Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques.
A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.
Fried
Johannes
J
Institut für Zoologie und Limnologie, Leopold-Franzens-Universität Innsbruck, Innsbruck, Austria. johannes.fried@uibk.ac.at
Ludwig
Wolfgang
W
Psenner
Roland
R
Schleifer
Karl Heinz
KH
D000818:Animals; D001483:Base Sequence; D016798:Ciliophora / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005404:Fixatives / Q000737:chemistry; D005453:Fluorescence; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017385:Sequence Homology; D016622:Silver Staining / Q000379:methods*; D013768:Tetrahymena / Q000302:isolation & purification; D014871:Water Microbiology; D018508:Water Purification / Q000379:methods
D005404:Fixatives; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S