DIAGNMICROBIOL INFECTDIS 203 1987;8:203-214 BACTERIOLOGY Effect of Age on the Sensitivity of Cell Cultures to Clostridium difficile Toxin Jo Tichota-Lee, Mary Jo Jaqua-Stewart, David Benfield, Jerry L. Simmons, and Richard A. Jaqua The effect of age on the sensitivity of four cell lines, human foreskin fibroblasts (HFS), CHO-K1, HEp-2, and WI-38 to detect Clostridium di~ficile toxin was tested. This study also addressed the sensitivity of these cell lines as expressed by early toxin detection. Twenty-eight positive and 13 negative patient specimens were tested. Cell cultures were inoculated at ages 3, 4, 5, 6, 7, 9 and 14 days and examined for cytopathic effects at 4, 24, and 48 hours post-inoculation. The sensitivity of three of the four cell cultures to C. difficile toxin decreased as the age of the cell cultures increased. However, the sensitivity of the HFS cell line was not influenced by the age of the culture or the time the assay was read in comparison to the other three cell lines. Five- to six-day-old HFS cell cultures detected 22/28 positive samples within 4 hr after inoc- ulation and 28/28 positive samples by 24 hr post-inoculation. INTRODUCTION Since the 1970s when Clostridium difficile was confirmed as the etiologic agent of some antimicrobial-associated diarrheas and of pseudomembranous colitis (PMC), laboratory studies have been performed to determine the most sensitive cell-culture system for the detection of C. difficile toxin. Various investigators have reported several cell lines susceptible to the toxin (Chang et al., 1979; Donta and Shaffer, 1980; Donta et al., 1982; Maniar et al., 1984; Murray and Weber, 1983). During pilot studies to establish the procedure for detection of C. difficile toxin in our laboratory setting, it was observed that older cells seemed to be less susceptible to the toxin than younger cells (24--48 hr after seeding). Age of cell lines was a technicality not addressed by earlier studies evaluating the sensitivity of various cell lines (Chang et al., 1979; Donta and Shaffer, 1980; Murray and Weber, 1983) with the exception of Maniar et al., (1984), who specified that cell cultures were used after overnight incubation if the monolayer was confluent. Virology laboratories per- forming C. difficle toxin testing may acquire cell cultures through in-house prepa- ration of primary or continuous cell lines, local suppliers, or suppliers geographically distant to the laboratory. This study was initiated to address the effect of age of cell From the University of South Dakota School of Medicine, Department of Laboratory Med- icine, Sioux Fails, South Dakota (J.T.-L., M.J.J.-S, J.L.S., R.A.J.), and Department of Veterinary Science, South Dakota State University, Brookings, South Dakota (D.B.). Address reprint requests to: Jo Tichota-Lee, University of South Dakota School of Medicine, Clinical Virology Laboratory, 2501 West 22nd Street, Sioux Falls, SD 57101. Received June 9, 1987; revised and accepted October 2, 1987. © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017 0732-8893/87/$03.50