MICROSCOPIC DEMONSTRATION OF MYCOPLASMA CONTAMINATION IN CELL CULTURES AND CELL CULTURE MEDIA Submitted by

T. R. CHEN Biology Department The University of Texas Houston, Texas 77025 I.

INTRODUCTION Various methods have been reported to demonstrate mycoplasma contamination in tissue cultures (1). Of these the procedure to be described here is comparable to the aceto-orcein staining method (A-O method) of Fogh and Fogh (2) but different in that it uses a highly specific fluorescent stain and is technically rapid, simple, and easy reproducible. In a properly stained slide from a mycoplasma-free culture, nuclei fluoresce a bright, whitish-yellow, whereas the cytoplasm does not fluoresce at all and is as dark as the background. Therefore, the DNAcontaining mycoplasmas, with or without membrane association, are readily distinguishable from others as the uniform-sized fluorescent extra-nuclear objects in the culture. So far, all mycoplasma species and strains we studied were readily demonstrable by the present method.

II.

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Miscellaneous: 1) Kronig cement or other alternative for sealing the edges of cover slip (Will3), 2) Sealing device (Glenco4), and 3) Blotting papers or filter papers Remarks: All equipment should be kept absolutely clean.

A. Sources of contamination in cells or media from cultures

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MATERIALS

B. Media: Specific medium that is used to culture cells being tested

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C. Culture flasks, glassware Petri dishes of various sizes in which a cover slip or microscope slide will fit. For example, use a 40x 22 mm cover slip in a 60x15 mm Petri dish or regular microscope slide in a 100x20 mm Petri dish. Both glass and plastic dishes can be used. Alternatives: 1) Leighton tubes 16 x 93 mm with cover slips, No. 1905-16095 Bellco 1 or 2) Flaskette Lab-Tek 4820 Fisher 2 Cover slips and microscope slides: 1) Any size glass slides can be used. Use No. 1 (or No. llA) grade thickness to improve the resolution in microscope observation. 2) There is no specification for the brand of slide to be used. Cells will grow on most commercially available glass slides. Generally these are acid washed before sterilization. Glassware: 1) Graduated cylinders or centrifuge tubes for centrifuging media, 2) Coplin jars (No. 8-816) or beakers, 50 or 100 rnl size, 2 and 3) Aspirator for decanting waste

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D. Chemicals and stains Fixative: Carnoy's fixative or a mixture of 3 parts absolute methanol (any brand) and 1 part glacial acetic acid (any brand). Mix before use. Rinsing solution: Filtered, distilled or deionized water preferred Stain: Bisbenzimidazol compound 33258 Hoechst Formula: 2 - [2 - (4 - hydroxyphenyl) 6 - benzimidazolyl] - 6 - (1 - methyl- 4 piperazyl) - benzimidazol - trihydrochloride This compound was a gift from Dr. H. Loewe, Pharma Synthese, Farbwerke Hoechst, 6230 Frankfurt (Main) 80, Postfach 800320, W. Germany. It is now available from Riedel de Haen AG, 3016 Seelze, Wunstorfer Strasse 40, Bundesrepublik Deutschland. Solvent: Sterile Hanks' solution, pH 7.0, without phenol red Stock and working staining solutions: Stock solution I: Dissolve 5 mg H-stain in 100 ml Hanks' solution (50 pg/ml). Stock solution II: Dilute 0.1 ml of stock solution I in 10 ml of Hanks' solution (0.5 pglml).

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1Bellco Glass, Inc., Vineland NJ 2Fisher Scientific Co., Houston, TX 3Will Scientific Co., Atlanta, GA 4Glenco Scientific Co., Houston, TX

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Working s o l u t i o n : D i l u t e 0.5 tion II in solution t r a t i o n .05

ml o f stock solu5 ml o f Hanks' ( f i n a l concenpg/ml).

IV.

PROCEDURE

A. Remarks: Stock Solution II and working solution should be clear, i.e.,transparent. Only a small amount of working solution is made each time because this solution has the highest possibility of being contaminated by microorganisms. All stain solutions should be stored under refrigeration (4 ~ C) and kept in the dark (wrapped in aluminum foil). Phosphate buffered saline (PBS) or any other isotonic solutions Citric acid mounting medium: Citric acidphosphate buffer is m a d e as follows: 22.2 ml of 0.1 M citric acid + 27.8 ml of 0.2 M disodium phosphate (Na~HPO4). The pH of this mixture should be close to 5.5 (or adjust to 5.5 with either solution above). All solutions should be kept under refrigeration and as germ-free as possible. (Generally, these solutions are filtered through millipore filter, and then stored in the sterile container. Antibiotics are not used for reducing germs. Instead, filtered solutions are stored at a smaller a m ount in each container and under refrigeration. Solution is discarded when the germ growth is detected.)

Demonstration of mycoplasmas by growing cells suspected of containing mycoplasmas on a cover slip or a microscope slide .

Plate approximately l 0 S cells/ml onto a 40 x 22 mm cover slip in a 60 x 15 mm Petri dish.

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Leave in CO2 incubator (5% CO2; 37~ for 4 hours to allow complete cell attachment onto the cover slip.

3.

Gently add 3 ml medium, return to incubator and grow cells for 1-2 days.

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After 1-2 days, remove from incubator, add about 10 drops of fixative directly to cells on cover slip. (Note: This is done without decanting medium or disturbing the cells.)

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After 2 minutes decant the entire solution mixture and add 3 ml of fixative for 5-10 minutes.

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Carefully remove the cover slip and air dry.

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Stain completely dried cell preparation by adding 3-4 drops of working solution (.05 pg/ml stain) to the cell surface, invert cover slip, and lay onto a clean microscope slide--cell surface is now facing down and i~ immersed in staining solution trapped in between cover slip and glass slide. Do not press out excess stain. Leave preparation in this position for 10 minutes.

F. Microscope Any kind of fluorescent microscope Use excitor filters BG 12 or BG 3 and two barrier filters in a combination of 53/44 (these are from Zeiss microscope system). Subject containing DNA will appear bright whitish yellow. Cytoplasmic portion of cells may be elucidated if a combination of excitor filter UG 1 and the barrier filter is used. Subjects appear whitish blue.

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Remove cover slip by dipping the whole preparation (cover slip plus glass slide) into distilled water in a Coplin jar, or 50 or 100 ml beaker. Rinse the cover slip several times more in distilled water.

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Mount the cover slip on a slide with the cell surface down using the citric acid mounting medium. Drain off excess mounting medium by placing blotting paper along the edge of cover slip.

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E. Other equipment COf-moisture chambered incubator Use desiccator, filled with mixed air conraining 5% CO2, and place in walk-in warm r o o m or dry incubator (37~

G. Photographing: Kodak second exposure

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H. Developing film: Use a 1:1 dilution of D-19 in distilled water; develop for 4 minutes at 70~ 230

Seal edges with Kronig cement and examine under microscope. (For immediate and quick examination, we do not seal cover slip. Dried cover slip can be reconstituted by adding a drop of mounting medium at an edge of cover slip to let medium seep inside by capillary action.)

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grain deposits (using autoradiography), and a special aceto-orcein staining procedure have been used previously. We have chosen to use 1) a fluorescent microscopic system that provides higher resolution than that obtained from ordinary light microscopy, 2) available fluorescent stains that bind specifically to DNA or chromatin, and 3) a staining procedure providing sufficient fluorescent expression by mycoplasmas to distinguish them from other cytoplasmic DNA components, e.g., mitochondrial DNA. The H-stain originally used by cytogeneticists is one which meets all the above-mentioned requirements (3, 4). This stain probably binds to DNA by intercalation and shows enhanced fluorescence at the repeated base pair A-T rich sites of the DNA-protein complex. We have tried other chromatin-specific stains such as quinacrine mustard and quinacrine dihydrochloride. H o w e v e r , t h e s e show brightly fluorescent mycoplasmas together with a greatly increased background fluorescence. Moreover, fluorescence is quenched very rapidly. Consequently, the H-stain is a better stain for this system because it not only stains mycoplasmas distinctly but also gives a bright and long-lasting fluorescence.

Demonstration of mycoplasma in culture medium in which cells have been grown 1.

Decant old medium from culture flask into a clean centrifuge tube.

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Centrifuge at approximately 1500 rpm for 15 minutes to concentrate the contents (detached cells, cell debris, suspending organisms including mycoplasmas, etc.).

3.

Decant supernatant carefully and then resuspend the pellet in 2-3 ml of isotonic saline (e.g., PBS). Treat pellet gently and a v o i d c l u m p formation during the resuspension.

4.

Centrifuge again. Decant supernatant, resuspend the pellet in 2-3 drops of isotonic saline, and then slowly add a few drops of fixative to the suspension and stir. Repeat this process until total volume is brought up to 3 ml.

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Follow standard procedures and also information suggested under Materials section (steps F through H) in this procedure for the necessary measures on microphotography.

B. Expectations In most instances, mycoplasmas appear microscopically as spherical or ellipsoid shapes, uniformly stained and without detectable components other than presumed DNAprotein complex. The diameter of the spherical form generally ranges from .1-.3 micron. They may be evenly distributed or aggregated in large clusters over the cell surface. Massive aggregates between cells are also detected. They may be in doublets, quadruplets, or chain strings. Some may appear threadlike, forming a network over the cell surfaces. Most mycoplasmas fluoresce brightly, but some are only dully expressed. These bright and dull types occur simultaneously in the same culture. It is as yet unknown whether they represent different species or the same species in different stages of development.

Let stand 5 minutes, spin down, and decant supernatant. Resuspend the pellet in fixative, adding enough to equal about twice the volume of the pellet.

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Spread a couple drops of the pellet suspension onto a clean microscope slide and air dry. Stain the slide by adding 3-4 drops of the working solution directly onto the slide. Carefully place a plain, clean cover slip over the stain drops to equally distribute the stain solution over the glass slide. Then follow steps A-7 through A-10.

IV. DISCUSSION A. Rationale

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C. Failures

Mycoplasmas, a group of the smallest existing organisms, are known to contain DNA. They may be freely suspended in culture medium or directly associated with host cells around the cell periphery. Other biochemical or microbiological characteristics of mycoplasmas will not be discussed here. Obviously mycoplasma detection requires a means which would provide higher resolution in localizing DNA specific components both around the cell periphery and in between cells. For this purpose, fluorescent expression of fluorescein~conjugated anti-serum, fluorescent Feulgen stain of DNA, membrane-bound silver

The following factors may cause confusion in demonstrating mycoplasmas: 1. Cell density. The best result in demonstrating mycoplasmas is obtained from preparations with nicely flattened cells with a broad cytoplasmic area. In densely populated sites with heavy cell density, cells are often so closely spaced that brightly fluorescing nuclei will interfere with detection of tiny mycoplasmas. Crowding creates depth of cellular preparation and, hence, lowered resolution power. Stain may easily be trapped in this cell mass, resulting in 231

cytoplasmic fluorescence. Also staining of these preparations is variable; often they will be poorly stained. Therefore, the number of cells per glass plate should be appropriately monitored to avoid overcrowding.

The method is simple, rapid and efficient. These points are illustrated by the following: 1. Cover slip cell growth can easily be set up for cell subcultures or for routine screening. 2. The method takes only 1/~ hour from fixation of cells to microscopic observation.

2. Micronuclei and other nuclear fragments. Occasionally micronuclei or other nuclear fragments from presumably dead cells appear as spherical bodies. However, they are distinguishable from mycoplasmas by their larger and variable size, as well as brighter fluorescent expression.

3. A quick microscopic scanning may easily cover thousands of cells, e.g., a field under a 25X objective and 12.5X eye lens may contain about 50-100 cells. 4. Both the number of mycoplasmas per cell and the number of infected cells per culture can readily be estimated.

3. Sterility. Use of unclean material such as glass slides, mounting medium, and old stain cause bacterial growth. These bacteria are often stained and will cover the cell surface. They also alter stain expression probably due to a pH change of the mounting medium, and therefore interfere with the demonstration of mycoplasmas.

5. Set amounts of cells or degree of infection are not required. 6. All handling procedures are simple. 7. The use of stain is minimal.

D. Safety and precautions

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Since the method is so simple, no special precautions are required except for proper handling of cultures to avoid cross-contamination. (In our experience, mycoplasma-contaminated and noncontaminated cultures in Petri dishes have been placed side by side in a CO~ incubator without cross-contamination. However, such an arrangement probably will greatly increase the chance of contamination and should be avoided.) CAUTION: The H-compound is known to bind to DNA by intercalation and is not easily removed; therefore, great care must be taken to avoid direct contact with the stain.

REFERENCES

1. Fogh, J., N. B. Holmgren, and P.P. Ludovici. 1971. A review of cell culture contaminations. In Vitro 7:26-41.

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2. Fogh, J., and H. Fogh. 1964. A method for direct demonstration of pleuropneumonia-like organisms in cultured cells. Proc. Soc. Exp. Biol Med. 117:899-901.

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3. Hilwig, I., and A. Gropp. 19 7 2. Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. Exp. Cell Res. 75:122-126.

E. Advantages and disadvantages The only disadvantage for the method is perhaps its failure to discriminate among mycop l a s m a species. Nevertheless, this becomes advantageous because it will indiscriminately stain all mycoplasma species (that we have studied thus far) and, therefore, can be used for routine mycoplasma screening. This method is also capable of detecting mycoplasmas in cell suspensions.

4. Hilwig, I., and A. Gropp. 1 9 7 3 . D e c o n d e n s a t i o n of constitutive heterochromatin in L cell chromosomes by a benzimidazole compound ("33258 Hoechst"). Exp. Cell Res. 81:474-477.

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