Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments. The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure. DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment. Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA 2 in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA 2 gene. Higher-contrast images were obtained with this method than with in situ PCR.

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Ha wobacterium-Cytophaga; Burkholderia~seudomonas (rRNA Ill)-authentic Alcaligenes; VibriwAeromonas; Pseudomonas (rRNA I); the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. A t the eutrophic site, 54-68% of all cells visualized by staining with DAPl (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 3 9 4 5 % of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flawobacteriu~Cytophaga group and BurkholderiaPseudomonas (rRNA Ill)-authentic Alcaligenes group. A t the oligotrophic site, only 2638% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.

Physiologic activity and community structure of planktonic and biofilm microbial communities in an urban river were analyzed using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Respiring bacteria estimated by CTC reduction were higher in biofilms (20%) than in stream water samples (12%). FISH analysis revealed that bacterial populations in both stream water and biofilms were dominated by L-Proteobacteria and Cytophaga^Flavobacterium cluster. Microbial community changes determined by multidimensional scaling analysis from DGGE patterns showed that microbial community structures in biofilms matured within 3^7 days of their formation and did not change further, while those in stream water changed continuously.