Chambers with membrane-filter side walls were designed for studies of the survival of coliform bacteria in natural and artificial waters. Experiments were carried out in the field and in the laboratory. The initial uptake rate of inorganic ions, total carbon, and glucose into the chamber was greater than twice as fast as the accumulation of each into dialysis tubing. When the survival of a waterisolated fecal coliform bacterium was examined in two adjacent mountain streams, it was found that the organism persisted longer in Bozeman Creek than in Middle Creek. These data may be a reflection of the water chemistry because the concentration of inorganic constituents of the former was greater. Laboratory studies of the survival of a fecal coliform bacterium in artificial and natural water with continuous flow were used to determine the effect of chemical composition, temperature, and pH. The relation of this type of data to the use of fecal coliform bacteria as indicators of health hazard in water is discussed.
The problems associated with recovery of pure cultures of Escherichia coli and Streptococcus faecalis from stream environments were examined utilizing membrane filter chambers. It was observed that upon exposure to the aquatic environment a significant proportion of cells lost their ability to produce colonies on a selective medium, yet retained this capability on a nutritionally rich, nonselective medium. Discrepancies in colony-forming units between nonselective and selective media indicated that a substantial portion of bacterial cells may become physiologically injured due to the environmental stress imposed by the aquatic environment. The extent of injury was observed to vary considerably among the eight different stream environments, since the amount of injury was not uniform for all types of water environments examined. It was observed that the injury acquired by a population of E. coli, during exposure to the aquatic environment, could be rapidly repaired in a nutritionally rich, nonselective medium. As the injured population of cells was exposed to the rich, nonselective broth, increasing proportions of cells were able to repair themselves such that they became insensitive to inhibitory agents in selective media.
The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.
A two-layer membrane filtration (MF) medium (injury-mitigating MF [IM-MF]) and a procedure for the enumeration of injured fecal coliforms are described. These procedures included the addition of glycerol and acetate plus reducing agents to both layers of a two-layer medium and rinsing of the filter with a rich resuscitation medium. Some changes in incubation time and temperatures were used. This method was compared with the multiple-tube fermentation most-probable-number procedure and the one-step M-FC agar-membrane filter method (direct M-FC) in terms of fecal coliform recovery from various aquatic environments that cause bacterial injury. With chlorinated sewage effluents, results of the IM-MF technique were equal to or greater than the most probable number in 9 of 18 trials and were 1.3 to 19 times greater than the M-FC method. When sewage samples were chlorinated in the laboratory, fecal coliform counts with IM-MF equaled or exceeded the most probable number in 7 of 15 trials and always exceeded the M-FC. M-FC was exceeded by IM-MF in 30 of 33 trials with clean mountain stream water. Fecal coliform bacteria that were exposed to low levels of an iodophore in the laboratory produced IM-MF counts 3 to 10 times greater than those with M-FC. A biochemical rationale for the formation of the IM-MF medium is discussed.
Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.
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