X-ray microanalysis of chlorine and phosphorus content in biguanide-treated Acanthamoeba castellanii.

Energy dispersive analysis of X-rays (EDAX) was used to study the effects of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) on Acanthamoeba castellanii. A high variation of elements occurred in untreated individual cells and only two elements, Cl (a biocide marker) and P, were investigated. X-ray dot mapping of untreated trophozoites and cysts revealed that Cl in cells was uniformly distributed throughout the cytoplasm, whereas P was less dense in the vacuoles. X-ray dots of Cl in biocide-treated trophozoites and cysts appeared denser and evenly distributed within the cells as the biguanide concentration increased. Quantitative analysis of either CHA or PHMB within the cells using Cl as an elemental marker was unsatisfactory because of the high Cl levels in untreated cells. The apparent increases of P in some experiments with treated cells might be associated with reduced permeability, protein coagulation or aggregation of phospholipids.

Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores.

Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water. More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware. This combined procedure also produced optimum and reproducible results for the synchronous growth of two B. subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation. The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.