Browsing by Author "Yu, Feipeng Philip"

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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.
A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection
(1993-04) Yu, Feipeng Philip
This report describes the adaptation of an situ direct viable count (in situ DVC) method in biolfilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kpl) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached celss did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25%C. Although being a weaker disinfectact, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.
Investigation of ciprofloxacin penetration into pseudomonas aeruginosa biofilms
(1994-09) Suci, Peter A.; Mittelman, M. W.; Yu, Feipeng Philip; Geesey, Gill G.
Bacterial infections associated with indwelling medical devices often demonstrate an intrinsic resistance to antimicrobial therapies. In order to explore the possibility of transport limitation to biofilm bacteria as a contributing factor, the penetration of a fluoroquinolone antibiotic, ciprofloxacin, through Pseudomonas aeruginosa biofilms was investigated. Attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry was employed to monitor bacterial colonization of a germanium substratum, transport of ciprofloxacin to the biofilm-substratum interface, and interaction of biofilm components with the antibiotic in a flowing system. Transport of the antibiotic to the biofilm-substratum interface during the 21-min exposure to 100 micrograms/ml was found to be significantly impeded by the biofilm. Significant changes in IR bands of the biofilm in regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating chemical modification of biofilm components. These results suggest that transport limitations may be an important factor in the antimicrobial resistance of biofilm bacteria and that ATR/FT-IR spectrometry may be used to follow the time course of antimicrobial action in biofilms in situ.
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.
Physiological assessment of bacteria using fluorochromes
(1995-01) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
Physiological assessment of bacteria using fluorochromes
(1995-01) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
Physiological heterogeneity and biofilm control," in: biofilms in the aquatic environment
(1999) McFeters, Gordon A.; Stewart, Philip S.; Huang, Ching-Tsan; Wentland, Eric Jon; Xu, Karen D.; Yu, Feipeng Philip
Physiological methods to study biofilm disinfection
(1995-10) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-μm) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.
Physiological responses of bacteria in biofilms to disinfection
(1994-07) Yu, Feipeng Philip; McFeters, Gordon A.
In situ enumeration methods using fluorescent probes and a radioisotope labelling technique were applied to evaluate physiological changes of Klebsiella pneumoniae within biofilms after disinfection treatment. Chlorine (0.25 mg of free chlorine per liter [pH 7.2]) and monochloramine (1 mg/liter [pH 9.0]) were employed as disinfectants in the study. Two fluorogenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride and rhodamine 123, and tritiated uridine incorporation were chosen for assessment of physiological activities. Results obtained by these methods were compared with those from the plate count and direct viable count methods. 5-Cyano-2,3-ditolyl tetrazolium chloride is an indicator of bacterial respiratory activity, rhodamine 123 is incorporated into bacteria in response to transmembrane potential, and the incorporation of uridine represents the global RNA turnover rate. The results acquired by these methods following disinfection exposure showed a range of responses and suggested different physiological reactions in biofilms exposed to chlorine and monochloramine. The direct viable count response and respiratory activity were affected more by disinfection than were the transmembrane potential and RNA turnover rate on the basis of comparable efficiency as evaluated by plate count enumeration. Information revealed by these approaches can provide different physiological insights that may be used in evaluating the efficacy of biofilm disinfection.
Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes
(1994-06) Yu, Feipeng Philip; McFeters, Gordon A.
Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values that those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.
Rapid in situ physiological assessment of disinfection in bacterial biofilms
(Montana State University - Bozeman, College of Agriculture, 1994) Yu, Feipeng Philip
Study of biofouling control with fluorescent probes and image analysis
(2000) Yu, Feipeng Philip; McFeters, Gordon A.