Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) ofAureobasidium pullulans
| Autor: | Shuxian Li, Russell N. Spear, Erik V. Nordheim, John H. Andrews |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Microbiology (medical)
Rhodosporidium toruloides Microbiology chemistry.chemical_compound Cell Wall Image Processing Computer-Assisted RNA Ribosomal 18S Fluorescence microscope medicine Fluorescein Molecular Biology In Situ Hybridization Fluorescence Analysis of Variance biology medicine.diagnostic_test biology.organism_classification Fluorescence Molecular biology Aureobasidium pullulans Biochemistry chemistry Mitosporic Fungi Oligonucleotide Probes Molecular probe Oligomer restriction Fluorescence in situ hybridization |
| Zdroj: | Journal of Microbiological Methods. 35:101-110 |
| ISSN: | 0167-7012 |
| DOI: | 10.1016/s0167-7012(98)00100-6 |
| Popis: | 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 M w 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. |
| Databáze: | OpenAIRE |
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