In this problem, an attempt was made to determine the fate of various species of bacteria which had previously been isolated from other sources when inoculated into Denton tap water.

This thesis investigates the susceptibility of various bacterial species to standard purification process.

Cheung, Man Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 81-93). / Abstracts in English and Chinese. / Abstract (English) --- p.I / Abstract (Chinese) --- p.III / Acknowledgements --- p.V / Table of contents --- p.VI / List of figures --- p.IX / List of tables --- p.XI / List of Appendices --- p.XII / Chapter Chapter 1. --- General introduction --- p.1 / Chapter 1.1. --- Picoeukaryotes --- p.1 / Chapter 1.2. --- Conventional characterization techniques --- p.1 / Chapter 1.3. --- Cloning and sequencing approach --- p.3 / Chapter 1.3.1. --- Applications in prokaryotic plankton --- p.3 / Chapter 1.3.2. --- Applications in eukaryotic picoplankton --- p.3 / Chapter 1.4. --- Variations in diversity with environmental factors --- p.5 / Chapter 1.5. --- Study site --- p.6 / Chapter 1.6. --- Objectives --- p.8 / Chapter Chapter 2. --- Materials and methods --- p.9 / Chapter 2.1. --- Study site --- p.9 / Chapter 2.2. --- Sample collection --- p.9 / Chapter 2.3. --- DNA extraction and 18S rRNA gene amplification --- p.11 / Chapter 2.4. --- Clone library construction and screening --- p.12 / Chapter 2.5. --- Sequencing and phylogenetic analysis --- p.13 / Chapter 2.6. --- Statistical analyses --- p.14 / Chapter Chapter 3. --- Results --- p.15 / Chapter 3.1. --- Hydrological parameters of study site --- p.15 / Chapter 3.2. --- Clone libraries --- p.15 / Chapter 3.3. --- Higher-level taxonomic distribution --- p.21 / Chapter 3.4. --- Phylogenetic affiliations of OTUs --- p.22 / Chapter 3.4.1 --- Alveolates --- p.35 / Chapter 3.4.2 --- Stramenopiles --- p.36 / Chapter 3.4.3 --- Rhizaria --- p.36 / Chapter 3.4.4 --- Other lineages --- p.37 / Chapter 3.4.5 --- Novel higher-level groups --- p.38 / Chapter 3.5. --- Diversity estimates of picoeukaryotes --- p.39 / Chapter Chapter 4. --- Discussion --- p.42 / Chapter 4.1. --- Picoeukaryotic diversity --- p.42 / Chapter 4.1.1 --- Overall diversity --- p.42 / Chapter 4.1.2 --- Diversity of individual taxonomic groups --- p.44 / Chapter 4.1.2.1 --- Most represented lineages --- p.44 / Chapter 4.1.2.2 --- Other photosynthetic lineages --- p.52 / Chapter 4.1.2.3 --- Other non-photosynthetic lineages --- p.55 / Chapter 4.1.2.4 --- Novel higher-level lineages --- p.56 / Chapter 4.2. --- Spatial and seasonal variations of picoeukaryotes --- p.58 / Chapter 4.2.1 --- Spatial variations --- p.59 / Chapter 4.2.1.1 --- Compositional variations --- p.60 / Chapter 4.2.1.2 --- Variations in diversity --- p.61 / Chapter 4.2.2 --- Seasonal variations --- p.65 / Chapter 4.2.2.1 --- Compositional variations --- p.65 / Chapter 4.2.2.2 --- Variations in diversity --- p.66 / Chapter 4.3. --- Methodological aspects --- p.67 / Chapter 4.3.1 --- Sample collection --- p.67 / Chapter 4.3.2 --- PCR amplification --- p.68 / Chapter 4.3.3 --- Cloning and RFLP screening --- p.69 / Chapter 4.3.4 --- Statistical estimates --- p.71 / Chapter 4.3.5 --- Future directions --- p.71 / Chapter Chapter 5. --- General conclusion --- p.73 / References --- p.81

The bacterial flora of groundwater obtained from wells and distribution sites was investigated from both an ecological and a public health perspective. A majority of the isolates were oxidase positive, non-fermentative, gramnegative bacilli. Extensive heterogeneity of groundwater microflora, as determined by biochemical characterization and antibiograms, was observed both within and among welldistribution (N-D) systems. Of the unique isolates (sorts) found in the W-D systems, 75% were resistant to 2 or more antibiotics at clinically significant concentrations. Community diversity within W-D systems was evaluated by rarefaction which failed to reveal a general trend. Standard plate counts in 2 of 3 wells were significantly higher on Standard Methods Agar diluted ten-fold than on the same medium at the standard concentration. Pseudomonas aeruginosa, Flavobacterium sp., Aeromonas hydrophila and Yersinia enterocolitica were detected in 21, 18, 7 and 3% respectively of water samples analyzed over a 12 month period. A selective medium was developed for the isolation of Flavobacterium sp. and was effective in suppressing 98% of the background flora when used in a membrane filtration (MF) procedure. Yersinia Selective Agar was employed in an MF procedure for the isolation of Y. enterocolitica. The use of anaerobic incubation in this procedure effectively suppressed background growth both in the presence and absence of an antimicrobic supplement. Anaerobic incubation of m-endo LES Agar (AN-MF) was effective in suppressing non-coliform growth in the total coliform MF test and markedly reduced the frequency of overgrown plates. The AN-MF appeared to obviate the need for selective chemical agents and thus has potential value in the isolation of stressed coliforms.

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The increased consumption of fresh produce has been shown to be related to increases in foodborne disease outbreaks and these have in many cases been ascribed directly to carry-over of pathogens from contaminated irrigation water. In South Africa, rivers are the main source of irrigation water but many have been found to be unsuitable for irrigation of fresh produce because of the unacceptably high levels of faecal contamination. The main aim of this study was to do a baseline evaluation of the microbiological quality of the Plankenburg and Eerste Rivers and to determine which bacterial contaminants are present. Two sampling sites were selected for the Plankenburg (Plank-1 and -3) and one for the Eerste River (Eerste-1). The microbiological analysis included aerobic colony count (ACC), aerobic and anaerobic sporeformers, Staphylococcus, Salmonella, Listeria, enterococci, coliforms, faecal coliforms and E. coli using standard methods. The faecal contamination levels for both rivers exceeded the DWAF and WHO guidelines of <1 000 E. coli per 100 mL water for irrigation of fresh produce intended to be consumed raw. The Plankenburg River sites always had higher coliform contamination levels (1 200 - 13 000 000 MPN per 100 mL water) than the Eerste River site (230 - 79 000 MPN per 100 mL water). There was also a high incidence of index organisms including Salmonella, Staphylococcus, Listeria and endosporeformers. The isolation of intestinal enterococci suggested the presence of potential pathogens that can cause disease outbreaks. The baseline data also showed large variations in microbial loads over the 15 month study with the faecal coliform counts ranging for Plank-1 from 1 200 to 7 000 000 MPN.100mL-1, Plank-3 from 10 to 460 000 MPN.100mL-1 and Eerste-1 from 28 to 79 000 MPN.100mL-1. The water temperatures at all three sites ranged from 12.1° to 21.7°C with COD values in most cases below 100 mg.L-1. As the baseline study showed large variations in microbial loads over the 15 month study period an assessment using the Colilert-18 system of the weekly, daily and hourly variations, for 6 weeks over a period of 4 months was conducted at site Plank-2. This site was specifically used as it is an irrigation source point for nearby fresh produce farmers and is about 2 km further downstream from an informal settlement. The weekly variation trend for total coliforms (TC) showed a decrease over the entire sampling period with the highest count of 3 200 000 MPN.100 mL-1 during the warmer period. The E.coli (Ec) counts showed a similar trend with the highest count of 440 000 MPN.100 mL-1 also in March. The daily variation trends were the same for both the TC and Ec and counts found to increase from Monday to Thursday followed by a decrease to Sunday. The highest counts were on Thursday with average TC and Ec counts of 1 900 000 and 160 000 MPN.100 mL-1, respectively. The hourly variation trends were similar for both TC and Ec with counts increasing from 06h00 to 12h00 followed by a decrease to 18h00. The increases in TC and Ec counts found during the weekly, daily and hourly variation trend studies clearly suggests that the 15 month sampling that was done once a month on Mondays at 08h00 could be considered an underestimation of the contamination levels of the Plankenburg and Eerste Rivers. The overall weekly variation trend for the water temperature showed a decrease over the sampling period while the daily and hourly variation trends showed an increase from 06h00 to 18h00. The overall weekly trend for pH differed from that of the temperature with an increase over the sampling period. The analysis of covariance showed no correlation (p < 0.05) between the physico-chemical (temperature and pH) and the microbial variables (TC and Ec). Therefore it was concluded that temperature and pH had no direct impact on either the total coliform or E. coli counts. Both the Plankenburg and Eerste Rivers were found to be unsuitable for the irrigation of fresh produce intended to be consumed raw due to the high levels of faecal contamination that exceeded DWAF and WHO guidelines. Irrigation with such water could pose a health risk because of presence of potential pathogens that could be carried-over to fresh produce. / AFRIKAANSE OPSOMMING: Die toenemende gebruik van vars produkte hou direk verband met die toename in voedseloordraagbare siektes. Alte dikwels kan dit toegeskryf word aan die teenwoordigheid van patogene in besproeiingswater. In Suid Afrika is riviere die hoofbron van besproeiingswater maar dit is al gevind dat meeste ongeskik is vir gebruik as besproeïngsbron as gevolg van die onaanvaarbare hoe vlakke van fekale besmetting. Die hoofdoel van hierdie studie was om ‘n basislyn evaluasie van die mikrobiologiese kwaliteit van die Plankenburg en Eerste Riviere te doen en ook vas te stel watter bakteriese kontaminante teenwoordig is. Twee bemonsteringpunte is geselekteer vir die Plankenburg (Plank- 1 en -3) en een vir die Eerste Rivier (Eerste-1). Mikrobiologiese analises met standaard metodes het die volgende ingesluit: aërobe kolonie telings (AKT), aërobe en anaërobe spoorevormers, Staphylococcus, Salmonella, Listeria, enterococci, koliforms, fekale koliforms en E. coli met gebruik van standaard metode. Die fekale besmettingsvlakke vir beide riviere het die DWAF en WHO leistreep van <1 000 E. coli per 100 mL water vir besproeiing van vars produkte wat rou geëet kan word oorskry. Die Plankenburg Rivier bemonsteringspunte het in alle gevalle ‘n hoër kolivorm besmettingsvlak (1 200 - 13 000 000 MPN per 100 mL water) as die Eerste Rivier punt (230 - 79 000 MPN per 100 mL water) gehad. Daar was ook ‘n hoër voorkoms van indeksorganismes insluitend Salmonella, Staphylococcus, Listeria en endosporevormers. Die voorkoms van ingewand enterococci was ‘n addisionele aanduiding van die voorkoms van patogene wat ernstige gesondheidsrisikos vir die verbruiker kan inhou. Die basislyn data het groot variasies in die mikrobe vlakke oor die 15 maand van studie getoon. Die faecal koliforms vir Plank- 1 het gewissel van 1 200 tot 7 000 000 MPN.100mL-1, vir Plank-3 van 10 tot 460 000 MPN.100mL-1 en vir Eerste-1 van 28 tot 79 000 MPN.100mL-1. Die water temperature het gewissel van 12.1° tot 21.7°C met die CSB waardes in meeste gevalle minder as 100 mg.L-1. Aangesien daar sulke groot variasies in mikrobe ladings oor die 15 maande tydperk voorgekom het, is die Colilert-18 sisteem gebruik om die weeklikse, daaglikse en uurlikse variasies vas te stel vir 6 weke oor ‘n periode van 4 maande by die Plank-2 bemonsteringspunt. Daar is spesifiek op die bemonsteringspunt gefokus omdat dit as ‘n besproeiingsbron gebruik word deur groente produsente. Dit is ook gelee ongeveer 2 km stroomaf van ‘n informele nedersetting. Die weeklikse variasies in totaal koliforms (TC) het ‘n afname oor die hele bemonsteringsperiode getoon, met die hoogstes telling van 3 200 000 MPN.100 mL-1 gedurende die warmer tydperk. Die E.coli (Ec) tellings het ‘n soortgelyke neiging getoon, met die hoogste telling van 440 000 MPN.100 mL-1 ook in Maart. Die daaglikse neigings was dieselfde vir beide die TC en Ec en die tellings het vermeerder van Maandag tot Donderdag, met ‘n afname tot Sondag. Die hoogste telling was op Donderdag met gemiddelde TC and Ec tellings van 1 900 000 and 160 000 MPN.100 mL-1, respektiewelik. Die uurlikse variasie profiel was soortgelyk vir beide TC and Ec met tellings wat vermeerder het van 06h00 tot 12h00 gevolg deur ‘n afname tot 18h00. Die toename in TC en Ec getalle soos vasgestel gedurende die weeklikse, daaglikse en uurlikse variasie het duidelik getoon dat die bemonsterings wat een maal per maand op Maandae om 08h00 gedurende die 15 maande tydperk uitgevoer is, tot ‘n erg onderskatting van die besmettings vlakke in die Plankenburg en Eerste Riviere gelei het. Die algehele weeklikse variasies vir die water temperatuur het ‘n verlaging oor die bemonsteringstydperk getoon terwyl die daaglikse en uurlikse variasie neigings ‘n verhoging van 06h00 tot 18h00 getoon het. Die weeklikse neigings vir pH het van die van die temperatuur verskil. Die analises van kovariante het geen korrelasie (p < 0.05) tussen die fisiese-chemiese (temperature en pH) parameters en die mikrobe veranderlikes (TC en Ec) getoon nie. Dus is daar afgelei dat temperatuur en pH geen direkte impak op die totale kolivorm of E. coli tellings gehad nie. Die data van die studie het duidelik getoon dat water van beide die Plankenburg en Eerste Riviere nie geskik is vir gebruik vir besproeiing van vars produkte wat rou geëet gaan word nie. In beide gevalle het die fekale besmettingsvlakke die DWAF en WHO leistreep oorskry. Besproeiing met sulke water hou ‘n gesondheidsgevaar in as gevolg van die teenwoordigheid van potensiële patogene wat oorgedra kan word na vars produkte.

A collaborative pilot study of the microflora on biological activated charcoal (BAC) filters employed in the tertiary treatment of drinking water revealed the principle bacterial genera to be Pseudomonas, Alcaligenes, Achromobacter, Bacillus, Micrococcus, Corynebacterium, Chromobacterium, Microcyclus and Paracoccus. The microbial population of the filters paralleled seasonal carbon dioxide production. Of particular interest were the effects of the BAC miroorganisms upon precursors of trihalomethanes (THMs). Mixed populations of BAC microorganisms were cultivated for 50 days in a mineral salts-humic acid medium. It was concluded that (1) the BAC microflora enhances the absorptive capacity of the filters; (2) chemico-physical and biological processes operate in concert to lower the concentration of precursors of THMs; and (3) few bacterial pathogens establish themselves on the filters.

Lui Yuk Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-148). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Abberviations --- p.v / Table of Contents --- p.vii / List of Tables --- p.xiv / List of Figures --- p.xvi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Bacteriological evaluation of water --- p.1 / Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2 / Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3 / Chapter 1.1.3. --- Example of common indicator organisms --- p.3 / Chapter 1.1.3.1. --- Total coliform group --- p.3 / Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4 / Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5 / Chapter 1.1.3.4. --- Klebsiella --- p.5 / Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6 / Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7 / Chapter 1.3.1. --- Bacteria --- p.7 / Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7 / Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11 / Chapter 1.3.1.3 --- Legionella pneumophila --- p.12 / Chapter 1.3.2. --- Protozoa --- p.14 / Chapter 1.3.3. --- Viruses --- p.15 / Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16 / Chapter 1.4.1. --- Examples of conventional detection methods --- p.17 / Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18 / Chapter 1.5. --- Novel approaches for pathogens detection --- p.19 / Chapter 1.5.1. --- Modifications of media --- p.19 / Chapter 1.5.2. --- Antibody-based methods --- p.20 / Chapter 1.5.3. --- Nucleic acid-based methods --- p.21 / Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22 / Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24 / Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26 / Chapter 1.9. --- Aims of this study --- p.28 / Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31 / Chapter 2.1. --- Introduction --- p.31 / Chapter 2.2. --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Bacterial strains --- p.35 / Chapter 2.2.2. --- Bacterial enumeration --- p.35 / Chapter 2.2.3. --- DNA extraction and purification --- p.36 / Chapter 2.2.3.1. --- Boiling method --- p.36 / Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36 / Chapter 2.2.3.3. --- Chelex extraction method --- p.37 / Chapter 2.2.4. --- Targeted sequences --- p.38 / Chapter 2.2.4.1. --- eaeA gene --- p.38 / Chapter 2.2.4.2. --- mdh gene --- p.39 / Chapter 2.2.4.3. --- flaR gene --- p.39 / Chapter 2.2.5. --- PCR amplification --- p.40 / Chapter 2.2.6. --- Gel electrophoresis --- p.41 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Optimization of the PCR --- p.42 / Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42 / Chapter 2.3.2.1. --- Boiling method --- p.42 / Chapter 2.3.2.2. --- Proteinease K method --- p.43 / Chapter 2.3.2.3. --- Chelex method --- p.43 / Chapter 2.3.3. --- Specificity of PCR detection --- p.43 / Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44 / Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44 / Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44 / Chapter 2.4. --- Discussion --- p.57 / Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and Methods --- p.66 / Chapter 3.2.1. --- Bacterial strains --- p.66 / Chapter 3.2.2. --- Bacterial enumeration --- p.66 / Chapter 3.2.3. --- Filtration --- p.67 / Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68 / Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68 / Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68 / Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70 / Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70 / Chapter 3.2.6. --- DNA extraction --- p.71 / Chapter 3.2.7. --- Multiplex PCR --- p.71 / Chapter 3.2.8. --- PCR amplification --- p.72 / Chapter 3.2.9. --- Gel electrophoresis --- p.72 / Chapter 3.3. --- Results --- p.73 / Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2. --- Detection limit of PCR --- p.73 / Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73 / Chapter 3.3.2.2. --- Influence of background flora --- p.73 / Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77 / Chapter 3.3.3. --- Multiplex PCR --- p.77 / Chapter 3.4. --- Discussion --- p.91 / Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94 / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Materials and Methods --- p.99 / Chapter 4.2.1. --- Bacteria strains --- p.99 / Chapter 4.2.2. --- Bacterial enumeration --- p.99 / Chapter 4.2.3. --- Primers and Probes --- p.100 / Chapter 4.2.3.1. --- eaeA gene --- p.101 / Chapter 4.2.3.2. --- mdh gene --- p.102 / Chapter 4.2.3.3. --- flaR gene --- p.102 / Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103 / Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103 / Chapter 4.2.4.2. --- Purification of PCR product --- p.104 / Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105 / Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105 / Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106 / Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107 / Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108 / Chapter 4.3. --- Results --- p.110 / Chapter 4.3.1. --- Determination of targeted sequences --- p.110 / Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110 / Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114 / Chapter 4.3.4. --- Specificity of real-time PCR --- p.121 / Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121 / Chapter 4.4. --- Discussion --- p.131 / Chapter 5. --- Conclusion and future perspectives --- p.133 / Chapter 6. --- References --- p.138

Chloramine is a widely used alternative disinfectant for chlorine in potable distribution water. This alternative was investigated and employed to show its effect for suppressing coliforms, trihalomethanes(THMs), disinfection by-products (DBPs), and corrosivity.Coliform analyses were performed with m-Endo(total coliform) and m-T7 agar(injured coliform) by using a standard Membrane Filtration method. Heterotrophic bacteria were monitored with HPC agar(PCA) and R2A agar (nutrient limited agar). EPA methods 502.2, 524.2, and 504 were used to determine levels of Trihalomethanes(THMs) and Disinfection by-products(DBPs).In our study, we observed no significant differences in coliform counts, that could be attributed to the switch in disinfectant. The most common coliform identified was Enterobacter cloacae. We also noted that m-T7 performed better than m-Endo in the detection of coliforms. We also observed a low level of corrosion (0.4-3.8 mils/year) in the distribution system (DS). Higher counts of heterotrophic bacteria were enumerated on R2A when compared to HPC. DBP values decreased two fold when compared with DBP values for the two previous years during which chlorine was used as the disinfectant. / Department of Biology

The bacterium Aeromonas hydrophila is a known fish and opportunistic human pathogen commonly occurring in surface waters supplying drinking water distribution systems. The major concern of government and drinking water providers is that A. hydrophila may invade and become established in the biofilm of a distribution system, thus potentially leading to outbreaks of disease. The purpose of this study was to survey source water, distribution system biofilm, and to establish a simulated distribution system to explore the possibility of A. hydrophila invading and becoming established under normal and disrupted treatment conditions. A. hydrophila (AH) medium and the API-20E system were used for identification, while Ampicillin-Dextrin Agar (ADA) was used for enumeration. Presumptive counts were high in source water approaching 103 CFU/ml during summer months. Biofiim from an actual distribution system showed the presence of A. hydrophila in 10 % of the samples. In the simulated distribution system A. hydrophila was never found in the bulk water or biofilm under normal treatment condition, showing disinfectant efficiency. Under disrupted treatment conditions A. hydrophila was not able to colonize a pre-established biofilm over a 14 week period. / Department of Biology

Tap water collected according to standard methods was examined for microbial presence. Epifluorescent diagnoses using redox probe 5-cyano-2,3ditolyl tetrazolium chloride (CTC), 4',6-diamidino-2-phenylindole (DAPI), and acridine orange (AO) were employed for direct evidence of microorganisms. Evidence of total (DAPI or AO), respiring (CTC) bacteria, and heterotrophic plate count (HPC) was determined on multiple occasions during the summer, fall, and winter 1996-1997. Pseudomonas aeruginosa, Acinetobacter sp., Bacillus licheniformis, and Methylobacterium rhodinum were isolated and identified by the API and Biolog system using GN and GP procedures. On the basis of comparisons presented in this study between the CTC method and the standard HPC procedure, it appeared that the number of CTC-reducing bacteria in the tap water samples was typically higher than that determined by HPC, indicating that many respiring bacteria detected by the CTC reduction technique fail to produce visible colonieson the agar media used. In the seasonal data obtained by the CTC method, no difference was shown among respiring bacterial counts obtained from June through January. In the examination of P. aeruginosa viability in presence of chlorine, the number of CTC-positive bacteria exceeded the number of CFU by more than 2 logs after exposure to chlorine, suggesting that reliance on HPC overestimate the efficacy of disinfection treatment. In inactivation assays using the Biolog MT plate, no sensitivity to chlorine or chloramine disinfectants was noted even at high concentration levels (5 mg/liter). Following initial drop, bacterial activities increased as contact time increased. Thus, it appears that the MT microplate provides too low a cell concentration, too great a contact time, and/or too low a concentration of tetrazolium dye within the well for successful analysis of disinfectant capability to selected bacterial strains isolated from distribution water. / Department of Biology