The Optimization of an Epifluorescence Microscopy Technique for the Direct Counting of Clumped Bacteria

The enumeration of heterotrophic bacteria is an important measure of water quality in mains'distribution systems. Conventional methods of enumerating bacteria by plate counts do not distinguish between single bacteria and clumps of cells; however, various methods have been reported which use epifluorescence microscopy to give a direct count of bacteria. There appears to be no standard method for the preparation of samples, with the choice of fluorochrome and staining technique varying considerably – even between workers using similar environmental samples. A number of the reported methods were tested, and a technique was developed which enabled a consistent total count of bacterial cells (occurring both singularly and in clumps) to be obtained.     

水中隐孢子虫和贾第鞭毛虫的替代检测指标

美国国家环保局（USEPA）制定并发布的1623方法是目前国际上常用的水中隐孢子虫和贾第鞭毛虫标准检测方法，该方法存在成本高、回收率低等缺陷。各国研究者试图通过考察其他水质指标与两虫的相关关系来寻找两虫密度和活性的有效替代指标，以间接反映两虫在水体中的存在水平，其中包括浊度、颗粒计数等物理学指标以及细菌、细菌孢子、病毒、噬菌体等生物学指标。虽然还未得出具有普遍性的结论，但研究表明颗粒计数、好氧孢子等有可能成为有效的两虫替代指标。     

New measurement principle and basic performance of high-sensitivity turbidimeter with two optical systems in series

An outbreak of Cryptosporidium infection from tap water has triggered the implementation of stricter controls for the turbidity of filtered water in Japan. Accordingly, a growing number of water purification plants have begun to measure the number of particles, which is more closely correlated to this kind of protozoan than turbidity. With this background we have developed a high-sensitivity turbidimeter based on the particle counting method that supports both turbidity measurement and particle counting to realize a compact and low-cost monitoring system of filtered water. This paper explains the basic principles of the turbidimeter, which uses both forward-angle light scattering and light obscuration methods in series and determines the turbidity from particle counting by applying Mie's theory of light scattering. In basic experiments on turbidity standards such as kaolin solution and formazine solution, we verified that the turbidimeter could determine the particle counts and turbidity of filtered water with sufficient accuracy.     

输配水系统中颗粒物与浊度演变特征研究

通过将颗粒物计数方法应用于城市输配水系统,研究分析了输配水系统中颗粒物数量与浊度的相关性以及其沿线变化规律.结果表明,随着水流方向,颗粒物数量有上升趋势;管网末梢颗粒物数量和浊度偏高;颗粒粒径越小,其含量越多;在一定范围内颗粒物数量和浊度有一定相关性;对颗粒物的研究比浊度更加准确和直观.此外,从颗粒物角度对水质进行特征分析,不仅为水质评价提供更多信息,还为颗粒物计数法的应用提供基础数据.     

配水管网中细菌再生长的预测研究

管网中细菌的再生长及其所带来的问题日益得到了人们的关注和重视，为此以某高校的校园管网为研究对象，建立了细菌总数的预测模型。算例结果表明，线性回归模型对细菌再生长的预测结果不理想，但创新性地选用Logistic回归模型并结合主成分分析法则可对细菌总数（二分变量）的超标概率进行准确预测，当将模型判别概率设为0．85时其精度〉90％，满足水质预警的要求。     

北方某高校管网水质生物稳定性的研究

目前关于饮用水安全性的研究大都集中在出厂水水质上，对给水管网中水质的变化则重视不够，而给水管网在整个供水体系中占有重要的地位，对保障水质安全起关键作用。为此，结合北方某高校校园管网的水质监测数据，分析了管网水中异养细菌的生长情况及其影响因素。结果表明，沿水流方向管网水质变得越来越差，至管网末梢时自由余氯、浊度、细菌总数等都劣于国家饮用水卫生标准；影响该管网水质生物稳定性的主要因素为AOC、自由余氯、浊度、有机物等。     

Overnight stagnation of drinking water in household taps induces microbial growth and changes in community composition

Drinking water quality is routinely monitored in the distribution network but not inside households at the point of consumption. Fluctuating temperatures, residence times (stagnation), pipe materials and decreasing pipe diameters can promote bacterial growth in buildings. To test the influence of stagnation in households on the bacterial cell concentrations and composition, water was sampled from 10 separate households after overnight stagnation and after flushing the taps. Cell concentrations, measured by flow cytometry, increased (2-3-fold) in all water samples after stagnation. This increase was also observed in adenosine tri-phosphate (ATP) concentrations (2-18-fold) and heterotrophic plate counts (4-580-fold). An observed increase in cell biovolume and ATP-per-cell concentrations furthermore suggests that the increase in cell concentrations was due to microbial growth. After 5 min flushing of the taps, cell concentrations and water temperature decreased to the level generally found in the drinking water network. Denaturing gradient gel electrophoresis also showed a change in the microbial composition after stagnation. This study showed that water stagnation in household pipes results in considerable microbial changes. While hygienic risk was not directly assessed, it emphasizes the need for the development of good material validation methods, recommendations and spot tests for in-house water installations. However, a simple mitigation strategy would be a short flushing of taps prior to use.     

Technique for measuring metallic salt effects upon the indigenous heterotrophic microflora of a natural water

The effects of several concentrations of the metallic salts Ag₂SO₄, NaAsO₃, BaCl₂2H₂O, CdCl₂2.5 H₂O, CrCl₂6H₂O, CuCl₂, HgCl₂, NaCl, NiCl₂6H₂O, PbCl₂ and ZnCl₂ upon the indigenous heterotrophic microflora of a natural water were studied. Effects of these salts upon the heterotrophic activity of the microbes were assayed using the heterotrophic technique whereas lethal effects upon the heterotrophic bacteria were determined using nutrient agar plate counts. The heterotrophic activity method is based upon the uptake and mineralization of a radioactively labeled metabolite (in these experiments ¹⁴C-glucose) by the indigenous aquatic microbes and data analysis is by Michaelis—Menten enzyme kinetics equations. Concentrations of metallic salts which resulted in bacterial death also caused erratic uptake and mineralization rates of ¹⁴C-glucose whereas sub-lethal concentrations, as determined by nutrient agar plate counts, caused a non-competitive inhibition of maximum heterotrophic activity and markedly increased the turnover time of the glucose substrate.     

低温低浊水处理技术影响因素及分析

指出低温低浊水处理是市政给排水处理工程的难题之一,对北方寒冷地区低温低浊水质难于处理的原因进行了分析,并结合絮凝动力学分析其沉淀,对今后低温低浊水处理技术发展作了展望,对具体市政给排水设计,科研有一定的指导意义。     

Quantitative biofouling diagnosis in full scale nanofiltration and reverse osmosis installations

Biofilm accumulation in nanofiltration and reverse osmosis membrane elements results in a relative increase of normalised pressure drop (ΔNPD). However, an increase in ΔNPD is not exclusively linked to biofouling. In order to quantify biofouling, the biomass parameters adenosine triphosphate (ATP), total cell count and heterotrophic plate count in membrane elements were investigated during membrane autopsies and compared with ΔNPD in test rigs and 15 full scale investigations with different types of feed water. The combination of biomass related parameters ATP and total cell count in membrane elements seem to be suitable parameters for diagnosis of biofouling, whereas plate counts were not appropriate to assess biofouling. The applied ΔNPD measurement was too insensitive for early detection of fouling. Measurements of biological parameters in the water were shown to be not appropriate in quantifying biofouling. Evidently, there is a need for a practical tool, sensitive pressure drop data and systematic research.     

The Microbiology of Lubricants and Oils for Water Supply Applications

Recent work in the US has shown that contact between lubricants and oils and drinking water can promote the growth of heterotrophic micro-organisms. It is unlikely that these species are pathogenic, and therefore harmful to man. However, the hygienic status of water distribution systems could be affected, leading to higher heterotrophic plate counts during routine sampling. Experimental work has been undertaken to study the microbiological impact of a range of lubricants and oils on drinking water. The methods have been based on an existing procedure for the assessment of the growth potential of drinking water, i.e. the 'assimilable organic carbon'test. The results of this work could serve as the basis of a future standard method for the rapid and inexpensive screening of oils and lubricants for their microbiological suitability in potable-water applications.     

Biostability and disinfectant by-product formation in drinking water blended with UF-treated filter backwash water

The overall objective of this study was to investigate the impact of blending membrane-treated water treatment plant (WTP) residuals with plant-filtered water on finished water quality in terms of biostability and disinfectant by-product (DBP) formation. Filter backwash water (FBWW) was treated with a pilot-scale ultrafiltration (UF) membrane to produce permeate that was blended with plant-finished water. The batch studies involved storing samples for a specified time with a disinfectant residual to simulate residence time in the distribution system. Both chlorinated and non-chlorinated FBWW streams were evaluated, and the experimental design incorporated free chlorine, monochloramine, and chlorine dioxide in parallel to a model system that did not receive a disinfectant dose. The results of the study found that blending 10% UF-treated FBWW with plant-filtered water did not have an impact on water biostability as monitored with heterotrophic plate counts (HPCs) or DBP concentrations as monitored by TTHM and HAA5 concentrations. However, the presence of preformed THM and HAA species found in chlorinated FBWW streams may result in higher levels of initial DBP concentrations in blended water matrices, and could have a significant impact on finished water quality in terms of meeting specific DBP guidelines or regulations.     

Biofilm responses to ageing and to a high phosphate load in a bench-scale drinking water system

The effects of ageing and of phosphate load on drinking water biofilms developed on a polycarbonate substratum in the pseudo-equilibrium state have been evaluated. Phosphate was added in an amount higher than the stochiometric nutrient requirements of bacteria, at concentrations commonly applied in a drinking water distribution system for corrosion control. Multiple parameters were monitored: heterotrophic plate counts (HPCs), total direct counts (TDCs) and potential exoproteolytic activity (PEPA) in order to characterise changes in bacterial biofilms. The total carbohydrate, amino acid and phosphate contents of biofilms were analysed to characterise and monitor the biochemical composition of the biofilm.The three enumeration methods showed that a pseudo-equilibrium state was reached after 7 weeks of colonisation after which, the bacterial growth rate in the biofilm was 0.1 log per week on average. Bulk phosphate addition doubled the phosphate in the biofilm, but did not affect the other biological, physiological or chemical parameters measured.Polysaccharides increased in the biofilm with ageing and the dynamics of individual carbohydrate synthesis also varied with the age of the biofilm. Once pseudo-equilibrium, it was found that the total proteins were globally constant, whereas the spectra of some individual amino acids of the proteins had significantly changed.     

Microbiological and Trihalomethane Responses to Booster Chlorination

Two zones of a water distribution supply area receiving treated surface water were used to study the effect of booster chlorination on bacterial regrowth. In the first year of the study general patterns of bacterial regrowth were observed, and in the second year one of the areas was booster chlorinated at the outlet from a surface reservoir. In both years of the study the heterotrophic plate counts showed a seasonal pattern. In the second year there was a large increase in plate counts from mid-July to October in both of the study areas. During this period, booster chlorination was unable to prevent bacterial regrowth. The addition of chlorine to produce an initial free chlorine residual of 0.5 mg/1 resulted in a reduction in bacterial numbers, but with further retention in the distribution system bacterial regrowth occurred. There was little increase in the trihalomethane (THM) concentration following booster chlorination, but booster chlorination appeared to increase the assimilable organic carbon concentration.     

Optimization of the extended terminal subfluidization wash (ETSW) filter backwashing procedure

The increased passage of particles and microorganisms through granular media filters immediately following backwashing is a common problem known to the water treatment community as filter "ripening" or maturation. While several strategies have been developed over the years to reduce the impact of this vulnerable period of the filtration cycle on finished water quality, this research involves a recently developed filter backwashing strategy called the extended terminal subfluidization wash (ETSW). ETSW is a method of terminating the backwash cycle with a subfluidization wash for a period of time sufficient to pass one theoretical filter-volume of water upward through the filter. ETSW was shown to remove significantly greater quantities of backwash remnant particles thereby reducing the magnitude of filter ripening turbidity and particle count spikes. Optimum ETSW flow rates were determined for deep-bed anthracite and granular activated carbon filters herein by monitoring filter effluent turbidities and particle counts during the filter ripening period. Optimality of the coagulation process was also shown to influence the magnitude of filter ripening particle passage. ETSW was found to be equally effective for biological and conventional deep-bed anthracite filters.     

The relationship between rainfall and nematode density in drinking water

A direct relationship exists between rainfall, streamflow and turbidity and the density of nematodes in drinking water. During rainy periods the density of nematodes increased from 1 to 15 gal⁻¹ of finished water. This supports the hypothesis that the nematodes are not breeding within the water treatment facility but are entering the municipal water system with the raw water.     

Monitoring source water for microbial contamination: evaluation of water quality measures

Watershed management programs often rely on monitoring for a large number of water quality parameters to define contaminant issues. While coliforms have traditionally been used to identify microbial contamination, these indicators cannot discriminate among potential contaminant sources. Microbial source tracking (MST) can provide the missing link that implicates the sources of contamination. The objective of this study was to use a weight-of-evidence approach (land use analysis using GIS, sanitary surveys, traditional water quality monitoring, and MST targets) to identify sources of pollution within a watershed that contains a raw drinking water source. For the study watersheds, statistical analyses demonstrated that one measure each of particulate matter (turbidity, particle counts), organic matter (total organic carbon, dissolved organic carbon, UV(254) absorbance), and indicator organisms (fecal coliforms, enterococci) were adequate for characterizing water quality. While these traditional parameters were useful for assessing overall water quality, they were not intended to differentiate between microbial sources at different locations. In contrast, the MST targets utilized (Rhodococcus coprophilus, sorbitol-fermenting Bifidobacteria, and male-specific coliphages) pinpointed specific sources of microbial pollution. However, these targets could not be used for routine monitoring due to a high percentage of non-detects.     

BAC滤池对浊度和颗粒数的控制研究

传统的贾第鞭毛虫和隐孢子虫（简称“两虫”）检测方法存在诸多不足，为此选用浊度和颗粒数作为“两虫”的替代指标，以对浊度和颗粒物的去除率来衡量生物活性炭（BAC）滤池对“两虫”的控制效果。试验结果表明：采用颗粒数表征滤后水水质比采用浊度更适宜。过滤初期颗粒数从峰值降到50个／mL以下所需的时间比浊度降到0．1NTU所需的时间多1h左右。正常过滤期间BAC滤池进水浊度一般在0．1NTU以下，经过BAC滤池处理后，浊度得到进一步降低，平均去除率为52．7％。炭层对浊度的去除率为56．4％，其出水浊度基本上都低于0．05NTU，而砂层对浊度不但没有去除能力，反而使出水浊度平均上升了约3．7％。炭层对颗粒物的平均去除率为33．3％，砂层对颗粒物的平均去除率为8．5％。     

Pathogenic or potentially pathogenic bacteria as contaminants of fresh water from different sources in Araraquara, Brazil

The microbiological quality of various fresh waters in the Araraquara region, state of São Paulo, Brazil was investigated. Ninety-nine water samples were taken from rivers, reservoirs, artesian and non-artesian wells, springs and tap waters, and these waters were analysed using: plate counts of heterotrophic microorganisms (per 1 ml); Most Probable Number (MPN) of fecal coliforms and E. coli (per 100 ml); tests for presence of Salmonella, Shigella, Yersinia, the E. coli pathogens of classes EPEC, ETEC and EIEC and Mycobacterium, Shigella, Yersinia and enteroinvasive E. coli (EIEC) were not isolated. The other types of microorganisms were isolated in varying proportions. We conclude that the waters investigated represent a potential microbiological health risk.     

Comparison of a novel MPN method against the yeast extract agar (YEA) pour plate method for the enumeration of heterotrophic bacteria from drinking water

This study compared the Quanti-Disc most probable number (MPN) test for heterotrophic bacteria from drinking water with the widely used yeast extract agar (YEA) pour plate method. The Quanti-Disc test module contains 50 reaction wells in which a medium has been pre-deposited. The medium contains a suite of three fluorogenic enzyme substrates selected for the detection of enzymes expressed widely by heterotrophic bacteria. The MPN of heterotrophic bacteria is calculated from the number of fluorescing reaction wells after incubation of a sample. Quanti-Disc and the YEA pour plate method were compared according to guidance on comparing methods given in United Kingdom national guidance and ISO 17994:2004. The two methods were also challenged with reference strains and isolates of heterotrophic bacteria from drinking water. This indicated that heterotrophic bacteria commonly encountered in drinking water are detected by both the YEA pour plate method and Quanti-Disc. Analysis of data from split water samples (723 for 37 degrees C tests and 872 for 22 degrees C tests) from nine geographically diverse laboratories in England and Wales demonstrated that the Quanti-Disc method is equivalent to the YEA pour plate method for the analysis of heterotrophic bacteria from drinking and similar waters at 37 degrees C, and superior to YEA for the analysis at 22 degrees C. The Quanti-Disc method is a simple and efficient alternative method for the enumeration of heterotrophic bacteria from drinking water.