Josée Coallier

LIVE/DEAD® BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.

Suspended bacterial biomass and activity in full-scale drinking water distribution systems: Impact of water treatment

Bacterial regrowth in drinking water distribution systems is a source of concern since it could result in non-compliance with water quality regulations, taste and odor problems, and may be associated with an increased risk of gastrointestinal illnesses. Bacterial regrowth is dependent on several factors, including the amount of substrate available for growth, such as biodegradable dissolved organic carbon (BDOC), oxidant residuals and the presence of corrosion. To investigate the impact of nutrients levels and oxidant residual maintenance, a full-scale investigation of two distribution systems was completed. This study presents data obtained from two distribution systems (DS) fed by different treatment trains: one DS has low BDOC concentrations and no free oxidant residual (St. Rose plant, Quebec, Canada), and the other has a high BDOC concentration and moderate concentrations of free oxidant residual (Pont Viau plant, Quebec, Canada). Monitoring included heterotrophic plate counts (HPC), total direct count by epifluorescence after acridine orange staining (AODC), direct viable counts (DVC-CTC method) and the measurement of bacterial production by 3H-thymidine incorporation. Results show that bacterial biomass (AODC and DVC) and bacterial production are lower in the DS fed by the treatment plant with a low BDOC concentration in the plant effluent. This difference is observed in warm water but not in cold water. The results suggest that HPCs are not a good indicator of bacterial regrowth in DSs. Finally, statistical analysis demonstrated that the treatment type and cumulative surface to volume ratio are the significant factors affecting regrowth in the distribution systems studied in warm waters.

Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family Enterobacteriaceae in Freshwater and Drinking Water

ABSTRACT Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 108 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.

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.

Examining the use of aerobic spore-forming bacteria to assess the efficiency of chlorination

Abstract Spores from aerobic bacteria were evaluated as an indicator of the efficiency of chlorination. Several inactivation experiments were performed. The influence of temperature and water type was evaluated. Resistance of spores to chlorination was higher compared with Giardia using the values given in the surface water treatment rule. Resistance was observed to increase with temperature. Both Chick–Watson and Hom models were found to well describe the inactivation of spores by chlorine.

Evaluating the risk of infection from the presence of Giardia and Cryptosporidium in drinking water.

Monte-Carlo simulations were performed in order to quantify the microbiological risk arising from the presence of Giardia and Cryptosporidium in drinking water. A two-year survey of 45 utilities located on a portion of the St. Lawrence River (Quebec, Canada) allowed us to collect the information required for this assessment: parasite occurrence data, treatment performances and daily unboiled water consumption. For a conventional treatment, the mean annual risk of infection with Cryptosporidium was found to be approximately eight times higher than with Giardia. The Monte-Carlo simulations confirm that the clarification process plays an important role in the reduction of the health risk associated with protozoan parasites. In fact, in the case of Cryptosporidium, the impact of chemical disinfectants (O3 or Cl2) appears to be minimal in the cold water treatment conditions prevailing in Quebec. According to the Monte-Carlo simulations, 45% (for conventional treatment+O3) and 51% (for conventional treatment) of individuals are exposed to a risk of infection with Cryptosporidium higher than the proposed USEPA guideline (1 infection per 10[emsp4 ]000 individuals per year).

Thiosulfate interference in the biodegradable dissolved organic carbon assay

Abstract Reducing agents, namely sodium thiosulfate, are used to eliminate oxidant residuals in samples for biodegradable dissolved organic carbon (BDOC) analyses. For the particular BDOC assay used in this study, samples are seeded using a natural bacterial inoculum. These bacteria grow in response to dissolved organic carbon (DOC) and the decrease in DOC measured during the thirty day batch incubation period determines the BDOC of the sample. Na 2 S 2 O 3 is usually added in excess, leaving a residual thiosulfate concentration which has been found in this study to interfere with the BDOC measurement. The effect of Na 2 S 2 O 3 residuals between 0 and 34 mg/l on the measured value of BDOC was tested. The interference increased with the amount of Na 2 S 2 O 3 added and above 20 mg/l, sodium thiosulfate caused a substantial underestimation of the BDOC. The natural bacterial inoculum was indirectly found to contain bacteria capable of metabolizing thiosulfate. At concentrations above 20 mg Na 2 S 2 O 3 l , these bacteria produced sufficient acid that the heterotrophic activity necessary in the BDOC assay was effected. More tests are required to determine if the growth of these sulfur-oxidizers effects BDOC values in the absence of pH change. The use of a phosphate buffer should be considered in samples of limited buffering capacity. Given the observed effects of Na 2 S 2 O 3 , residuals should be limited and, where possible, the use of Na 2 S 2 O 3 should be avoided.