The Mechanical Destruction of Bacteria

Abstract

SUMMARY: A number of different organisms were subjected to violent shaking with minute round glass particles. Vegetative bacteria, spores, and acid-fast species were killed by this treatment, though a t varying rates.Curran & Evans (1942) described the destruction of bacterial spores by violent agitation yvith small inert particles. Several workers (e.g. Gale, 1947) have applied this technique to the extraction of enzymes from vegetative cells. Unlike other mechanical methods (e.g. sonic disintegration or the use of bacterial mills) it requires no elaborate equipment, and the risk of damage to proteins or other labile cell constituents is less than with methods involving autolysis or the use of chemical agents. Cuiran & Evans (1942) obtained large decreases in the viable count of spore suspensions by this method and claimed (though without giving experimental data) that suspensions of Bacterium coli may be completely sterilized. We have investigated a wider range of both sporing and non-sporing bacteria. In confirmation of Curran & Evans's findings we obtained a rapid exponential fall in the number of viable organisms: but a small proportion of the cells appeared to be much more resistant to this treatment than the others present in the same bacterial suspension. METHODSBacterial suspensions. The organisms tested were mainly from our laboratory stock. They were grown for 24 hr. (48 hr. for Mycobacterium megmatis) a t 37' on the surface of nutrient agar, with addition of 0.1 yo glucose and 10 % horse blood for the pneumococcus, washed off with physiological saline, washed twice with saline, and resuspended in the fluid (usually saline) in which they were to be shaken. The cultures of Bacillus subtilis, B. mesentericus and B. anthracis were allowed to stand a t room temperature for a few days before preparing the suspensions, which consequently consisted mainly if not entirely of spores. Suspensions of yeast were prepared from commercial brewers' yeast by repeated washing with saline.All suspensions were adjusted to an opacity equal to Brown's tube no. 3, which corresponds to about los organisms/ml. in most cases. Exact adjustment of the initial count was not practicable since, for reasons stated later, the 'initial' was taken after a short period of shaking during which a variable degree of killing took place. All precautions to avoid extraneous contamination were taken throughout.Particles. Curran & Evans (1942) employed several types of particles and found small smooth glass beads to be the most suitable. We obtained these from Messrs Chance Bros. Ltd., Smethwick, England, under the trade name ' ballotini '. Three grades were supplied whose mean diameters, measured

Penicillins and Cephalosporins

“…DAWSON ( 1949), following a report by KING and ALEXANDER ( 1948) on the mechanical killing of bacteria by shaking with glass beads, demonstrated a relatively clean separation of cell walls from protoplasmic material using this technique. A number of workers have since contributed knowledge concerning cell wall preparations; one of the principal investigators in this field, M. R. J.…”

The Study of Cell Rupture in Staphylococcus aureus

“…Therefore the turbidity of a staphylococcal suspension after partial mechanical rupture with glass beads would be expected to consist of the sum of the turbidity due to unchanged cells and the turbidity of the cell walls produced from those bacteria which have been ruptured. The rate of killing of bacteria by mechanical rupture follows a logarithmic law (King & Alexander, 1948) so that, if complete rupture is the sole cause of killing, the turbidity of a given suspension after t minutes shaking should equal…”

“…aureus 205 As the rate of fall of viable counts has been found by other workers (King & Alexander, 1948) to be logarithmic, it was assumed above that death only occurs simultaneously with complete rupture in order to deduce a logarithmic fall in turbidity. The fact that the observed results followed the theoretical equations and that viable and electron micrographic counts agreed closely with the production of cell walls calculated by this method confirm this assumption.…”

“…When bacterial suspensions are shaken with small glass beads the turbidity decreases (Curran & Evans, 1942) and the viable count steadily drops according to a logarithmic law (King & Alexander, 1948). This appears to be due to mechanical rupture of the organisms, as the cell walls of the burst bacteria can be separated from the soluble cytoplasmic contents by centrifugation (Cooper, Rowley & Dawson, 1949;Salton & Horne, 1951).…”

On the mechanism of microbial cell destruction during ballistic disintegration processes

“…The change in the number of intact cells during ballistic disintegration is for a wide range of microbial cell types as follows (King & Alexander, 1948): (1)…”