New possibilities for bacterial cytochemistry: light microscopical demonstration of beta-galactosidase in unfixed...

The possibility of applying light microscope cytochemical techniques in order to determine the identity and physiological state of microbes, particularly bacteria, has received little attention in recent years. The technical obstacles have perhaps been thought too great and the potential rewards too small. In order to demonstrate the feasibility of the cytochemical approach to problems in microbiology, an indoxyl method was developed for the demonstration of beta-galactosidase activity in unfixed bacteria. Cells were immobilized on 3-aminopropyltriethoxysilane-treated glass, permeabilized by air drying, then incubated in an indoxyl beta-D-galactopyranoside substrate plus a ferri-ferrocyanide reaction mix. Specific enzyme activity was demonstrated in Escherichia coli and a strain of Bacillus subtilis containing the LacZ gene. In the former, activity was inducible both before and after immobilization. These findings indicate that the basic prerequisites for light microscopical demonstration of bacterial intracellular activities, i.e. immobilization without disruption and reagent access without loss of localization, can now be fulfilled. Further development of this approach is desirable because it allows rapid demonstration of specific microbial activities without an intervening period of in vitro cultivation, thus avoiding the time delays and adaptive changes associated with propagation on laboratory media.