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Colorimetric Indicators of Microbial Contamination in Corneal Preservation Medium

Chu, Yvonne I. M.D.; Penland, Rebecca L. M.(A.S.C.P.); Wilhelmus, Kirk R. M.D., M.P.H.

Clinical Sciences

Purpose. To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media.

Methods. Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4°C, 22°C, or 35°C. The pH or percent reduction were determined hourly for eight hours, then daily for one week.

Results. The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4°C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22°C and 35°C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2–3 days at 22°C and 35°C.

Conclusion. Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.

From the Sid W. Richardson Ocular Microbiology Laboratory, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, U.S.A.

Submitted August 19, 1999.

Revision received December 4, 1999.

Accepted December 13, 1999.

Address correspondence and reprint requests to Dr. K.R. Wilhelmus, Department of Ophthalmology, 6565 Fannin Street, NC-205, Houston, TX 77030, U.S.A. E-mail:

Presented in part at the 38th annual scientific session of the Eye Bank Association of America, Orlando, FL, October 23, 1999.

© 2000 Lippincott Williams & Wilkins, Inc.