X-ray microanalysis of chlorhexidine-treated cells of Saccharomyces cerevisiae

Abstract

The use of energy dispersive analysis of X-rays (EDAX) to identify and quantify the presence of chlorhexidine diacetate (CHA) within Saccharomyces cerevisiae cells was examined. Chlorine was used as the elemental marker tag. Saccharomyces cerevisiae cells exposed to 1000 micrograms ml-1 CHA took up increasing amounts of CHA over a time period of 30 s to 30 min. Electron probe micro-analysis was employed to examine the specific accumulation of CHA across the treated cells. These results showed that CHA was distributed evenly between the cell wall, cytoplasm and vacuoles of cells pre-treated with this concentration of CHA for 30 min. The EDAX system therefore provides a useful tool for examining the qualitative and quantitative effects of chlorhexidine on yeast cells, although quantitative data must be interpreted with caution.

Antiseptics and Disinfectants: Activity, Action, and Resistance

“…The effects of chlorhexidine on yeast cells are probably similar to those previously described for bacteria (204)(205)(206)(207). Chlorhexidine has a biphasic effect on protoplast lysis, with reduced lysis at higher biguanide concentrations.…”

“…Similar techniques have been used to study the activity of antiseptics and disinfectants against fungi, in particular yeasts. Additionally, studies on cell wall porosity (117)(118)(119) may provide useful information about intracellular entry of disinfectants and antiseptics (204)(205)(206)(207)(208).…”

“…In one form of intrinsic resistance, the cell wall presents a barrier to reduce or exclude the entry of an antimicrobial agent. The evidence to date is somewhat patchy, but the available information links cell wall glucan, wall thickness, and relatively porosity to the susceptibility of Saccharomyces cerevisiae to chlorhexidine (Table 13) (204)(205)(206)(207)(208). Protoplasts of this organism prepared by glucuronidase in the presence of ␤-mercaptoethanol are lysed by chlorhexidine concentrations well below those effective against "normal" (whole) cells.…”

Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

“…Phosphorus (P), which is present in the phospholipid bilayers, the sugar-phosphate backbone of nucleic acids, and the nucleotide pool in the cytosol, could be used as an elemental marker to reflect damages on the cell wall and membranes. Energy dispersive X-ray analysis (EDAX) is a technique that can be used to determine the elemental composition in a small area observed under scanning electron microscope [12]. We utilized this technique to measure the cellular contents of Cl and P treated with 0, 0.3 and 0.75 mg/L of CHX.…”

“…The percentage composition of the detected elements was calculated. Weight percentages of chlorine and phosphorus reflect the cellular content of CHX and phospholipids in the cells, respectively [12]–[14].…”