Testing for Mycoplasma by Indirect DNA Stain (Hoechst 33258 stain)

Aim
DNA staining methods such as Hoechst staining techniques are quick with results available within 24 hours, which compares favorably with 4 weeks for detection by culture. However the staining of cultures directly with a DNA stain, results in a much-reduced sensitivity (~10₆cfu/ml). This may be improved by co-culturing the test cell line in the presence of an indicator cell line such as Vero (Prod.No. 84113001-1v1). This enrichment step results in a sensitivity of 104 cfu/ml of culture. This step also improves sensitivity by increasing the surface area upon which mycoplasma can adhere. Like detection by culture, DNA staining methods are suitable for the detection of mycoplasma from cell cultures or cell culture reagents.

Materials

• Media– pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct medium)

• 70% ethanol in water (Prod. No. R8382)

• Methanol (Prod. No. 175)

• Acetic Acid Glacial (Prod. No. A6283)

• Hoechst 33258 stain solution (Prod. No. H6024)

• Vero cells (Prod. No. 84113001-1v1)

• Mountant (Autoclave 22.2ml 0.2M citric acid with 27.8ml 0.2M disodium phosphate. Add 50ml glycerol. Filter sterilize and store at 4ºC) (Prod. No. M1289)

• Mycoplasma hyorhinis NCTC* 10112

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)

• Waterbath set to 37ºC

• Microbiological safety cabinet of appropriate containment level

• Centrifuge

• CO₂ Incubator set at 37ºC

• Microscope (uv Epi-Fluorescent.)

• 35mm plastic tissue culture dishes (Prod. No. C6296)

• Multidish 24 well (Prod. No. M9655)

• Cell scraper

• Microscope slides and 22mm cover slips

• Aluminum foil (Prod. No. Z185140)

Procedure Equipment

1. For each sample and control sterilize 2 cover slips in a hot oven at 180ºC for 2 hours or by immersing in 70% ethanol (Prod. No. R8382) and flaming in a blue Bunsen flame until the ethanol has evaporated. Also sterilize 2 cover slips to use as a negative control.

2. Place the cover slips in 35mm culture dishes (Prod. No. C6296) (1 per dish).

3. Store until needed.

4. To prepare the Vero (Prod. No. 84113001-1v1) indicator cells add 2x104 cells in 2ml of antibiotic-free growth medium to each tissue culture dish.

5. Incubate at 37ºC in 5% CO₂ for 2 – 24 hrs to allow the cells to adhere to the cover slips.

6. Bring attached test cell lines into suspension using a cell scraper. Suspension cell lines may be tested directly.

7. Remove 1ml of culture supernatant from duplicate dishes and add 1ml of test sample to each. Inoculate 2 dishes with 100cfu M. hyorhinis and 2 with 100cfu M. orale.. org.uk

8. Leave duplicate tissue culture dishes un-inoculated as negative controls.

9. Incubate dishes at 37ºC in 5% CO₂ for 1-3 days.

10. After 1 day observe one dish from each pair for bacterial or fungal infection. If contaminated discard immediately. Leave the remaining dish of each pair for a further 2 days.

11. Fix cells to cover-slip by adding a minimum of 2ml of freshly prepared fixative (1:3 glacial acetic acid: absolute methanol) to the tissue culture dish and leave for 3 to 5 minutes.

12. Decant used fixative to toxic waste bottle. Add another 2ml aliquot of fixative to cover-slip and leave for a further 3 to 5 min. Decant used fixative to toxic waste.

13. Air dry cover-slip by resting it against the tissue culture dish for 30-120 min.

14. Replace cover-slip in dish and add a minimum of 2ml Hoechst stain (Prod. No. H6024). Leave for 5 minutes shielded from direct light by aluminum foil (Prod. No. Z185140).

15. Decant used and unused stain to toxic waste.

16. Add 1 drop of mountant to a pre-labeled microscope slide and place cover-slip (cell side down) onto slide.

17. Keep slide covered with aluminum foil (Prod. No. Z18514-0), allowing it to set for at least 15 min at 37ºC or for 30 min at room temperature.

18. Observe slide under uv Epi-Fluorescence at x1000.

Notes

1. DNA stains such as Hoechst stain (Prod. No. H6024) bind specifically to DNA. In all cultures cell nuclei will fluoresce. Uncontaminated cultures will show only fluorescent nuclei whereas mycoplasma positive cultures contain small cocci or filaments which may or may not be adsorbed onto the cells (see figure 16).

2. Hoechst stain is toxic and should be handled and discarded with care.

3. Culture dishes should be placed in a sealed box or cultured in large petri dishes to reduce evaporation.

4. Positives should obviously be handled in a laboratory remote from the main tissue culture laboratory.

5. Control organisms (M. hyorhinis) are available from the National Collection of Type Cultures (UK).

6. In some instances results may be difficult to interpret for the following reasons:

• Bacterial/yeast/fungal contamination

• Too much debris in the background

• Broken nuclei as cells are all dead

• Too few or no live cells

Although this procedure recommends the setting up of positive controls, this may not necessarily be feasible nor desirable in a cell culture facility with limited resources. If positive controls are to be set up they should be done so in a separate laboratory from the main tissue culture facility. If this is not possible then positive slides can be purchased from ECACC. If positive controls are not being used then it is strongly recommended that you get an independent testing laboratory to periodically test your cell lines.