Sunday, February 3, 2019

Procedure for staining cell sensitivity plate


1.    Scope
·       This SOP explains procedure to stain the cell sensitivity plate.

2.    Definition
  • ART: Aerosol Resistant Tips

3.    Material
  • 0.025% Crystal Violet stain prepared in 10% formalin
  • Distilled water
  • 200 µl multichannel micropipette or Single channel 50-200 µl pipette
  • 200 µl ART tips
  • Plates to be stained
  • Discarding jar with freshly prepared 1% Na hypochlorite solution
  • Trough
  • Tray
  • Kimwipes
  • Gloves

4.    Procedure
i.            Observe and take reading of cell sensitivity plate microscopically on 7th day before staining.
ii.          All the steps of staining should be performed in BSC-II.
iii.        Carefully remove the medium from the plates taking care not to touch the monolayer with the help of multichannel pipette (adjusted to 200µl).
iv.        Pour the stain in the trough and with the help of multichannel pipette add 50 µl of 0.025% crystal violet stain in all wells touching the side of wells.
v.          Incubate the plates with stain for 40 minutes at room temperature.
vi.        After incubation carefully remove the stain from the plates taking care not to touch the monolayer with the help of multichannel pipette.
vii.      Pour distilled water in fresh trough and add 200µl in all wells and carefully remove the water from the plate taking care not to touch the monolayer with the help of multichannel pipette (adjusted to 200µl).
viii.    Repeat the above step twice (step no.vii).
ix.        After the plate is washed, invert it on Kimwipes in a tray and keep for drying in BSC-II.
i.            The wells which are violet are negative and which are clear are considered as positive.
ii.          Compare the result with microscopically taken readings of the plate.
iii.        Plates can be wrapped with paper and stored at room temperature for longer periods for record.


1.    Safety conditions
·       To avoid creation of aerosols transfer the fluid gently during procedure.
                        
2.    Documentation  
  • Cell line sensitivity worksheet-

3.    References used to draw SOP
·       WHO Provided Kit Literature

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