Scholarly Work - Center for Biofilm Engineering
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Item 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, NickLaboratory 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.Item Bacterial characterization of toilet bowl biofilms(1998-08) Pitts, Betsey; Stewart, Philip S.; McFeters, Gordon A.; Hamilton, Martin A.; Willse, Alan Ray; Zelver, NickMethods 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.Item Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability(1998-10) Xu, Karen D.; Stewart, Philip S.; Xia, Fuhu; Huang, Ching-Tsan; McFeters, Gordon A.Item The study of microbial biofilms by classical fluorescence microscopy(1998) Huang, Ching-Tsan; Stewart, Philip S.; McFeters, Gordon A.Item Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation(1998-04) Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.Item Fluorescent probes applied to the physiological characterization of bacterial biofilms(1999) Lisle, John T.; Stewart, Philip S.; McFeters, Gordon A.Item 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.Item Physiological stress in batch cultures of pseudomonas putida 54g during toluene degradation(1997-06) Mirpuri, Rajesh G.; Jones, Warren L.; McFeters, Gordon A.; Ridgway, H. F.Physiological stress associated with toluene exposure in batch cultures of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were enumerated on non-selective (R2A agar plates) and a selective minimal medium incubated in the presence of vapor phase toluene (HCMM2). Differential recovery on the two media was used to evaluate bacterial stress, culturability and loss of toluene-degrading capability. A majority of the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol− variants. Numbers of stressed organisms increased with duration of toluene exposure and toluene concentration and coincided with accumulation of metabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indicating a possible relationship between respiring and injured cells. Rate expressions for injury, for formation of Tol− variants and for growth of Tol− variants were determined by calibrating a theoretical model to the results obtained. These rate expressions can be used to calibrate bioreactor models, and provide a basis for better design and control of bioremediation systems.Item 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.Item Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm(1996-06) Wentland, Eric Jon; Stewart, Philip S.; Huang, Ching-Tsan; McFeters, Gordon A.The use of acridine orange to visualize and quantify spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm was investigated. Bacterial colonies supported on polycarbonate filter membranes were grown on R2A agar plates. Some colonies were sampled for cell enumeration, while others were cryoembedded, sectioned, and stained with the fluorescent nucleic acid stain acridine orange. Spatial patterns of fluorescent color and intensity with depth in the colony were quantified using confocal microscopy and image analysis of stained cross sections. Colonies sampled in the midexponential phase were thin (20 μm), had high average specific growth rates (>1 h−1), and had all the cells stained bright orange. Colonies sampled after more than 24 h of growth were thick (>200 μm) and were growing slowly (μ < 0.15 h−1). These older colonies were characterized by distinct bands of orange at the colony edges and a dark green center. Stained biofilm cross sections displayed a similar orange band at the biofilm−bulk fluid interface and a green interior. Colony-average specific growth rates, determined by calculating the local slope of the cell accumulation versus time data, were correlated with colony-average fluorescence intensities. There was no correlation betweeen average specific growth rate and orange or green intensity individually, but growth rate did correlate with the orange:green intensity ratio (r2 = 0.57). The resulting regression was used to predict specific growth rate profiles within colonies. These profiles indicated that bacteria were growing rapidly near the air and agar interfaces and more slowly in the center of the colonies when thicker than about 30 μm. The dimension of the orange bands ranged from 10 to 30 μm, which may indicate the thickness of growing regions. The inherent variability associated with this technique suggests that it is best applied in single species systems and that the results should be regarded as qualitative in nature.