The use of human fibroblasts with an agarose overlay medium was found to be a reliable and practical method for the plaque assay of Herpesvirus hominis (1). This technique has now been modified for use in assaying cytomegalovirus (CMV) strains of human origin. Two methods for the quantitation of cytomegalovirus have been described previously, in addition to the traditional determination of 50% tissue culture infectivity doses by tube dilution. The first of these, the microscopic counting of infected cells on coverslip preparations after either immunofluorescent (2) or Giemsa (3) staining had obvious limitations and technical difficulties. Plummer and Benyesh-Melnick (4) then described a plaque assay technique using methyl cellulose in the overlay medium. Since agarose overlays are more easily handled than methyl cellulose and afford a firm overlay to prevent viral spread, we investigated the use of an assay with two sequential agarose overlays. The criteria for a useful plaque assay described by Cooper (5) were used to evaluate this technique.

Materials and Methods. Cells. The FT line of human fibroblasts was derived in our laboratory from embryonic tonsillar tissue. Routine cultures of this cell line have never yielded any mycoplasmata when incubated both aerobically and anaerobically during the period of the study.

Media. Cells were grown in Eagle's minimum essential medium with 10% fetal calf serum, 6.6 mM sodium bicarbonate and appropriate antibiotics (1). Virus was diluted in maintenance medium containing 2% “GG-free” calf serum in place of fetal calf serum (MEM2). Overlay medium consisted of equal parts of double strength MEM2 and 0.6% agarose (SeaKem, Bausch and Lomb Inc., Rochester, N.Y.) in distilled water. The sodium bicarbonate was increased to a final concentration of 8.0 mM in the complete medium.