Possible mechanisms for the revival of glutaraldehydetreated spores of Bacillus subtilis NCTC 8236

Abstract

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.

Bacterial spores and chemical sporicidal agents

“…Some revival of glutaraldehyde-treated spores can be achieved by means of a posttreatment heating (76,154), but the extent of this revival (maximum, two-to threefold increase in viable count achieved at 57°C) is less than by alkali treatment and also markedly less than that reported with formaldehyde (201). Coat-removing agents fail to achieve any revival (154) despite reports that glutaraldehyde-treated spores damaged by one such treatment are capable of germination (76 Spores have been recovered after 24-h exposure to the dialdehyde, whereas the Association of Official Analytical Chemists test (8) recommends a 10-h exposure and much shorter exposure periods are commonly encountered, particularly in the hospital environment where time is often at a premium.…”

“…Coat-removing agents fail to achieve any revival (154) despite reports that glutaraldehyde-treated spores damaged by one such treatment are capable of germination (76 Spores have been recovered after 24-h exposure to the dialdehyde, whereas the Association of Official Analytical Chemists test (8) recommends a 10-h exposure and much shorter exposure periods are commonly encountered, particularly in the hospital environment where time is often at a premium. An additional point is that in our studies (76,153) only freshly activated glutaraldehyde solutions were used, whereas in practice older solutions are frequently used, with some deterioration occurring (155).…”

“…(An exception to this general statement is glutaraldehyde, low concentrations of which inhibit both processes [154]). What is not clear is why this should be so.…”

“…At higher glutaraldehyde concentrations (01. to 1%, wt/vol), uptake of L-alanine is significantly reduced, presumably the result of a sealing effect by the aldehyde on the spore surface. Spores do not concentrate L-alanine and uptake proceeds rapidly without the necessity for an energy-dependent active transport system, demonstrating that the dormant spore is freely permeable to the amino acid, which enters by simple diffusion (53,154).…”

“…The major initial effect of bicarbonate is believed to be on the outer layers of spores of bacterial cell walls (79-81, 84, 137), although it will also inhibit germination of Bacillus spores (15,45). Glutaraldehyde is thus likely to seal the outer layers of spores, an action that would also be of importance in inhibiting spore germination (154), with penetration at alkaline pH into the spore. Glutaraldehyde combines with amino acids (94,182,235) and has been found to interact strongly with the cortex and spore protoplast, the latter prepared by Fitz-James' technique (61).…”