I. INTRODUCTION

Members of the genera Mycoplasma and Acholeplasma of the order Mycoplasmatales (hereinafter referred to as mycoplasmas) are parasitic prokaryotes and are the smallest (0.2 to 2m in diameter) and simplest free-living parasitic organisms known (6). They lack a cell wall and possess the smallest recorded genome in living cells, 0.5 x 10⁹ to 1.0 x 10⁹ Da (11, 12). They naturally infect warm-blooded animals, arthropods, and plants and are capable of causing a variety of diseases. In humans they are most often involved in pneumonias, which accounts for their earlier name, pleuropneumonialike organisms, but they also play a role in postpartum fever, pelvic inflammatory disease, pyelonephritis, and nongonococcal urethritis. Little is known of their biology in vivo; however, it appears that most species do not invade cells but rather remain extracellular, found in association with the cell membrane or glycocalyx.

Mycoplasmas often contaminate cell cultures. It is estimated that 5 to 35% of cell cultures worldwide are contaminated with one or more species of mycoplasma (6). Of the 18 most common species recognized as culture contaminants, Mycoplasma orale, M. hyorhinis, M. arginini,M. fermentans , and Acholeplasma laidlawii are the most frequently isolated, representing 80 to 90% of isolates (2, 5, 10). In cell cultures, many of these organisms, especially the pathogenic mycoplasmas, grow in association with, and attached to, the host cell membranes, but in most cases remain extracellular. With some species, growth is best in the surrounding medium of cell cultures. It is surprising that most infections of cell cultures remain cryptic despite the fact that they can reach titers of 10⁸/ml (6), exceeding the cultured cells by over 100:1. In contrast, a few produce severe cytopathology with total destruction of the cell monolayer (1). Those that are not cytopathic nonetheless subtly interfere in numerous biochemical and cytogenetic events in the infected cultures (9, 10). Besides the lack of overt cytopathology, in most cases of culture contamination, some remain cryptic because mycoplasmas are not easily cultured away from the infected cultures, and in laboratories in which no further testing for their presence is done (6), they go unnoticed. Noteworthy in this regard are the difficult-to-cultivate strains of M. hyorhinis and certain strains of M. orale and M. fermentans , which are often referred to as noncultivable, growing very poorly or not at all in broth or agar but to high titers in infected cultured cells (3, 4, 7). Recently, Lo et al. (8, 13) isolated a strain of M. fermentans from human AIDS Kaposi's sarcoma DNA and suggested that the agent may play a role in the pathogenesis of AIDS. The agent could not be recovered by using broth or agar cultivation procedures. Because the noncultivable mycoplasmas so readily invade the cell culture environment and grow so well once there, infected cell cultures are ideal for use as model systems to study their pathogenesis, genetics, and molecular biology.

II. MATERIALS

1. Stock Hoechst 33258 stain (1mg/ml in Hanks Balanced Salt Solution)  

    a.  Working Hoechst stain: Just before use, dilute stock 1:4000 (25ul/100ml or 12.5ul/50ml) in CMF-PBS IN THE DARK.  Wrap the bottle in which the stain is diluted in tin foil. This should be enough for one wide-mouth jar.

(YOU SHOULD BE AWARE THAT HOECHST IS A MUTAGEN AND POSSIBLE CARCINOGEN. THEREFORE, BE CERTAIN TO WEAR GLOVES WHEN WORKING WITH HOECHST. IT IS SAFE TO WORK WITH AS LONG AS YOU TAKE THE PROPER PRECAUTIONS.)

2. (CMF-PBS)

3. Carnoy's Fixative (1 part Glacial Acetic Acid : 3 parts absolute methanol). This is the same fixative used in the karyology lab exercise.

4. Mountant - glycerine in citrate buffer:
    a. Solutions for making mountant:
        1.) 0.1M citric acid [2.1g citric acid monohydrate (210.14 mw) in 100ml water]
        2.) 0.2N dibasic sod. phos. (Na ₂HPO₄ - mwt.141.94) - 2.84 g in 100 ml water
    b. Final mountant:
        1.) 1ml citric acid : 1.27ml Na ₂HPO_4, at pH 5.5, [AND THIS IS CRITICAL]. To get this pH, add one or the other of the above until the pH is correct.
        2.) Then add an equal volume of glycerol. Assuming the pH is correct, the final mountant is:  
            1ml citrate : 1.27 ml Na2HPO4 : 2.27ml glycerol

5. Cells planted on coverslips.

6. Coverslip carrier

7. Four widemouth jars
 

III. PROCEDURE

Cell cultures:

In preparation for this exercise, for at least TWO passages, plant cells in a T-25 WITHOUTANTIBIOTICS in the medium. Then 2-3 days prior to when you plan to stain for mycoplasmas, plant the cells in petri dishes containing 2-4 coverslips. Coverslips must be prewashed in alcohol, dried, placed on filter paper in glass Petri dishes and autoclaved. Place 2-4 coverslips in the dish in which you are going to grow the cells, carefully add medium so that the coverslips do not float up and then add the correct number of cells so that, in the 2-3 days of incubation, the cell monolayers will be lessthan 3/4. In this case, you want spaces between areas of cell growth so that you can see perimeters of areas of growth. Gently swirl the dish to disperse the cells evenly and then incubate until you are ready for staining. Cells could also be grown on slides, prepared as above. The only difficulty here is that the entire slide will contain cells so handling becomes somewhat difficult.

Staining:

1. Fix the culture by removing the medium and adding back that volume of fixative.

2. Fix for 5 minutes.

3. Remove fixative/medium and replace with pure fixative for 5 minutes.

4. Using forceps (no need for sterility here) remove the coverslip from the dish and place it in the carrier, making certain you orient the coverslip in the carrier in such a way that you know on which side the cells are located.

5. Place the carrier containing the coverslips into a wide mouth jar containing fresh fixative and fix for 10 minutes.

6. At this point, after removal from the fixative and drying, they can be stored for later staining. They should be stored in a desiccator. If not stored in a desiccator, they should be redried 30 minutes in an oven at 60 ^o C for 30 minutes.

7. Prepare working Hoechst stain in a wide-mouth jar (see above #1a). Mix and allow to come to room temp.

8. Place the coverslip into the porcelain carrier and stain the coverslips, in the dark for 30 minutes. That is, cover the wide mouth jar containing the stain with tinfoil. (Why is this done?)

9. Remove the carrier from the stain and wash the coverslips with 3 one-minute washes in distilled water contained in three wide mouth jars also in the dark..

10.  In the dark, allow slides/coverslips to drain off water and shake off the excess.

11. Add a drop or two of mountant onto a clean, dry slide and invert the coverslip (i.e. cellsidedown) onto the drop(s) of mountant. Remove excess by blotting.

12. If the preparation is to be saved for future use, one can seal around the coverslip with clear nail varnish.

13. Examine in the fluorescent microscope using the Hoechst filter.
 

IV. RESULTS/DISCUSSION

A. If the cells are not infected, you will see only regular-shaped nuclei (smallish round discs) stained a brilliant robin's-egg blue color.

B. If the cells are infected, you will see the same as above only the nuclei will be surrounded by an outline of the cytoplasm in dots stained the same robin's-egg blue.

Why do the slides appear as they do if the cells are infected with mycoplasma?
 

V. REFERENCES

1. Bartle, M. F. 1973. Mycoplasma contamination of cell cultures: mycoplasma-virus-cell culture interactions, p. 131-172. In J. Fogh (ed.), Contamination in tissue culture. Academic Press, New York.

2. Barile, M. F. 1979. Mycoplasma-tissue cell interactions, p. 425-474. In J. G. Tully and R. F. Whitcomb (ed.), The mycoplasma, vol. 2. Academic Press, New York.

3. Barile, M. F., and M. W. Grabowski. 1983. Detection and identification of mycoplasmas in infected cell cultures by direct immunofluorescence staining, p. 173-182. In J. G. Tully and S. Razin (ed.), Methods in mycoplasmology, vol. 2. Academic Press, New York.

4. Barile, M. F., and G. J. McGarrity. 1983. Special techniques for isolation and identification of mycoplasmas from cell cultures, p. 155-210. In J. G. Tully and S. Razin (ed.), Methods in mycoplasmology, vol. 2. Academic Press, New York.

5. Del Giudice, R. A., and R. S. Gardella. 1984. Mycoplasma infection of cell culture: effects, incidence, and detection, p. 104-115. In E. M. Levine and R. E. Stevenson (ed.), Uses and Standardization of vertebrate cell cultures, vol. 5. In Vitro, monograph no. 5. Tissue Culture Association, Gaithersburg, Md.

6. Hay, R. J., M. L. Macy, and T. R. Chen. 1989. Mycoplasma infection of cultured cells. Nature (London) 339:487-488.

7. Hopps, H. E., B. C. Meyer, M. F. Barile, and R. A. Del Giudice. 1973. Problems concerning "noncultivable" mycoplasma contaminants in tissue cultures. Ann. N.Y. Acad. Sci. 225:265-276.

8. Lo, S.-C., J. Shih, P. B. Newton, D. M. Wong, M. M. Hayes, J. R. Benish, D. J. Wear, and R. Wang. 1989. The virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma. Am. J. Trop. Med. Hyg. 41:586-600.

9. McGarrity, G. J., and H. Kotani. 1985. Cell culture mycoplasmas, p. 353-390. In S. Razin and M. F. Barile (ed.), The mycoplasmas: pathogenesis of mycoplasma diseases, vol. 4. Academic Press, New York.

10. McGarrity, G. J., V. Vanaman, and J. Sarama. 1982. Cytogenetic effects of mycoplasmal infection of cell cultures: a review. In Vitro 20:1-18.

11. Razin, S. 1985. Molecular biology and genetics of mycoplasmas (Mollicutes). Microbiol. Rev. 49:419-455.

12. Razin, S. 1989. Molecular approach to mycoplasma phylogeny, p. 33-70. In R. F. Whitcomb and J. G. Tully (ed.), The mycoplasmas: spiroplasmas, acholeplasmas and mycoplasmas of plants and arthropods. vol. 5. Academic Press, New York..

13. Saillard, C., P. Carle, J. M. Bove, C. Bebear, S. C. Lo, J. W. Shih, R. Y. H. Wang, d. L. Rose and J. G. Tully. 1990. Genetic and serologic relatedness between Mycoplasma fermentans and a mycoplasma recently identified in tissues of AIDS and nonAIDS patients. Res. Virol. 141:385-395.

14. Schaeffer, W. I., L. D. Olson, M. F. Barile and F. W. Sun. 1991. Analysis of Mycoplams hyorhinis DNA in the presence of host cells without growing the mycoplasma axenically. J. Bact. 173:1382-1387.