Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans.
Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) 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 fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.
Spéar
R N
RN
Department of Plant Pathology, University of Wisconsin Madison 53706, USA.
Li
S
S
Nordheim
E V
EV
Andrews
J H
JH
D000704:Analysis of Variance; D002473:Cell Wall / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D003904:Mitosporic Fungi / Q000235:genetics*; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S / Q000235:genetics
D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S