Rn. Spear et al., Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans, J MICROB M, 35(2), 1999, pp. 101-110
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
.