Procedure for Testing Mycoplasma in Cell - Culture

1.    Scope

·       This procedure is done using a DNA specific fluorochrome; it can detect Mycoplasma Contamination in tissue culture.

2.    Definition

• DAPI   -           (4, 6 diamidino -2- phenylindole dihydrochloride)
• PBS     -           Phosphate Buffer Saline
• DNA    -           Deoxyrebpnucleic acid.

3.    Material

• 2 – 3 Days old cover slips Cell Cuture.
• Discarding bottle.
• Pipettes/Pasture pipettes.
• Pipetting aid.
• Forceps.
• Slides (75mm x 25mm x 1.25mm)
• Fluorescent Microscope
• Working solution of DAPI
• Camoy’s Fixative.
• PBS without Ca⁺⁺ & Mg⁺⁺
• Methanol (BDH – xxxx)
• Mountant
• Sealing material – Nail paint

4.    Procedure

• Prepare monolayer of cell cultures to be examined on coverslips in Petri plates (SOP no.6.9)
• At about 70% - 80% cell growth the coverslips are ready for staining.
• Remove the medium from the Petri plate with a pipette and discard in discarding bottle.
• Add 5ml PBS to the petriplate to wash the coverslips. Gently swirl. Remove and discard PBS.
• Add 5 – 7ml of Carnoy’s fixative. Close the lid of the petriplate. Allow 5min. for fixative of cells.

• Remove fixative, discard.
• Wash with 5ml of methanol. Allow coverslips to air dry. The cells are now fixed.
• Remove the required number of coverslips to be stained to another petriplate. (In case all are not to be stained. Otherwise the same petriplate can be used.)
• Cover the coverslips with DAPI stain. Close the petriplate. Allow standing for 15min. see that DAPI dose not evaporate.
• Remove DAPI to a separate bottle and store for re use.
• Label the bottle as DAPI working solution, number of times used, and date.
• Wash with methanol (2-3 times).
• Allow drying.
• Take a clean slide. (75mm×  25mm×  1.25mm )
• Place a drop of mountain at the center of the slide.
• Slowly invert the coversilp (Cell on the lower side of the coverslip) on to the mountain, take care that there are no bubbles trapped between the coverslip and slide.
• Wipe away the extra mountant with the help of tissue paper.
• Seal the slides of coverslip using nail paint.
• Label correctly with cell line, passage number, sate of seeding, and staining on the slide.
• Slide is now ready for observation.

1.    Safety conditions

• Close the lid of petriplate properly during fixation of cells so fixative does not evaporate.
• All reagents are discarding carefully.
• Staining time should be proper. (Not more or less than 15 min.)
• During staining step see that DAPI does not evaporate.
• Take care during mounting of coverslip, there are no bubbles trapped between coverslip and slide.

                         

2.    Documentation  

• Cell line sensitivity maintenance register PR-39.

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.