This technique is rapid (less than 30 minutes), but requires heavy contamination (106 mycoplasma/ml) to produce a clear positive result. If the suspect cells are co-incubated for 2–4 days with an “indicator” cell line (such as 3T3) that is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased. Cell cultures are stained with Hoechst 33258, a fluorescent stain, which binds specifically to DNA. Mycoplasma contain DNA and can be detected readily by their characteristic particulate or filamentous pattern of fluorescence in the cytoplasm or between cells. Mycoplasma-negative cells will show only brightly fluorescent cell nuclei. The Hoechst stain also detects bacterial and fungal contamination, although these are usually obvious from the visible turbidity of the culture medium of infected cells.
Have available the following materials:
· 25 cm2 flask with cell culture to be evaluated for Mycoplasma. Cells should have been grown to confluence for at least one passage in antibiotic free growth medium.
· balanced salt solution (BSS) without phenol red (BSS-PR), pH 7.0;
·
Hoechst 33258 stain(2-2(4-hydroxyphenol)-6-benzimidazolyl-6-/1-methyl-4-
pierpazyl)-lbenzimidalol-trihydrochloride: make up as 1 mg/ml (w/v) stock
solution in BSS-PR and store at -20°C until ready for use. For use dilute
10 μl in 200 ml BSS-PR. (Note: Hoechst stain may be carcinogenic
and must be handled with care);
· deionized or distilled water;
· fresh acetic acid/methanol fixative (cold):
Add glacial acetic acid to absolute methanol in the ratio of 1:3 (make up the day before use and refrigerate overnight);
· mountant: 50% glycerine in 0.044M citrate, 0.111 M phosphate buffer, pH 5;
· coverslips;
· 8-well chamber slide;
· adjustable pipettors;
· incubator at 36°C;
· refrigerator at 4–8°C;
· epi-illumination fluorescence microscope with 330/380 nm excitation filter and 440 nm barrier filter.
Test procedure
1) Trypsinize cell culture and prepare a cell suspension containing 0.5–1 x 105 cells/ml in antibiotic-free cell culture growth medium (see Section 4.2.5).
2) Using a sterile pipette place 0.5 ml cell suspension in each of the eight wells of a chamber slide. Incubate at 36°C and observe daily until the cell culture reaches approximately 20–50% confluence.
3) Remove and discard the growth medium from each well.
4) Gently rinse the monolayers with BSS-PR and discard rinse.
5) Add to each well 0.5 ml of freshly made BSS-PR diluted with acetic acid/methanol 50:50 v/v. Rinse the monolayers and discard rinse.
6) Add 0.5 ml acetic acid/methanol fixative to each well. Rinse and discard rinse.
7) Add 0.5 ml acetic acid/methanol fixative to each well and leave for 10 minutes.
8) Remove and discard acetic acid/methanol fixative from each well.
9) Wash off acetic acid/methanol with distilled water and discard wash.
10) Add 0.1 ml Hoechst 33258 stain in BSS-PR to each well, ensuring that the stain covers the complete surface of the well, and leave for 10 minutes at room temperature.
11) Remove stain and discard.
12) Rinse monolayer with water and discard rinse.
13) Peel off plastic chamber and silicone seal. Add a minimal volume of mountant, sufficient to just cover each monolayer, and place a coverslip on the slide.
14) Examine the slide using an fluorescence microscope with 330/380 nm excitation filter and LP 440 nm barrier filter. At least 500 x magnification (10 x ocular and 50 x objective) will be required to find evidence of Mycoplasma staining. Examine for evidence of extranuclear fluorescence. Mycoplasmas give pinpoint or filamentous fluorescence over the cytoplasm and, if heavily infected, fluorescence may also be seen in between cells. Since the Hoechst stain will stain all DNA, the nucleus of all cells should also stain brightly. Mycoplasma negative cells will show brightly staining nuclei and no staining in the cytoplasm.