Figure 1 schematically illustrates a laboratory apparatus that provides quantitative information on the density and on the rate of increase (or decrease) of density at which bacteria attach themselves to solid surfaces in a potable-water-supply system. These biofilms are potentially pathogenic and/or phytotoxic and can adversely affect processing of the water under some circumstances. The apparatus was devised to assess the abilities of biocide chemicals in water to suppress the biofilms in potable-water subsystems within closed life-support systems of spacecraft, but may also be adaptable to similar investigations of biofilms in terrestrial water supplies.

Figure 1. The Microscope and Video Camera generate images of water-borne microbes that have attached themselves to a solid surface. The images are digitized and processed to yield cell counts as a function of time.

The apparatus implements a recently developed method of automated image analysis for continuous, real-time monitoring of attachment of microbes to (or detachment of microbes from) a surface of interest that is immersed in water containing the microbes and chemical(s) to be investigated. The surface of interest is located in a flow-chamber/optical-cell (FOC), which is a modified fungal-growth chamber with interior dimensions of 30 by 5 by 2 mm. Monitoring is performed by use of a video camera mounted on a microscope that is focussed on the surface of interest.

Water is recirculated through the FOC at a rate of 9.8 mL/min by a high-speed micropump. The pump produces a pulsating flow; the pulses are damped by a device of 1-mL volume upstream of the FOC. The influent and effluent tubes are positioned to achieve turbulent flow in the middle of the FOC, where the monitored surface is located.

The video images of the monitored surface are preprocessed into binary images. Then a frame grabber grabs two successive images and stores an average of them on an optical disk. The averaged image is processed to extract information on the number and total area of objects (attached bacteria) in the field of view. This process can be repeated at intervals, yielding cell counts that can be plotted as a function of time, to determine rates of attachment and/or detachment.

Figure 2. These Plots of Cell Counts Versus Time were obtained in an experiment in which the microbes were Pseudomonas cepacia.

In a typical experiment, water containing a suspension of microbes is first circulated through the FOC for 18 to 24 hours, during which time some microbes attach themselves. Images are analyzed during this time to acquire data on the rate and density of attachment. Then the FOC and its recirculation loop with the high-speed micropump are isolated from the source of the cell suspension and washed with 300 mL of deionized water. Next, water containing the biocide to be tested is pumped into the FOC and recirculated through the FOC for 5 to 6 hours, during which time more images are analyzed and data collected. This is followed by another wash cycle, which is followed by another cycle of recirculation of the cell suspension and analysis of images, to measure the rate and degree of attachment on the surface of interest after the treatment with the biocide (see figure). Images are acquired at a frequency of at least once an hour, to measure the rate of attachment, and at least once every 20 minutes, to evaluate the effect of the biocide on the size of microbes and on the rate of detachment.

This work was done by Duane. L. Pierson of Johnson Space Center and David W. Koenig and Saroj K. Mishra of KRUG Life Sciences, Inc. For further information, access the Technical Support Package (TSP) free on-line at www.nasatech.com/tsp  under the Bio-Medical category.

This invention is owned by NASA, and a patent application has been filed. Inquiries concerning nonexclusive or exclusive license for its commercial development should be addressed to

the Patent Counsel
Johnson Space Center
(281) 483-0837.

Refer to MSC-22679.


NASA Tech Briefs Magazine

This article first appeared in the August, 2000 issue of NASA Tech Briefs Magazine.

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