Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

First Advisor

David A. Benfield

Second Advisor

Mary Jo Taqua


Clinical microbiology laboratories performing C. difficile toxin assays usually elect to maintain only cell lines shipped from suppliers. In this study, four cell lines, primary human foreskin (HFS), Chinese Hamster Ovary (CHO-Kl), HEp-2, and WI-38 were tested for sensitivity to the effects of C. difficile cytotoxin. The influence of the age of the culture and duration of the assay, 4 versus 24 hours, was also examined. Twenty-eight positive and 13 negative patient specimens were tested. Tubes of cells were inoculated at ages of 3,4,5,6,7,9, and 14 days and examined for cytopathic effects at 4 and 24 hours post inoculation. The four cell cultures detected fewer fecal samples positive for C. difficile toxin when the assay was read at 4 hours compared to 24 hours after inoculation. However, the HFS cell line was the most sensitive in detecting C. difficile toxin compared to the other three cell lines. Five to 6-day-old primary HFS cells were 14 to 17% more sensitive than the other three cell types. Sensitivity of all four cell cultures to C. difficile toxin decreased as the age of the cell cultures increased. The HFS cells detected C. difficile toxin, in fewer fecal samples, but were still 12-18% more sensitive at_ 7 days of age, at 9 days of age, 13-20% more sensitive and 12-20% more sensitive at 14 days of age than were the HEp-2, CHO-Kl and Wl-38 cells. The sensitivity of all four cell lines to C. difficile toxin was greater if the cytotoxicity assay was observed at 24h after inoculation. This was especially true for fecal samples containing low levels of C. difficile toxin. The HFS cells were 3-7% more sensitive at 24h after inoculation compared to the other three cell cultures. However, the HFS cell line remained 100% sensitive except on days 3 and 7, when one true positive was not detected. In conclusion, HFS, CHO-Kl, HEp-2 and WI-38 cells are less sensitive to the effects of C. difficile toxin as the cultures age. Primary HFS cells are more sensitive than CHO-Kl, HEp-2 and WI-38 cells for detection of C. difficile toxin in feces, regardless of the age of the cells and the time of incubation of the cytotoxicity assay.

Library of Congress Subject Headings

Clostridium difficile -- Toxicology
Cell culture
Cytochemical bioassay



Number of Pages



South Dakota State University


No Copyright - United State