IMPROVEMENTS IN THE QUANTIFICATION OF AIRBORNE MICRO-ORGANISMS IN THE FARM ENVIRONMENT BY EPIFLUORESCENCE MICROSCOPY

摘要

Micro-organisms in samples collected in different farm environments were analysed by staining with a fluorochrome and counting with an epifluorescence microscope. Errors from preparation losses and non-specific fluorescence of non-microbial particles were found when the CAMNEA method was used. These errors could be reduced by modifications of the CAMNEA method including the use of a destaining technique, and the use of DAPI instead of acridine orange as the fluorochrome. Non-specific fluorescence 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 discriminated and counted and the number of micro-organisms in such aggregates was estimated by linear regression. The occurrence of such aggregates was lowest when the samples were destained. Significantly higher concentrations of micro-organisms were found in samples from three of four farm environments with the modified CAMNEA method, using an improved resuspension and destaining technique, even if preparation losses were included and micro-organisms in uncountable aggregates were estimated.