Preliminary study on the potential utility of GFP as a biosensor for drug stability in parenteral solutions

In the health care setting, drugs added to large volume parenteral solutions (LVPS) are routinely administered to improve therapeutic effects and provide a faster clinical response. The development of analytical techniques that permit the detection of incompatibilities between drugs and parenteral solutions is necessary to guarantee their correct association with minimum adverse effects. Green fluorescent protein (GFP) has been used as a biological indicator of sterilization and disinfection processes because it exhibits a high thermal stability and is easily detected using UV light and spectrofluorometry. The response of GFP structure and/or protonation state to physicochemical changes in the solution favors its potential use as a biosensor for drug stability in parenteral solutions. The stability of the diuretic drugs furosemide and aminophylline, individually or combined, added to parenteral solutions of 20% mannitol and 0.9% NaCl was monitored by absorbance and RP-HPLC immediately and after 20 h of storage at room temperature, with and without 1 h exposure to a strong oxidant, H2O2. Changes in GFP fluorescence intensity were evaluated under the same conditions for purified GFP added to aliquots of the drug/LVPS solutions. Results show that GFP fluorescence intensity was proportional to the loss in drug stability over time and thus may potentially be added to a lot sample of a drug/parenteral solution as an immediate on-site test for defective product.     

Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.     

Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.     

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve >95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving >99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d post-exposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.     

Effectiveness of chlorine washing disinfection and effects on the appearance of artichoke and borage

AIM: Optimal conditions for chlorine application to obtain a reasonable decrease in the microbial counts without damaging the appearance of artichoke and borage have been established. METHODS AND RESULTS: The influence of chlorine concentration (0-200 mg l(-1)), pH, addition of organic acids, contact time and presence of protective structures on the microflora and vegetal appearance were studied. When pH was not controlled the effect of chlorine depended on its concentration until the pH increase caused by addition of chlorine reached 8.8. Any further increase in chlorine concentration was nullified by the pH increase. When pH was adjusted to 4.5 with acetic acid, the effectiveness increased with concentration. However, the use of citric acid to control pH caused a sharp decrease in effectiveness at concentration about 250 mg l(-1). The higher effectiveness of chlorine on homogenized plant extracts compared with the whole plant showed the impact of the vegetal structures on the resistance of the microorganisms. For artichoke, a relationship between the effectiveness of chlorine disinfection and its structures was also found. Extended washing times did not affect the total counts. However, in both vegetables, the appearance was affected by the extended contact times. CONCLUSIONS: The solutions rendering the highest microbial reduction with minimum damages were: 50 mg l(-1) free chlorine without pH control for artichoke and 100 mg l(-1) free chlorine at pH 7.0 for borage. SIGNIFICANCE AND IMPACT OF THE STUDY: Specific conditions for chlorine disinfection of artichoke and borage were determined to reduce the microorganisms in minimally processed artichoke and borage without damaging their appearance.     

Influence of water chlorination on the counting of bacteria with DAPI (4',6-diamidino-2-phenylindole).

Counting bacteria in drinking water samples by the epifluorescence technique after 4',6-diamidino-2-phenylindole (DAPI) staining is complicated by the fact that bacterial fluorescence varies with exposure of the cells to sodium hypochlorite. An Escherichia coli laboratory-grown suspension treated with sodium hypochlorite (5 to 15 mg of chlorine liter-1) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better and DAPI diffusion. At chlorine concentrations greater than 25 mg liter-1, DAPI-stained bacteria had only a low fluorescence. Stronger chlorine doses altered the DNA structure, preventing the DAPI from complexing with the DNA. When calf thymus DNA was exposed to sodium hypochlorite (from 15 to 50 mg of chlorine liter-1 for 90 min), the DNA lost the ability to complex with DAPI. Exposure to monochloramine did not have a similar effect. Treatment of drinking water with sodium hypochlorite (about 0.5 mg of chlorine liter-1) caused a significant increase in the percentage of poorly fluorescent bacteria, from 5% in unchlorinated waters (40 samples), to 35 to 39% in chlorinated waters (40 samples). The presence of the poorly fluorescent bacteria could explain the underestimation of the real number of bacteria after DAPI staining. Microscopic counting of both poorly and highly fluorescent bacteria is essential under these conditions to obtain the total number of bacteria. A similar effect of chlorination on acridine orange-stained bacteria was observed in treated drinking waters. The presence of the poorly fluorescent bacteria after DAPI staining could be interpreted as a sign of dead cells.     

Note: susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0.1, 0.2 and 0.5 mg l-1), pH (6, 7 and 8) and temperature (4, 21 and 32 degrees C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH6 than at pH8 for both strains. Under the standard conditions of temperature 21 degrees C, pH7 and chlorine concentration 0.2 mg l-1, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r² > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml⁻¹ and 500 μg ml⁻¹, respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r² > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.     

甲醛固定对GFP发光特性的影响

绿色荧光蛋白(GFP)能够作为报告分子对活体细胞中特定基因的时空表达进行实时追踪,因此广泛应用于生物学研究领域。在用GFP对细胞活动进行追踪的实验中,常有无法在取样后及时对样品进行荧光观察的情况,此时需要先将样品进行固定以便对其进行观测。然而,不恰当的细胞固定方法会导致胞内GFP荧光信号强度减弱、位置改变等后果。甲醛是最常用的细胞固定剂,也常被用于固定表达GFP蛋白的细胞样品。但对甲醛固定GFP样品的报道多是针对于真核细胞,且固定效果也存在较大差异。文章系统地探索了甲醛浓度、固定时间、固定缓冲液种类对两种细菌(E.coli及鱼腥蓝细菌Anabaena PCC7120)胞内GFP信号的影响。结果显示,较低浓度(≤1%)的甲醛处理2h后,细胞的荧光强度在1d后仍可保持80%以上,胞内荧光点无弥散现象发生。具有相近pH的几种常见缓冲液对荧光强度的影响无显著差异。随着甲醛浓度的增加、固定时间的延长、溶液pH的增加(中性至偏碱性),细胞中的荧光强度会逐渐降低。     

Control of biofouling by Cordylophora caspia in freshwater using one-off, pulsed and intermittent dosing of chlorine: laboratory evaluation

Cordylophora caspia is a hydrozoan which causes biofouling in power plants and is an increasing problem in UK drinking water treatment works. Thermal control is not usually feasible without a ready source of hot water so laboratory experiments were conducted to assess whether using pulsed doses of chlorine is an alternative solution. C. caspia polyps disintegrated after a single 20?min dose (the length of one backwash cycle in water treatment work filter beds) of 2.5?ppm chlorine. Without further treatment colonies regenerated within 3 days, but repeated dosing with chlorine for 20?min each day inhibited this regeneration. The resistance of surviving colonies to chlorine increased over time, although colony size and polyp regeneration continued to fall. These results suggest pulsed treatment with chlorinated backwashes at 2?ppm could be used to control C. caspia biofouling in rapid gravity filters and this may have relevance to other settings where thermal control is not feasible.     

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.     

Green fluorescent protein as a noninvasive intracellular pH indicator.

It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.     

A Rapid and Sensitive Fluorimetric Protocol for the Quantification of Green Fluorescent Protein in Soluble Protein Extracts from Transgenic Arabidopsis

Green fluorescent protein (GFP) is a popular qualitative reporter protein used to study different aspects of plant biology. However, to be used as a reliable quantitative reporter in expression studies using fluorescence based assays, methods to eliminate interfering endogenous molecules must be considered. Therefore, a standard curve based solid phase fluorescent immunoassay that eliminates the effects of interfering endogenous molecules was developed to quantify the GFP levels in soluble green extracts prepared from plants. Microtiter plates coated with anti-GFP were used to capture GFP from soluble plant extracts, interfering endogenous molecules was eliminated by washing without disturbing the anti-GFP binding of GFP, and then the fluorescence intensity of bound GFP was measured using a spectrofluorometer. We report in this study the use of this method to quantify the expression levels of soluble modified GFP in transgenic Arabidopsis thaliana.     

Physical and chemical effects on viability of the Myxobolus cerebralis triactinomyxon

Various chemical and physical methods for destroying the triactinomyxon (TAM) stage of the myxozoan parasite Myxobolus cerebralis were tested. The fluorescent stains propidium iodide and fluorescein diacetate were used as indicators of viability. Physical variables tested included freezing, drying, high temperature, sonication, and pressure of 6.2 x 10(7) Pa (9000 psi). Chemicals evaluated included chlorine bleach, povidone-iodine, and hydrogen peroxide. Freezing or drying for 1 h was effective in killing TAMs, but pressure was not. Temperatures above 75 degrees C for at least 5 min were also effective. Sonication with a laboratory instrument cleaner for 10 to 13 min killed and ruptured TAMs, resulting in <1.9% recovery. However, among the surviving TAMs, 39 to 58% were still viable. Chlorine concentrations of 130 ppm for 10 min were also effective at temperatures ranging from ice-water to room temperature and total hardness ranging from 10 to 500 mg l(-1). Lethal concentrations of hydrogen peroxide and povidone-iodine (10% solution) were quite high: 10% for 10 min, and 50% (5000 ppm active iodine) for 60 min, respectively. The stain results indicating TAM death were verified in 2 tests in which rainbow trout Oncorhynchus mykiss were exposed to TAMs that had been either frozen for 1 h or treated with 66 ppm chlorine as sodium hypochlorite for 1 min. None of the fish exposed to the treated TAMs became infected. These results should provide disinfection guidelines to prevent transfer of M. cerebralis TAMs to uninfected areas and provide information on the risks of parasite transfer under various treatment scenarios.     

Quantitative measurement of green fluorescent protein expression

Summary Data presented here shows a time course analysis of E. coli shake flask cultures expressing the reporter gene green fluorescent protein (GFP) with simultaneous comparison of microbial fluorescence intensity measurements and GFP concentration measured by Western blot. There is an apparent lag between the presence of GFP and its fluorescence due to the time required for formation of the chromophore. We demonstrate that GFP fluorescence can be used as a quantifiable reporter gene, provided the cyclization time for chromophore formation is considered.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of fluorescence change-based, reagent-less optic immunosensor

A reagent-less, regenerable and portable optic immunosensor was developed. A model sample, immunoglobulin G (IgG), was detected with this system based on changes in fluorescent intensity of fluorescent labeled protein A with specific reactivity to IgG depending on a reaction between the proteins. A glass plate immobilized with Qdot-labeled protein A was placed on the top of optic fibers designed for both excitation and fluorescence emission. The optic fibers with the Qdot-labeled protein A-immobilized glass plate were inserted into a solution of pH 7.4 phosphate buffered saline. After stabilization of the fluorescence intensity, IgG was added and the time-course of the fluorescence intensity was measured on a fluorometer connected with the optic fibers. Furthermore, the fluorescence response of a transient state was evaluated with the same system. When the Qdot-labeled protein A bound to IgG, fluorescence intensity decreased because of the inhibition by IgG. The degree of fluorescence decrease depends on the IgG concentration at a steady state and also in a transient state.     

Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.     

Evaluation of bacterial aerotaxis for its potential use in detecting the toxicity of chemicals to microorganisms

Bacterial aerotaxis (the movement of a cell toward oxygen) was evaluated for its potential use in detecting the toxicity of chemicals to microorganisms. The level of toxicity was determined by the concentration of test chemicals resulting in a 50% inhibition of aerotaxis of Pseudomonas aeruginosa PAO1 after 40 min of exposure. The aerotactic responses of P. aeruginosa were measured by using chemotaxis well chambers. Each clear acrylic chamber had a lower and upper well separated by a polycarbonate filter with a uniform pore size of 8.0 microm. To automatically detect bacterial cells that crossed the filter in response to a gradient of oxygen, P. aeruginosa PAO1 was marked with green fluorescent protein (GFP), and the GFP fluorescence intensity in the upper well was continuously monitored by using a fluorescence spectrometer. By using this technique, volatile chlorinated aliphatic compounds, including trichloroethylene (TCE), trichloroethane, and tetrachloroethylene, were found to be inhibitory to bacterial aerotaxis, suggesting their possible toxicity to microorganisms. We also examined more than 20 potential toxicants for their ability to inhibit the aerotaxis of P. aeruginosa. Based on these experimental results, we concluded that bacterial aerotaxis has potential for use as a fast and reliable indicator in assessing the toxicity of chemicals to microorganisms.     

Effects of Source Water Quality on Chlorine Inactivation of Adenovirus,Coxsackievirus, Echovirus,and Murine Norovirus

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15°C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration × contact time (CT) values (mg·min/liter) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5°C ranging from ≤0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log₁₀ CT values at 5°C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15°C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA''s Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.Disinfection processes are critical for the reduction of infectious virus concentrations in source water, because viruses are less efficiently removed by primary treatment of drinking water (e.g., coagulation and filtration) than are other pathogen types of concern (e.g., bacteria and protozoa). Over the years, many disinfection studies have focused on the inactivation of viruses in purified and buffered, demand-free, reagent-grade water (RGW). However, relatively few investigators have examined the impact of water quality during the disinfection process, even though water quality has been found to be a significant factor for inactivation of viruses.Several researchers found that the inactivation rate of poliovirus by free chlorine increased as the ionic concentration of water increased. In one study, poliovirus 1 was inactivated three times faster in boric acid buffer than in purified water (3). In addition, several investigators found that when the ionic content of buffered water was raised by the addition of NaCl or KCl, poliovirus 1 was inactivated two to four times faster than in the buffered water alone (2, 16, 17). In another study, poliovirus 1 was inactivated 10 times more rapidly in drinking water than in purified water (4).Studies conducted with natural waters have demonstrated both increased and decreased disinfection efficacy of chlorine in these waters compared to purified or buffered waters. In a study comparing chlorine disinfection in purified water and Potomac estuarine water, coxsackievirus A9 was inactivated more rapidly in the source water. The remaining study viruses (coxsackievirus B1, echovirus 7, adenovirus 3, poliovirus 1, and reovirus 3) were all inactivated more slowly in the source water (13). Bacteriophage MS2 was inactivated more slowly by free chlorine in two types of surface water than in buffered, demand-free water. However, there was no difference between the inactivation rates of this virus in the buffered water and groundwater (10). In another study, both feline calicivirus and adenovirus 40 were inactivated more slowly in treated groundwater than in buffered, demand-free water (21).The United States Environmental Protection Agency''s (USEPA) Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources (Guidance Manual) recommends disinfectant concentration × contact time (CT) values of 4, 6, and 8 to achieve 2-, 3-, and 4-log₁₀ inactivation, respectively, with chlorine at 5°C and pH 6 to 9 (23). These CT values, which incorporate a safety factor of 3, were obtained from inactivation experiments conducted with monodispersed hepatitis A virus (HAV) in buffered, demand-free water. As water quality can significantly affect the disinfection efficacy of chlorine, it is unclear whether these CT value recommendations are sufficient for inactivation of viruses in source water. More information is needed to systematically examine the role of water quality in chlorine disinfection of viruses.The objective of the present study was to examine the disinfection efficacy of free chlorine on selected viruses from USEPA''s Contaminant Candidate List (CCL) (22) in one untreated and two partially treated source waters from distinct geographical regions. By comparing the efficacy of chlorine disinfection in the source water types to disinfection in buffered, chlorine-demand-free RGW (7), the impact of water quality could be examined. The four representative CCL viruses selected for this study included human adenovirus 2 (HAdV2), echovirus 1 (E1), coxsackievirus B5 (CVB5), and murine norovirus (MNV), a surrogate for human norovirus (22). The viruses were selected because they were previously found to be the least effectively inactivated viruses of their type in RGW (6). Disinfection experiments were carried out in duplicate in pH 7 and 8 source water at 5 and 15°C using 0.2 and 1 mg/liter free chlorine. Inactivation curves were plotted using Microsoft Excel, and CT values were calculated using the efficiency factor Hom (EFH) model (9).