A digital imaging procedure for seven-probe-labeling FISH (Rainbow-FISH) and its application to estuarine microbial communities.
For multi-probe-labeling fluorescence in situ hybridization (FISH), a digital imaging procedure was developed consisting of systematic background noise reduction and target signal equalization using a hue, saturation, value color partitioning technique. By the combined application of seven DNA probes, each labeled with three fluorochromes at maximum, seven kinds of cultured type strains were distinguished in a microscopic field simultaneously. Using this seven-probe-labeling FISH (Rainbow-FISH), several phylogenetic groups of microbes that occur frequently in aquatic environments, such as Alpha-, Beta- and Gammaproteobacteria, Cytophaga-Flavobacterium and Actinobacteria, were identified and quantified. The total counts of cells specified by Rainbow-FISH were in the range of 96-108% of those of general FISH, showing that the method is highly reliable for quantitative population analysis. Analyzing samples obtained at points along a river to a sea, we found a reverse population change in two groups: apparent decreases in Betaproteobacteria but gradual increases in Gammaproteobacteria. This method provides a platform toward the improvement of semiautomatic analysis of aquatic microbes under various metabolic conditions.
Sunamura
Michinari
M
Biological Resources and Functions, National Institute of Advanced and Industrial Science and Technology, AIST, Tsukuba, Ibaraki, Japan.
Maruyama
Akihiko
A
D039903:Actinobacteria / Q000235:genetics / Q000302:isolation & purification; D001105:Archaea / Q000235:genetics; D015169:Colony Count, Microbial / Q000379:methods; D003201:Computers; D003589:Cytophaga / Q000235:genetics / Q000302:isolation & purification; D005618:Fresh Water / Q000382:microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007564:Japan; D008386:Marine Biology / Q000379:methods*; D015345:Oligonucleotide Probes; D020560:Proteobacteria / Q000235:genetics / Q000302:isolation & purification; D014871:Water Microbiology
D015345:Oligonucleotide Probes