Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans

Image and multifactorial statistical analyses were used to evaluate the int ensity of fluorescence signal from cells of three strains of A. pullulans a nd one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in di rect or indirect FISH reactions. In general, type of fixation (paraformalde hyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for va rious times, though needed to admit high M-w detection reagents (avidin-FIT C) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluores cence about fourfold over unilabelled probes. Overall, direct FISH was pref erable to indirect FISH and is recommended especially for studies of microb es on natural substrata. (C) 1999 Elsevier Science B.V. All rights reserved .