IMPROVEMENTS IN THE QUANTIFICATION OF AIRBORNE MICROORGANISMS IN THE FARM ENVIRONMENT BY EPIFLUORESCENCE MICROSCOPY

Micro-organisms in samples collected in different farm environments we re analysed by staining with a fluorochrome and counting with an epifl uorescence microscope. Errors from preparation losses and non-specific fluorescence of non-microbial particles were found when the CAMNEA me thod was used. These errors could be reduced by modifications of the C AMNEA method including the use of a destaining technique, and the use of DAPI instead of acridine orange as the fluorochrome. Non-specific f luorescence was substantially reduced if samples were destained using a citrate buffer at pH 3 followed by iso-propanol. Preparation losses were reduced to approximately 6% if the samples were resuspended in 0. 1% Tween-80 by 3 min agitation in an ultrasonic bath. Aggregates were also disrupted by ultrasonic treatment. Nevertheless, aggregates were observed using all the methods where micro-organisms could not be disc riminated and counted and the number of microorganisms in such aggrega tes was estimated by linear regression. The occurrence of such aggrega tes was lowest when the samples were destained. Significantly higher c oncentrations of micro-organisms were found in samples from three of f our farm environments with the modified CAMNEA method, using an improv ed resuspension and destaining technique, even if preparation losses w ere included and micro-organisms in uncountable aggregates were estima ted. Copyright (C) 1996 British Occupational Hygiene Society.