Recognition errors in the quantification of micro-organisms by fluorescence microscopy

Objectives. An interlaboratory comparison of fluorescence microscopic count ing of microorganisms was carried out to assess counting errors in the meas urement of micro-organisms in bioaerosols generated during handling of muni cipal waste. Methods. Series of 27 replicate samples were collected in the work environm ent with a modified field exposure chamber. The preparation methods of thre e Scandinavian laboratories were compared. Four microscopists from these la boratories performed the counts which were also compared. Duplicate countin g of identical microscopic fields allowed the assessment of recognition err ors. Results. The field exposure chamber collected replicate samples with a rela tive standard deviation of 5% when particles less than or equal to 15 mum a erodynamic diameter were collected. Storage time of 40-200 days had no sign ificant influence on the total micro-organism count. Differences between pr eparation methods were from 2 to 35% for bacteria, and from 15 to 35% for f ungal spores when samples were analysed in Oslo; the results for fungal spo re counts were significantly different (P <0.01). These differences were no t confirmed when samples were analysed in Ume (a) over circle, Copenhagen a nd Oslo using those laboratories methods. These results can be explained by less efficient redispersion of aggregates when the Ume (a) over circle and Copenhagen methods were recreated in Oslo yielding a greater number of inn umerable aggregates. Differences between microscopists were minor for funga l spores (2-12%) but substantial for bacteria (4-53%). A major source of er ror was the recognition of bacteria which had a relative standard deviation (rsd) of 37% although a lower size limit of 0.75 mum was adopted for count ing of bacteria. Fungal spores were recognised with much better precision ( rsd 9%). Conclusions: Recognition errors of bacteria may be substantial and more spe cific fluorochromes are needed for fluorescence microscopic counting of mic ro-organisms. (C) 2001 British Occupational Hygiene Society. Published by E lsevier Science Ltd. All rights reserved.