Date of Award

Fall 1997

Document Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Dentistry

First Advisor

Kos, William L.

Second Advisor

Sohnle, Peter G.

Third Advisor

Ward, Timothy

Abstract

In this study, two stainjng methods, uptake of acridine orange (AO) and reduction of 3-[4,5-dimethylthiazol-2-yl]-2.5 diphenyltetrazolium bromide (MTT), and a microscopic proliferation assay were compared with the viable plate count (VPC) method for enumeration of viable Candida albicans. Cells were either freshly harvested from slant cultures, damaged by prolonged incubation in distilled water, or killed by boiling. In initial assessments of the three assays using various proportions of untreated and heat-killed C. albicans, the AO and MTT methods consistently yielded significantly higher values for viability than did the viable plate count. This difference was even greater when using organisms incubated for 3, 7, 10 or 14 days in distilled water. Repeated assays using various stainjng patterns as criteria for viability in the AO and MTT tests appreciably improved the correlation between the staining methods and the viable plate count. In additional experiments using an overnight culture, it was found that approximately 95% of the cells were capable of dividing at least once in a microscopic proliferation assay, whereas only 69% were capable of forming colonies. Assays comparing AO uptake and MIT reduction were in agreement with the microscopic proliferation assay. In tests using organisms undergoing prolonged incubation in distilled water, much lower viability was obtained with the viable plate count method at 7 and 10 days than with the microscopic proliferation assay or the two staining methods. These results indicate that the AO and MTT assays correlate well with the ability of C. albicans to divide at least once, but may not accurately indicate the percentage of organisms actually able to form colonies on solid media.

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