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Cell staining to aid in visualizing viral plaques. Used with MNV-1, FCV and HAV plaque assays.
- 37% formaldehyde
- Prepare a 3.7% solution by diluting (1:10) in PBS.
- Don't use water in this step. pH buffering is needed.
- crystal violet stain
- 2.5 g crystal violet powder
- 50 ml methanol
- 197.5 ml Milli-Q DI water
- Remove liquid medium from agarose layers on virus-infected cell monolayers in 60 mm dishes (skip this step if no liquid is present).
- Add 2-3 ml of 3.7% formaldehyde to each dish.
- Incubate at room temp for at least 2 hr in the fume hood.
- In the fume hood, pour off formaldehyde from each dish into chemical hazardous waste container.
- Remove the agarose layer from the fixed monolayer using a pipette tip (10-200 ul tip), taking care to not touch or disturb the underlying cells.
- Stain with crystal violet, adding enough stain to cover the cell monolayer.
- Cells will be stained within 30 sec.
- Remove crystal violet stain, reserving in a waste container (stain can be filtered and reused).
- Rinse dishes in DI water. We use two buckets to sequentially rinse the plates.
- Let the dishes air dry facing down on an absorbent cloth. This prevents pooling of liquid on the cells, potentially creating holes in the monolayer.
- Count plaques.
Relevant papers and books
- Who has experience with this protocol?
or instead, discuss this protocol.