Rapid direct methods for enumeration of specific, active bacteria in waterand biofilms

Conventional methods for detecting indicator and pathogenic bacteria in wat er may underestimate the actual population due to sublethal environmental i njury, inability of the target bacteria to take up nutrients and other phys iological factors which reduce bacterial culturability. Rapid and direct me thods are needed to more accurately detect and enumerate active bacteria. S uch a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this p resentation is to describe novel approaches we have formulated for the rapi d and simultaneous detection of bacteria plus the determination of their ph ysiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane f iltration or immunomagnetic separation and combines an intracellular fluoro chrome (CTC) for assessment of respiratory activity plus fluorescent-labell ed antibody detection of specific bacteria. This approach has also been suc cessfully used to demonstrate spatial and temporal heterogeneities of physi ological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory acti vity, membrane potential, membrane integrity, growth rate and cellular enzy matic activities. Results obtained thus fat indicate that immunomagnetic se paration can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The cap tured and stained target bacteria are then enumerated by either conventiona l fluorescence microscopy or ChemScan(R), a new instrument that is very sen sitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Pari s, France) provides the detection of individual fluorescently labelled bact erial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the Che mScan and traditional plate counts of mixed natural bacterial populations i n water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.