A combination detergent wash-optical staining technique was developed to determine the concentration of virus polyhedral inclusion bodies (PIBs) present on samples of common forest substrates. Experiments were performed to determine the efficacy of the procedure for estimating the abundance of applied virus of gypsy moth, Lymantria dispar (L). (Lepidoptera: Lymantriidae), from samples of bark, leaf litter, and soil. Detection and quantification of inclusion bodies present in the wash supernatant was accomplished by staining the inclusion body protein and observing the stained particles using phase contrast microscopy. Positive identification of the virus particles and the determination of virus activity was accomplished through the bioassay of the wash supernatants. The mean recovery rate of virus applied to substrates, after washing and staining, was high (76.5%) but differed between bark (65%), leaf litter (68%), and soil (79%). No difference in recovery rates was detected when different concentrations of virus were applied to the same substrates. Bioassays were performed for virus concentrations ranging from 103 to 107 PIBs per ml by applying virus directly to artificial diet, and by applying virus to forest substrates, subjecting these substrates to the washing procedure, then applying the wash supernatant to artificial diet. An analysis of covariance was performed on the doseresponse curves of control virus and virus washed from the three substrates. The analysis revealed no significant differences between the two curves, indicating that the washing and staining procedure itself has no effect on virus viability. The optical enumeration technique, therefore, provides a reliable and inexpensive method for the assessment of virus abundance on substrates in the environment.
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Taverner et al. (1992) studied this question.