related products for mycoplasma contamination. With such a sustained level of expertise in mycoplasma detection, Bionique's staff will work with labs to choose the right assay for their particular needs. Bionique performs DNA staining, direct culture assays, FDA-compliant testing, species identification, and antibiotic susceptibility studies of infected samples. No review of mycoplasma detection technologies is complete without mentioning how to eliminate contamination once detected. If the cultures can be replaced, conventional wisdom suggests autoclaving the contaminated cells, decontaminating the cell culture facility, and starting over with fresh cultures.1,4,6 At this point, knowledge of speciation is useful; if one can identify the route of infection, one can minimize the chances of introducing the same type of contamination into fresh cultures. For irreplaceable cell lines, the use of a commercially available antibiotic specific for mycoplasma may be the best solution. Three such products are Mycoplasma Removal Agent (ICN Biomedicals), M-Plasmocin™ (InvivoGen), and BM-Cyclin (Roche Molecular Biochemicals). A second approach would be to test cultures for antibiotic susceptibility and then design an appropriate antibiotic cocktail treatment. Bionique can both perform the susceptibility test and, if desired, decontaminate the cells for investigators. One recent study suggested that antibiotic treatment for mycoplasma contamination is successful almost three-fourths of the time.9 For hybridomas, Maine Biotechnology Services (MBS), offers cell passage in mice as a decontamination option. Mycoplasma-contaminated cells are injected into mice and decontaminated cells are retrieved, subcloned, and rescreened to assure the absence of mycoplasma before being returned to the investigator. MBS clients who have mycoplasma-positive cultures identified by the company's screening services can request the decontamination service for an additional fee. Linda S. Raab can be contacted at raabx004@maroon.tc.umn.edu. C. Lincoln et al., "Cell culture contamination: sources, consequences, prevention, and elimination," Methods in Cell Biology: Animal Cell Culture Methods, vol. 57, San Diego, Academic Press, 1998, pages 49-65. D. McKenzie et al., "A new PCR-based mycoplasma detection method," Strategies, 11[1]: 21-3, Stratagene Inc., 1998. . B.M. Martin, Tissue Culture Techniques: An Introduction, Boston, Birkhauser, 1994. M.A. Harrison et al., Handbooks in Practical Animal Cell Biology: General Techniques of Cell Culture, Cambridge, U.K., Cambridge University Press, 1997. R.J. Hay, "Testing cell cultures for microbial and viral contami- nants," Cell Biology: A Laboratory Handbook, San Diego, Academic Press, 1994, pages 25-62. Stacey et al., "Routine testing of cell cultures and their products for mycoplasma contamination," Methods in Molecular Biology: Basic Cell Culture Protocols, vol. 75, Totowa, N.J., Humana Press Inc., 1997, pages 305-11. "The mycoplasma story and ... how to detect this major problem in cell culture," Biochemica, 1:23-4, Roche Molecular Biochemicals, 1999. R. Hay et al., "Mycoplasma infection of cultured cells," Nature, 339:487-8, 1989. E. Fleckenstein, H.G. Drexler, "Elimination of mycoplasma contamination in cell cultures," Biochemica, 1:48-51, Roche Molecular Biochemicals, 1996. Mycoplasma Detection Kits Mycoplasma Testing Services

Cultural Revolution: Mycoplasma Testing Kits and Services

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