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Colorimetric indicators of microbial contamination in corneal preservation medium
Y I Chu1, R L Penland, K R Wilhelmus
1Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.
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 degrees C, 22 degrees C, or 35 degrees 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 degrees 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 degrees C and 35 degrees 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 degrees C and 35 degrees 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.
Insights
AlamarBlue, a redox indicator, is more sensitive than phenol red for detecting microbial contamination in corneal media. However, neither indicator reliably detects Candida albicans, especially in buffered media at refrigeration temperatures.
Area of Science:
- Microbiology
- Biochemistry
- Ophthalmology
Background:
- Microbial contamination of corneal preservation media poses a significant risk to transplant success.
- Accurate and timely detection of contamination is crucial for maintaining the viability and safety of preserved corneas.
- Colorimetric indicators offer a potential method for monitoring microbial growth in these media.
Purpose of the Study:
- To compare the efficacy of acid-base (phenol red) and oxidation-reduction (AlamarBlue) indicators in detecting microbial growth.
- To evaluate the impact of buffer (HEPES) and temperature on indicator performance in corneal preservation media.
- To assess the detection limits for common microbial contaminants, including bacteria and Candida albicans.
Main Methods:
- Corneal preservation media with and without HEPES buffer were prepared using phenol red or AlamarBlue indicators.
- Media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans.
- Incubation was performed at 4°C, 22°C, and 35°C, with pH or percent reduction monitored over time.
Main Results:
- Indicator response varied significantly with microorganism type, temperature, and buffering.
- At 4°C, AlamarBlue detected some bacteria within 4 hours, while pH changes were not observed within 24 hours in buffered media.
- AlamarBlue demonstrated higher sensitivity than phenol red for bacterial detection, but neither reliably detected Candida albicans, particularly in HEPES-buffered media.
Conclusions:
- Refrigerated corneal preservation media may not show acidic shifts upon contamination by certain microorganisms.
- AlamarBlue (redox indicator) is more sensitive than phenol red (acid-base indicator) for detecting specific bacterial contaminants.
- Neither pH nor redox indicators reliably detect Candida albicans contamination in corneal preservation media under tested conditions.