WinterVomitingLab:Protocols/HAV plaque assay

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Overview

Quantify infectivity of Hepatitis A Virus (HAV).

Materials

• 2X Complete HAV DMEM
  • DMEM without phenol red (Sigma- cat# D2902)
  • 4% Atlanta Bio Fetal Bovine Serum (Atlanta Biologicals- cat# S11050H)
  • 2% Penicillin/Streptomycin (VWR- cat# 16777-164)
  • 2% Non-essential amino acids (VWR- cat# 12001-634)
  • 5% Sodium Bicarbonate (VWR- cat# 45000-704)
  • 2% Nystatin (Sigma- cat# N1638)
• Lonza SeaKem LE Agarose (Fisher- cat# BMA 50004)

Procedure

1. At least 45 minutes prior to infection, place infection media, 2X HAV MEM, and a sterile 500 ml glass bottle in the 37°C water bath, and place 1% SeaKem agarose (that has been previously autoclaved and re-melted) in the 45°C water bath.
2. After making sample dilutions, aspirate growth medium from the 60 mm dishes, and add 0.4 ml of infection media to each dish.
3. Plate 100 ul of sample dilution. Place trays in 37°C incubator with 5% CO2 for 1 hour, rocking the trays every 15 minutes.
4. After 1 hour, aspirate the inoculum and media from the cells.
5. Immediately add agarose overlay as follows- mix equal amounts of 1% SeaKem agarose and 2X HAV MEM, and add 5 ml to each dish.
6. Allow agarose to solidify (should not take longer than 10 min).
7. Leave trays in incubator for 7-8 days (checking for plaques at days 6, 7 & 8).

 * When working with a large number of 60 mm dishes, we have started adding 1 ml of 1X HAV MEM on 
   top of the solidified agarose layer.  This prevents the underlying cells from drying out.

If plaques can be visualized, proceed to staining procedure with crystal violet or neutral red staining.

References

Relevant papers and books

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