Browsing by Author "McFeters, Gordon A."

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Bacteria associated with granular activated carbon particles in drinking water
(1986-09) Camper, Anne K.; LeChevallier, Mark W.; Broadaway, Susan C.; McFeters, Gordon A.
A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barries and enter potable water supplies.
Bacterial characterization of toilet bowl biofilms
(1998-08) Pitts, Betsey; Stewart, Philip S.; McFeters, Gordon A.; Hamilton, Martin A.; Willse, Alan Ray; Zelver, Nick
Methods have been developed and applied for sampling, characterizing and quantifying naturally occurring toilet bowl biofilms. Ceramic porcelain disks mounted in neoprene rubber strips were sealed in place in toilet bowls in three residences in Bozeman, Montana. In each bowl, duplicate strips were placed above, at and below the water level. In 7 consecutive weeks, duplicate disks from each zone in each bowl were removed. Surface biofouling was measured by viable cell areal density. Specific fouling rates were calculated and variability among toilet bowls and water levels was assessed. Specific fouling rates ranged from 0.0 to 0.46d‐1. Average areal cell densities at the end of 7 weeks ranged from 103 to 107cfu cm‐2. The extent of fouling was highest below the water line. Neutralization of the chlorine residual (typically 0.9 mg l‐1) in one toilet did not increase the extent of fouling compared to the controls. Biofilm areal viable cell densities and bowl water viable counts were positively correlated (r = 0.78). The visual threshold for detection of toilet bowl biofilm by the naked eye was approximately 105 cfu cm‐2. In a heavily fouled toilet bowl, the biofilm was up to 20 μm thick. Microorganisms were isolated from the biofilm and identified. Of the 32 organisms that were further characterized, 10 were identified as Pseudomonas, Sphingomonas or Chryseomonas species.
Biofilm control by antimicrobial agents
(2000) Stewart, Philip S.; McFeters, Gordon A.; Huang, Ching-Tsan
Biogenic production of cyanide and its application to gold recovery
(2001-03) Campbell, Scott C.; Clark, T. R.; McFeters, Gordon A.
Chromobacterium violaceum is a cyanogenic (cyanide-producing) microorganism. Cyanide is used on an industrial scale to complex and recover gold from ores or concentrates of ores bearing the precious metal. A potentially useful approach in gold mining operations could be to produce cyanide biologically in relatively small quantities at the ore surface. In this study, C. violaceum grown in nutrient broth formed a biofilm and could complex and solubilize 100% of the fold on glass test slides within 4-7 days. Approximately 50% of cyanide-recoverable gold could be mobilized from a biooxidized sulfidic-ore concentrate. Complexation of cyanide in solution by gold appeared to have a beneficial effect of cell growth- viable cell counts were nearly two orders of magnitude greater in the presence of gold-coated slides or biooxidized ore substrates that in their absence. C. violaceum was cyanogenic when grown in alternative feedstocks. When grown in a mineral salt solution supplemented with 13.3% v/v swine fecal material (SFM), cells exhibited pigmentation and suspended cell concentrations comparable to cultures grown in nutrient broth. Glycine supplements stimulated production of cyanide in 13.3% v/v SFM. In contrast, glycine was inhibitory when added at the time of inoculation in the more concentrated SFM, decreasing cell numbers and reducing ultimate bulk-solution cyanide concentration. However, aeration and addition of glycine to stationary phase cells grown on 13.3% v/v SFM anaerobically resulted in rapid production and high concentrations (up to 38 mg I-1) of cyanide. This indicates that biogenesis of cyanide may be supported in remote areas using locally produced and inexpensive agricultural feedstocks in place of commercial media.
Color measurement as a means of quantifying surface biofouling
(1998-11) Pitts, Betsey; Hamilton, Martin A.; McFeters, Gordon A.; Stewart, Philip S.; Willse, Alan Ray; Zelver, Nick
Laboratory reactors fitted with removable ceramic porcelain growth surfaces were inoculated with a consortium of biofilm forming environmental isolates. A Minolta colorimeter CR-200 (Minolta Camera Co., Ltd, Ramsey, NJ) was used in conjunction with a specially designed adapter to evaluate the reflective color of the porcelain disks as biofilm accumulated on them. Areal viable cell counts were monitored over a period of eleven days in two separate experiments and direct color measurements of the untreated, microbially fouled test surfaces were collected. This colorimetric assay was both non-destructive and immediate. A strong linear relationship between log cell density and log color change was observed. The Pearson product moment correlation coefficient for all 45 observations combined was r=0.95. Separate regression lines for each experiment were not significantly different (P=0.19). When adjusted for time, the (partial) correlation coefficient between log cell density and log color change was r=0.87, which suggests that the relationship between the two measures can not be explained by their mutual dependence on time. Reflective color measurement provided a rapid, non-destructive and quantitative measure of biofilm accumulation.
Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water
(2004-09) Lisle, John T.; Hamilton, Martin A.; Willse, Alan Ray; McFeters, Gordon A.
Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter–1. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.
Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms
(1994-05) Stewart, Philip S.; Griebe, Thomas; Srinivasan, Rohini; Chen, Cheng-Sao; Yu, Feipeng Philip; de Beer, Dirk; McFeters, Gordon A.
Biofilm bacteria challenged with monochloramine retained significant respiratory activity, even though they could not be cultured on agar plates. Microbial colony counts on agar media declined by approximately 99.9% after 1 h of disinfection, whereas the number of bacteria stained by a fluorescent redox dye experienced a 93% reduction. Integrated measures of biofilm respiratory activity, including net oxygen and glucose utilization rates, showed only a 10 to 15% reduction. In this biofilm system, measures of microbial respiratory activity and culturability yielded widely differing estimates of biocide efficacy.
Cryosectioning of biofilms for microscopic examination
(1994) Yu, Feipeng Philip; Callis, G. M.; Stewart, Philip S.; Griebe, Thomas; McFeters, Gordon A.
A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.
Effects of carbon source, carbon concentration, and chlorination on growth related parameters of heterotrophic biofilm bacteria
(1999-11) Ellis, B. D.; Butterfield, Phillip W.; Jones, Warren L.; McFeters, Gordon A.; Camper, Anne K.
To investigate the growth of heterotrophic biofilm bacteria, a model biofilm reactor was developed to simulate a drinking water distribution system. Controlled addition of three different carbon sources (amino acids, carbohydrates, and humics) at three different concentrations (500, 1,000, and 2,000 ppb carbon), in the presence and absence of chlorine, was used in separate experiments. An additional experiment was run with a 1:1:2 mixture of the above carbon sources. Biofilm and effluent total and culturable cells, in addition to total and dissolved organic carbon, were measured in order to estimate specific growth rates (SGRs), observed yields, population densities, and bacterial carbon production rates. Bacterial carbon production rates (mg C/L day) were extremely high in the control biofilm communities (range = 295-1,738). Both growth rate and yield decreased with increasing carbon concentrations. Therefore, biofilm growth rates were zero-order with respect to carbon concentrations used in these experiments. There was no correlation between growth rate and carbon concentration, but there was a significant negative correlation between growth rate and biofilm cell density (r = -0.637, p = 0.001 control and r = -0.57, p = 0.021 chlorinated biofilms). Growth efficiency was highest at the lowest carbon concentration (range = 12 - 4.5%, amino acids and humics respectively). Doubling times ranged from 2.3 - 15.4 days in the control biofilms and 1 - 12.3 days in the chlorinated biofilms. Growth rates were significantly higher in the presence of chlorine for the carbohydrates, humics, and mixed carbon sources (p = 0.004, <0.0005, 0.013, respectively). The concept of r/K selection theory was used to explain the results with respect to specific growth rates and yields. Humic removal by the biofilm bacteria (78% and 56% for the control and chlorinated biofilms, respectively) was higher than previously reported literature values for planktonic bacteria. A number of control experiments indicated that filtration of drinking water was as effective as chlorination in controlling bacterial biofilm growth.
Effects of culture conditions and biofilm formation on the iodine susceptibility of legionella pneumophila
(1992-01) Cargill, Kari Lisa; Pyle, Barry H.; Sauer, R. L.; McFeters, Gordon A.
Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.
Effects of culture conditions and biofilm formation on the iodine susceptibility of legionella pneumophila
(1992-05) Cargill, Kari Lisa; Sauer, R. L.; McFeters, Gordon A.
The susceptibility of Legionella pneumophila to iodination was studied with cultures grown in well water, on rich agar media, and attached to stainless-steel surfaces. Legionella pneumophila grown in water cultures in association with other microorganisms were less sensitive to disinfection by chlorine and iodine than were agar-passaged cultures. Differences in sensitivity to disinfection between water-cultured and agar-grown legionellae were determined by comparing C × T values (concentration in milligrams per litre multiplied by time in minutes to achieve 99% decrease in viability)and CM × T values (concentration in molarity). Iodine (1500×) gave a greater difference in CM × T values than did chlorine (68×). Iodine was 50 times more effective than chlorine when used with agar-grown cultures but was only twice as effective when tested against water-grown Legionella cultures. C × T × S values (C × T multiplied by percent survivors), which take into consideration the percent surviving bacteria, were used to compare sensitivities in very resistant populations, such as those in biofilms. Water cultures of legionellae associated with stainless-steel surfaces were 135 times more resistant to iodination than were unattached legionellae, and they were 210 000 times more resistant than were agar-grown cultures. These results indicate that the conditions under which legionellae are grown can dramatically affect their susceptibility to some disinfectants and must be considered when evaluating the efficacy of a disinfecting agent.
Evaluation of physiological staining, cryoembedding and autofluorescence quenching techniques on fouling biofilms
(1996-07) Huang, Ching-Tsan; McFeters, Gordon A.; Stewart, Philip S.
Physiological staining, cryoembedding, cryosectioning and autoftuorescence quenching techniques were evaluated for their applicability to undefined mixed population biofilms collected from environmental or engineered systems. Four different biofilms from two cooling towers, a paper mill machine and the effluent ditch of a wastewater treatment plant were tested. The redox dye 5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC) was used in combination with the DNA stain 4’,6‐diamino‐2‐phenylindole (DAPI) to distinguish respiring and nonrespiring cells. Positive CTC staining, as evidenced by the development of pink or red color, was successful in all samples examined except for paper mill biofilm. The structural integrity of frozen sections deteriorated when biofilms contained rigid or fibrous material. Autofluorescence generally impaired the ability to distinguish specific staining from natural background fluorescence. Two physical and three chemical methods were tested to quench autofluorescence. Quenching with crystal violet reduced most of the autofluorescent interference and still maintained physiological staining intensity, but contrast between CTC staining and residual autofluorescence was poor. Autofluorescence and the difficulty of sectioning thick biofilms containing abiotic materials limit the applicability of cryoembedding/staining techniques to fouling biofilms.
Factors affecting the determination of respiratory activity on the basis of cyanoditolyl tetrazolium chloride reduction with membrane filtration
(1995-12) Pyle, Barry H.; Broadaway, Susan C.; McFeters, Gordon A.
Fluorescent probes applied to the physiological characterization of bacterial biofilms
(1999) Lisle, John T.; Stewart, Philip S.; McFeters, Gordon A.
Gene expression and protein levels of the stationary phase sigma factors, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms
(2001-05) Xu, Karen D.; Franklin, Michael J.; Park, C-H; McFeters, Gordon A.; Stewart, Philip S.
Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.
Growth and persistence of pathogens on granular activated carbon filters
(1985-07) Camper, Anne K.; LeChevallier, Mark W.; Broadaway, Susan C.; McFeters, Gordon A.
Three enteric pathogens Yersinia enterocolitica 0:8, Salmonella typhimurium, and enterotoxigenic Escherichia coli, were examined for their ability to colonize granular activated carbon (GAC) in pure cultures and in the presence of autochthonous river water organisms. All three organisms readily colonized sterile GAC and maintained populations of ca. 105 to 107 CFUg-1 for 14 days when suspended in sterile river water. Exposure of pathogen biofilms on GAC to unsterile river water resulted in a gradual decline in pathogens on the carbon (0.08 to 0.14 log day-1). When pathogens were introduced to sterile GAC in the presence of heterotrophic plate count organisms, they attached at levels similar to those in the pure cultures and then decreased (0.10 to 0.22 log day-1). When added with heterotrophic plate count bacteria to GAC supporting a mature biofilm of native river water bacteria, they attached at a lower level (1.0 x 104 to 4.6 x 104 CFUg-1) and decreased at a more rapid rate (0.11 to 0.70 log day-1).
Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems
(1991-08) Camper, Anne K.; McFeters, Gordon A.; Characklis, William G.; Jones, Warren L.
The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (<1 mg liter-') of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.
Mechanisms of int (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and ctc (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in escherichia coli k-12
(1997-06) Smith, James J.; McFeters, Gordon A.
The tetrazolium salts INT and CTC are frequently used as indicators of bacterial respiration. Mechanisms of tetrazolium reduction to formazan in eukaryotic cells have been proposed, however those in prokaryotes remain largely unresolved. We undertook studies to determine the sites, and degree of coupling for INT and CTC reduction in the aerobic Escherichia coli K-12 respiratory chain using both intact cells and inverted membrane vesicles. In addition, reduction in whole cells was assayed under anaerobic conditions which elicit different electron transport pathways. Results of experiments with inhibitors of specific electron transport components indicated that both CTC and INT were reduced prior to ubiquinone in the E. coli respiratory chain by the primary aerobic [succinate and NAD(P)H] dehydrogenases. INT was also reduced at ubiquinone and possibly cytochromes b555, 556. Quantitative CTC reduction was more closely correlated with respiration in whole cells than INT, but the reverse was true in inverted membrane vesicles. Both tetrazolium salts were reduced to significant degrees under anaerobic conditions, particularly glucose fermentation. Mid-point reduction potential of CTC was determined to be −200 mV by redox titration. However, it appears that CTC forms a weakly fluorescent, poorly-localized intracellular formazan at redox potentials higher than ca. −200 mV. Inhibition of the aerobic terminal oxidases with KCN or azide strongly increased INT-, but significantly decreased CTC reduction in whole cells, indicating that these agents may not be useful for optimizing CTC-formazan or CTC (+) cell numbers in some bacteria. However, several other chemical agents stimulated CTC and INT reduction. These results suggest strategies for optimizing methods using these tetrazolium salts for assessment of bacterial respiratory activity.
Nonuniform spatial patterns of respiratory activity within biofilms during disinfection
(1995-06) Huang, Ching-Tsan; Yu, Feipeng Philip; McFeters, Gordon A.; Stewart, Philip S.
Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4*,6-diamidino2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.
Nutritional Relationships Among Microorganisms in an Epilithic Biofilm Community
(1982-10) Haack, T. K.; McFeters, Gordon A.
Previous studies of an epilithic algal-bacterial community in a pristine mountain stream suggested that heterotrophic bacteria were responding to the metabolic activities of the phototrophic population. Subsequent studies were performed to follow the flow of labeled carbon, from its initial inorganic form, through the trophic levels of the mat community. A majority of primary production metabolites were excreted by the algal population during active growth; this shifted to an incorporation into cellular material as phototrophic activity declined. Results suggest that there was a direct flux of soluble algal products to the bacterial population, with little heterotrophic utilization of dissolved organics from the overlying stream water. Both phototrophic productivity and bacterial utilization of algal products peaked at approximately the same time of year. Activity of the diatom-dominated algal population declined as silica concentrations in the stream water dropped, leading to a situation in which the sessile bacteria were substrate limited. These events resulted in an almost complete disappearance of the community in early September.