Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Membrane Filter Method for Enumeration of Acinetobacter calcoaceticus from Environmental Waters

A membrane filter method was developed and evaluated for the quantitative recovery of Acinetobacter calcoaceticus from environmental waters. The procedure utilized a mineral medium, with sodium acetate and potassium nitrate as the carbon and nitrogen sources, respectively. Formic acid was included to enhance the recovery of A. calcoaceticus and to inhibit background growth. The medium was incubated for 46 h at 30°C, after which fermentation and cytochrome oxidase tests were performed on the colonies as they appeared on the membrane. Background microbial growth decreased on the average by 1.77 orders of magnitude. An essentially quantitative recovery relative to that on nutrient agar spread plates was obtained from freshly prepared suspensions of eight A. calcoaceticus strains in filter-sterilized pond water and from suspensions of five of the strains held for up to 96 h in filter-sterilized pond water at 15 and 22°C. Markedly reduced relative recoveries were obtained with the three remaining strains. However, these three strains, in contrast to the first five, not only did not grow, but also decreased in number in the eutrophic, filter-sterilized pond water. The confirmation rate of presumptive A. calcoaceticus colonies was 95%, whereas 8% of the presumptively negative colonies were A. calcoaceticus. The precision of the method did not exceed that expected from random error alone. Densities of A. calcoaceticus in freshwaters ranged from <1 to 7.9 × 10⁴ organisms per 100 ml and were about 10⁶ organisms per 100 ml in raw sewage.