Intracellular solutes were released from growing or resting cells of Bacillus megaterium as a consequence of hexachlorophene treatment. The effect was dose dependent, with the optimum at a concentration about sevenfold greater than the minimal lethal dose. The effects of pH and temperature on the leakage process also were inconsistent with the killing effects of the drug. The types of materials released appeared to be the same with or without hexachlorophene treatment. The released materials were smrall molecules which apparently derived from preexisting ribonucleic acid and protein, but not from deoxyribonucleic acid. Compared to the effects of other representative surface-active agents and other bis-phenols, hexachlorophene was superior in ability to cause leakage of intracellular materials. Different microorganisms varied in their susceptibility to hexachlorophene, with Pseudomonas aeruginosa and a paracolon isolate the most resistant of the vegetative cells examined. It was concluded that the release of intracellular solutes was an effect secondary to the lethal event and presumably arose from hexachlorophenemediated stimulation of degradative enzymes.

Hexachlorophene is a soap-compatible bisphenol that has been widely used as an antiseptic, yet its mechanism of action is undefined. The relative threshold concentration for bactericidal effect on a susceptible test organism, Bacillus megaterium , was established to be about 10 μg/mg of cell dry weight. At this or at high (≥100 μg/mg) concentration, adsorptive uptake by cells displayed saturation kinetics. At about 30 μg/mg, the time course of adsorption occurred in three distinct stages. The triphasic pattern was interpreted to represent successive penetration of and adsorption by the cell wall, the protoplast membrane, and the cytoplasm. This interpretation was substantiated by determinations of hexachlorophene adsorption by isolated cell components. Electron microscopy disclosed cytopathology, evidenced as gaps or discontinuities, in the protoplast membrane (but not in the cell wall or cytoplasm) at > 30 μg of hexachlorophene per mg of cell dry weight. Similarly, treatment with > 30 μg/mg allowed a fluorescigenic dye (tolyl-peri acid) to penetrate into the protoplast. However, no detectable cytological manifestations were discerned at the minimum lethal concentration of 10 μg/mg. Apparently, hexachlorophene is physically disruptive at intermediate or high relative concentrations but acts in a more subtle fashion at the minimal lethal concentration.

Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 ug of drug per mg of original cell dry weight were required to lyse 4.4 x 109 protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.