C-199. Temperature Optimization in Preparation of Slides for AFB Smear Microscopy

S. Jones, J. A. Westerling, R. Droste, P. Elvin, A. Sloutsky;
Massachusetts Dept. of Pub. Hlth., Jamaica Plain, MA.

AFB SmearFluorescent microscopy remains an important means of mycobacterial diseases diagnostics. Optimization of each step can improve the sensitivity of this method and increase its programmatic value. Heat fixation is a universal step in all types of AFB smear preparation. It is unclear however, what is the optimal temperature is which allows for visualization of thefor this step which is permissible for the highest quantity of AFB per slide. Using an infrared thermometer, wWe have tested several commercial slide warmers by using an infrared thermometer and demonstrated that the temperature in different parts areas of each of them varied by 10-20º C. Therefore, for the purpose of this study we used one of thea precision instruments slide warmer with the an accuracy of + 0.1º C. A set of slides was prepared using non-infectious sputum mixed with the culture of non-heat inactivated TB bacilli which was diluted adequately to obtain slides with the a bacterial load less thanof approximately 1 AFB per low power field. The entire slide set was validated via by testing a representative sample read by different readers to insure the consistency of the set to within two standard deviations of the average. . Slides from the set were heat fixed for 2 two hours using different temperatures ranging from 55º to 95º C. Slides were then stained using a standard protocol, air dried and evaluated by five independent operators. The sSignificance of our findings was verified using statistical analysis. It was demonstrated that the optimal temperature was 75º C. The amount number of AFB seen at this temperature was about 14% higher than at 55º C and 100% higher than at 85 º C. Similarly, heat fixation by flame will be optimized by means of a specially designed slide holder with variable height. Optimization of other steps of smear preparation is currently underway.