Characteristics of plaque formation in cell culture of primary chick embryo (PCE), have been found useful as a biological marker in differ en t ia t ing Herpes virus hominis (H V H ) oral type 1 from genital type 2 strains (1, 2). Type 1 strains appear to or have been reported to produce small plaques and to have lower efficiency of plaque formation than type 2 strains in PCE cell culture. We describe a simple microtest for differentiation of HVH types based on the apparent differential sensitivity of PCE cell culture in microplates. The type of cytopathic effect (CPE) and the efficiency with which the virus infects the PCE monolayer are used as criteria for typing. The results of microtyping in PCE are compared with kinetic neutralization (K) and immunofluorescence (IF) typing.Materials and Methods. Tissue Culture. Ten per cent fetal calf serum in Eagle's minimal essential medium (MEM) with 100 IU/ml of penicillin, 100 pg/ml of streptomycin, 50 pg/ml of mycostatin, 6.6 mM sodium bicarbonate and 2.0 mM L-glutamine was used throughout.Primary chick embryo cells were obtained from minced 9-to 10-day old embryos by dispersion with 0.2% trypsin. Strains of golden Syrian hamster embryo cells (HE) and rabbit kidney cells (RK), originated in our laboratories. Human diploid fetal tonsil (FT) fibroblasts, were kindly supplied by Dr. Berttina Wentworth, Seattle. The passage numbers were as follows: HE 15-17, RK 1-3; and FT 15-25.Viruses. Herpesvirus hominis strains used in these experiments included four that were of known type and 106 clinical isolates from Taipei and Seattle. The reference strains were: UW-168 (3) and KOS (4), type 1; UW-268(3) and 196-2(4), type 2. The UW-168 and UW-268 strains had been passed approximately 50 times in HEp-2, HeLa and FT cells. Of the 106 clinical isolates, 59 were genital in origin; 57 were from the cervix of patients attending venereal disease clinics, and two were urinary isolates. The nongenital isolates included, 45 from throat swabs (from a study of respiratory infection), one from typical herpes labialis, and one from a brain specimen obtained at autopsy. The previously untyped herpes strains were in the 2nd through 10th passage at the time of typing, initially isolated in FT, HEp-2 or primary monkey kidney cells and passed in FT and/or HEp-2 cells. Virus pools for all strains were prepared in HE, RK or FT cells and have titers ranging from lO4mo to TCID50/ml as assayed in a microtest system using HE or FT cells. Two of the untyped clinical isolates, NHS-17 and 23, were passed an additional nine times in RK and then either ten times in FT (NHS-17) or 21 times in HE cells .Virus rnicrotyping. Serial tenfold dilutions of the stock virus to be typed were made in MEM. Diluted virus (0.05 ml) was delivered to two wells per dilution in each of two 324

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