Cell monolayers were inoculated with 169 fresh and 76 previously frozen clinical specimens and examined for the presence of herpes simplex virus (HSV) by noting the appearance of characteristic cytopathic effect (CPE) and by using a direct immunoperoxidase (IP) stain for viral antigen. HSV was detected by IP staining in 40 of 169 (23.7%) monolayers and by CPE in 39 of 169 (23.1%) monolayers inoculated with fresh specimens. All 40 isolates were detected and confirmed by IP staining within 24 h. Although 39 of 40 isolates were detected by CPE, only 9 of 39 (23%) were positive within 24 h. CPE was observed at 2.7 days on the average, but 4 days were required before 90% of the cultures were positive and more than 5 days were required before all HSV isolates were recognized. Similar results were observed for frozen specimens. HSV was detected earlier with IP staining, which demonstrated more extensive infection of cell monolayers inoculated with titrated fresh culture isolates and clinical specimens than did CPE. IP staining reduces the amount of time required for detection and identification of HSV in culture, is readily adaptable for use in the clinical laboratory, and permanent stained preparations can be made.

One hundred fifty-two blood specimens, largely from immunocompromised patients, were collected in heparinized Vacutainer tubes and divided into paired aliquots of equal volume. Buffy-coat preparations, containing mixed leukocyte and separate mononuclear and polymorphonuclear leukocyte populations were obtained by treatment of blood with conventional and Ficoll-Paque/Macrodex (F-P/M) methods. The development of cytopathic effect in monolayers of WI-38 fibroblasts inoculated with cell suspensions derived from the two methods was used to assess virus infectivity. Twice as many virus isolations were obtained using F-P/M. Of those viruses isolated by both conventional and F-P/M, the development of cytopathic effect was more extensive using the latter method. Moreover, a greater variety of viruses was isolated using F-P/M method, as compared to the conventional method. The F-P/M method is no more time consuming than conventional procedures, is readily adaptable for use in the diagnostic virology laboratory, requires only minimal additional cost, and is a particularly suitable and effective means of monitoring viremia.

Cytomegalovirus and varicella-zoster virus recovery from sucrose phosphate (0.2 M SP) and 70% sorbitol (sorbitol) was compared after storage at -70, 4, and 20 degrees C over time. Recovery from 0.2 M SP was uniformly better. More tissue culture infective doses and infectious foci were recovered in cell monolayers inoculated with 0.2 M SP virus stocks as compared with viruses stored in 70% sorbitol. Although both viruses were isolated from diluted fresh stocks (10(-1) through 10(-4], freezing diluted virus suspensions generally resulted in diminished recovery. Similar stabilizing effects on respiratory syncytial virus and herpes simplex virus type 1 infectivities were observed when stocks were preserved in 0.2 M SP, as compared with 70% sorbitol. Overall, 0.2 M SP was better than 70% sorbitol for stabilizing cytomegalovirus, varicella-zoster virus, respiratory syncytial virus, and herpes simplex virus type 1 infectivities under the conditions tested.

The effect of ozonation of supply water for one wing of an unoccupied hospital building which had positive cultures for Legionella pneumophila from multiple potable water fixtures was studied in a prospective, controlled fashion. Mean ozone residual concentrations of 0.79 mg/liter eradicated L. pneumophila from the fixtures, but so did nonozonated water in the control wing fixtures. The efficacy of the nonozonated water was most probably due to a mechanical flushing effect and to an unexpected rise in the chlorine content of the supply water. Determination of the in vitro activity of ozone against L. pneumophila did not predict the efficacy of its eradication from water fixtures treated with ozone.

Although semen is a particularly rich source of cytomegalovirus (CMV) and is useful for monitoring CMV shedding, its culturing is associated with extensive monolayer toxicity, isolation failures, and lengthy detection times. Inoculation of fractionated semen with immunoperoxidase staining of monolayers eliminated virtually all toxicity, increased isolation rates and monolayer infectivity, and greatly reduced detection times. Semen specimens (n = 73) were processed conventionally (C) or separated into supernatant (SF) and cellular pellet (PF) fractions, and 35% of C and SF inocula produced extensive toxicity. In contrast, virtually no toxicity was observed in monolayers inoculated with PF. C and SF isolation rates were 41 of 73 and 38 of 73, respectively, whereas that for PF was 51 of 73. Although monolayer infectivity at initial CMV detection was often <10% for C and SF, it was as much as 25% for PF. Average detection times were reduced from 13 days for C and SF to 6 days with PF and were further reduced to 3 days when PF inoculation was combined with immunoperoxidase staining. Thirty percent of specimens negative by C were positive by PF.