Practical Technique- Fluorescence Staining for Mycoplasma

Fluorescence Staining Technique for detection of Mycoplasma Contamination:

The cultures are stained with Hoechst 33258, a fluorescent dye that binds specifically to DNA. Because mycoplasmas contain DNA, they can be detected readily by their characteristic particulate or filamentous pattern of fluorescence on the cell surface and if the contamination is more fluorescence pattern is observed in surrounding areas. Monolayer cell cultures can be fixed and stained directly, but after centrifugation, the medium from cells growing in suspension will need to be added to an indicator cell (i.e., another monolayer) known to be free of mycoplasma but also known to be a good host for mycoplasma and to spread well in culture, with adequate cytoplasm to reveal any adherent mycoplasma. The cell lines that can be commonly used include epithelial cell lines from mammals like the Normal Rat Kidney (NRK), 3T6, Vero cells and A549. Using an indicator cell line is suitable in the technique as it helps avoid problem of false positives that arise due to debris during the assay of primary cultures or of cells right after thawing. In case of more debris, we can filter the medium by a sterile 5µm filter membrane or centrifuged at about 1000 rpm.

PROTOCOL FOR FLUORESCENCE DETECTION OF MYCOPLASMA CONTAMINATION

Culture cells in the absence of antibiotics for at least one week, transfer the supernatant medium to an early log phase-indicator culture, incubate 3–5 days, fix and stain the cells, and look for fluorescence other than in the nucleus.

Materials:

Sterile or Aseptically Prepared:

• Supernatant of medium that do not contain antibiotics, from a seven day monolayer or centrifuged suspension cell culture 
• Petri dishes 
•  Indicator cells (e.g. NRK, 3T6, Vero cells, A549)

Non-sterile:

• Hoechst 33258 stain, 50ng/ml in BSS without phenol red (BSS-PR) or D-PBSA 
• Buffered glycerol mounting mixture (substance used to mount microscopic specimens) 
• BSS-PR: Hank's Buffer Saline Solution without Phenol Red 
• D-PBSA: Dulbecco’s Phosphate Buffer Saline without Ca²⁺ and Mg²⁺ 
• Deionized water 
• Freshly prepared acetic acid - methanol (1:3) used as fixative.

Procedure:

1.    Place the given indicator cells into Petridishes without the use of antibiotics; use sufficient cells to yield about 60% confluence (cells growing and coming together to cover the medium) in about 5 days (e.g. 10⁵ A549 or 20⁵ NRK, in 5 ml of medium).

2.    Take 1.5ml of medium from the test culture.

3.    Keep the culture for incubation till 60% confluence is attained by the cells.

Note: Confluence of cells should not be attained at the end of assay otherwise the staining will be inhibited and the resulting visual for mycoplasma could be unclear.

After every rinsing, the rinse should be discarded properly.

4.    Discard the medium.

5.    Monolayer should be rinsed with BSS-PR or D-PBSA.

6.    Rinse the monolayer again with fresh BSS-PR or D-PBSA diluted 50:50 with fixative.

7.    Rinse with pure fixative.

8.    Take more fixative ∼0.5 ml/cm² over the monolayer, and allow fixation to continue for 10 minutes.

9.    Discard the fixative after the 10 minutes fixation (of previous step).

10. Monolayer must be completely dried for storage.

(Samples can be stored at this stage and can be used later for staining)

11. For staining directly, the fixative must be washed using deionized water.

12. Hoechst 33258 mixed with BSS-PR or D-PBSA is added to the cells and allowed it to stain at room temperature for 10 minutes.

13. Rinse the monolayer with water to remove the stain.

14. Take a coverslip, one drop buffered glycerol mounting mixture and mount it for observation, blot out excess mounting mixture from the edge of coverslip.

15. Observe monolayer under epi-fluorescence microscope with a 330-380nm excitation filter and a Long pass (LP) 440nm barrier filter.

Analysis:

Observe for extra nuclear fluorescence. Mycoplasma gives pinpointed or filamentous fluorescence on the cytoplasm and sometimes in intracellular space also. The location of pinpoints is close to the limit of resolution with a 50x objective (0.1–1.0µm) usually of regular size and shape. All cells will may not be infected, so very careful observation must be done to finally reach a conclusion and confirming any solution as uninfected.

At few times, a light, uniform staining on cytoplasm is seen, which could be due to RNA.

If there doubts to interpret the fluorescence test result, the test should be repeated further after sub-culturing test cells in absence of antibiotics.

In case of repeated unclear results, PCR or ELISA can be performed.

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Did you read ? 
Maintenance of Sterility in Animal Tissue Culture.

Detection of Mycoplasma Contamination in Animal Cell Cultures.

Also read:

Viral Contamination in Animal cell cultures.