Browsing by Author "Loetterle, Linda R."

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A laboratory hot tub model for disinfectant efficacy evaluation
(2007-01) Goeres, Darla M.; Loetterle, Linda R.; Hamilton, Martin A.
This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (± the repeatability standard deviation of log densities) were 7.2 (± 0.31) for the bulk water (density in units of cfu ml− 1), 5.3 (± 0.56) for the coupons (density in units of cfu cm− 2), and 6.6 (± 0.50) for the filters (density in units of cfu cm− 2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p = 0.016), biofilm bacteria on the coupons (p = 0.09) and biofilm bacteria on the filter (p = 0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.
A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor
(2009-04) Goeres, Darla M.; Hamilton, Martin A.; Beck, Nicholas A.; Buckingham-Meyer, Kelli; Hilyard, Jackie D.; Loetterle, Linda R.; Lorenz, Lindsey A.; Walker, Diane K.; Stewart, Philip S.
This protocol describes how to grow a Pseudomonas aeruginosa biofilm under low fluid shear close to the airâ€“liquid interface using the drip flow reactor (DFR). The DFR can model environments such as food-processing conveyor belts, catheters, lungs with cystic fibrosis and the oral cavity. The biofilm is established by operating the reactor in batch mode for 6 h. A mature biofilm forms as the reactor operates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences a low shear as the media drips onto a surface set at a 101 angle. At the end of 54 h, biofilm accumulation is quantified by removing coupons from the reactor channels, rinsing the coupons to remove planktonic cells, scraping the biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration. The entire procedure takes 13 h of active time that is distributed over 5 d.
Statistical assessment of a laboratory method for growing biofilms
(2005-03) Goeres, Darla M.; Loetterle, Linda R.; Hamilton, Martin A.; Murga, Ricardo; Kirby, D. W.; Donlan, R. M.
Microbial biofilms have been grown in laboratories using a variety of different approaches. A laboratory biofilm reactor system, called the CDC biofilm reactor (CBR) system, has been devised for growing biofilms under moderate to high fluid shear stress. The reactor incorporates 24 removable biofilm growth surfaces (coupons) for sampling and analysing the biofilm. Following preliminary experiments to verify the utility of the CBR system for growing biofilms of several clinically relevant organisms, a standard operating procedure for growing a Pseudomonas aeruginosa biofilm was created. This paper presents the results of a rigorous, intra-laboratory, statistical evaluation of the repeatability and ruggedness of that procedure as well as the results of the experiments with clinically relevant organisms. For the statistical evaluations, the outcome of interest was the density (c.f.u. cm-2) of viable P. aeruginosa. Replicate experiments were conducted to assess the repeatability of the log density outcome. The mean P. aeruginosa log10 density was 7·1, independent of the coupon position within the reactor. The repeatability standard deviation of the log density based on one coupon per experiment was 0·59. Analysis of variance showed that the variability of the log density was 53% attributable to within-experiment sources and 47% attributable to between-experiments sources. The ruggedness evaluation applied response-surface design and regression analysis techniques, similar to those often used for sensitivity analyses in other fields of science and engineering. This approach provided a quantitative description of ruggedness; specifically, the amount the log density was altered by small adjustments to four key operational factors – time allowed for initial surface colonization, temperature, nutrient concentration, and fluid shear stress on the biofilm. The small size of the regression coefficient associated with each operational factor showed that the method was rugged; that is, relatively insensitive to minor perturbations of the four factors. These results demonstrate that the CBR system is a reliable experimental tool for growing a standard biofilm in the laboratory and that it can be adapted to study several different micro-organisms.