WinterVomitingLab:Protocols/crystal violet

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Overview

Cell staining to aid in visualizing viral plaques. Used with MNV-1, FCV and HAV plaque assays.

Materials

• 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

Procedure

1. Remove liquid medium from agarose layers on virus-infected cell monolayers in 60 mm dishes (skip this step if no liquid is present).
2. Add 2-3 ml of 3.7% formaldehyde to each dish.
3. Incubate at room temp for at least 2 hr in the fume hood.
4. In the fume hood, pour off formaldehyde from each dish into chemical hazardous waste container.
5. 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.
6. Stain with crystal violet, adding enough stain to cover the cell monolayer.
7. Cells will be stained within 30 sec.
8. Remove crystal violet stain, reserving in a waste container (stain can be filtered and reused).
9. Rinse dishes in DI water. We use two buckets to sequentially rinse the plates.
10. 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.
11. Count plaques.

References

Relevant papers and books

Contact

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