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Mycoplasma-contaminated cells. Stained with Hoeschst stain. (Photograph courtesy of Dr. Gerald Massover)
Electron micrographs of mycoplasma growing on cell cultures.
Mycoplasma, the smallest free-living organisms, are too small to be seen with a light microscope, although fluorescent staining allows one to see them at high power (previous figure). (A) SEM and (B) TEM of Mycoplasma pneumonia on mammalian cells. The mycoplasma are about the same size as microvilli, but can be distinguished by being able to see both ends. (C) a more bulbous-shaped mycoplasma and (D) spiral spiroplasma growing on insect cells.
that depends on cultivation of mycoplasma or detection of mycoplasma using specific antibodies or polymerase chain reaction (PCR) (for example, the "Panverra" kit for PCR). Out of 3409 samples screened in one facility, 3.7% were positive by culture and an additional 0.7% positive by both Hoechst stain and PCR. No contamination was detected by culture that was not also positive using PCR and DNA staining detection methods (M. Roy, unpublished data). It would therefore seem advisable to use DNA staining or PCR for mycoplasma detection if the equipment necessary for these techniques is available.
Method for Fluorescent Detection of Mycoplasma
1. Hoechst 33258, 1 mg/ml stock solution (protect from light at 4jaC)
2. Acidic acid:methanol (1:3) fixative
3. Mounting solution (50% glycerol in water)
4. Nunc two-well or four-well chamber slides
6. Microscope with UV light source and 100X oil immersion objective suitable for use with fluorescence (Note: Some lenses will not transmit UV light)
1. Seed the culture so that 50% confluence can be reached in 48"C72 hr.
2. Incubate at 37jaC, 5% CO2, humidified. Place the chamber slides in a 150-mm tissue culture dish, if available, to facilitate handling and avoid spillage.
3. Remove the medium.
4. Wash 1 x with PBS.
5. Add PBS:fixative (1:1); leave at room temperature, 5 min.
6. Remove PBS:fixative and add straight fixative.
7. Remove and add fresh fixative; leave at room temperature for 5 min.
8. Remove fixative and rinse with distilled or purified water.
9. Make up a working solution of 100 ng/ml Hoechst 33258 in PBS.
10. Add working solution to chamber slide and leave at room temperature 10 min.
11. Remove the dye and rinse with water.
12. Remove the upper chamber from the slide. Make sure no silicon adhesive is still attached.
13. Mount a coverslip with a drop of mounting solution and examine the slide under epifluorescence with a 360-nm excitation 490 barrier filter.
Electron micrographs of virus infected cell cultures. Viruses, the smallest living organisms, must replicate inside cells, using part of the cell's protein synthesis molecules. They can only be seen using transmission electron micrography, but they can significantly alter cellular functions.
(A, B) Adenovirus. (C) Human herpes virus 6. (D) HIV on the surface of a human T cell.
Endogenous and contaminant viruses present in cell culture systems can be difficult and expensive to identify. Representative problem viruses include Sendai virus, simian virus, murine or human hepatitis virus, EpsteinCBarr virus, human cytomegalovirus, and human immunodeficiency viruses (HIVs) and other endogenous retroviruses. Protocols have been established by organizations such as the American Type Culture Collection (ATCC) and government agencies to screen for latent and chronic viruses. Additional protocols have been developed where cell lines are to be used for production of biological pharmaceuticals (Lubiniecki, 1990) (see Chapter 12). Most exogenous retroviruses are undetectable in cell cultures, except by serological or biochemical methods, or by the use of electron microscopy. Several electron micrographs of virally contaminated cells are shown in Fig. 7.5. Infected cultures do not necessarily display overt morphological change.
In general, all human cells should be handled as if they were a biohazard. Cells should be handled in a contained hood with air HEPA-filtered going into and out of the hood. All cell culture waste should be autoclaved after use and spent media should be treated with a viricide and disposed of appropriately. If primary tissues are needed, they can be tested for hepatitis and HIV when they are removed from the donor. Unless the laboratory is equipped for handling these agents, such infected tissues and cell lines should not be used, since viruses from cells and tissues can infect human handlers. Likewise, mouse viruses can spread from cell cultures to an animal colony. Therefore, many animal facilities require viral tests of cultures before the cells are injected into animals in a colony. If primary cultures are to be studied or used to establish new cell lines, always obtain animals from a reliable source that tests for viruses and mycoplasma in the animal colonies. If human sera are to be used in culture, use only sera that have been screened for viral contaminants.