In a 2016 PLOS ONE experiment with E. coli, neither 0.675% phenoxyethanol nor 0.075% ethylhexylglycerin alone reduced culturable cell counts after 30 minutes. Combining them at those concentrations reduced surviving counts by more than five logarithmic units.
Ethylhexylglycerin is abbreviated as EHG. The researchers used E. coli ATCC 11229, generally suspended in sodium phosphate buffer at pH 7.5. Unless otherwise stated, experiments were repeated on three separate days using freshly prepared cultures and solutions.
Culture counts first establish the combined response
The researchers tested EHG, phenoxyethanol and their combination in separate treatment groups. Each component in the combined treatment corresponded to its concentration in the relevant individual group. Matching exposure time allowed the researchers to compare culturable counts after treatment with one material and after treatment with both.
After exposure, the antibacterial agents were neutralized and samples were spread on culture medium. Colony-forming units were counted after incubation. This measurement concerns cells still able to form colonies following treatment and provides the basis for the bactericidal results in Figure 1.
At the stated concentrations, the 30-minute comparison establishes a marked difference between individual and combined treatment. Explaining EHG’s potentiating role then requires observations of the cells beyond the culture-count result.
EHG changes the membrane barrier
The cell membrane restricts movement between the cell interior and its surroundings. The researchers assessed membrane integrity through measurements including dye entry and leakage of intracellular material into the surrounding solution. These observations follow movement across the barrier in two directions: material entering the cell and material leaving it.
Dye staining was examined after three and twenty-four hours. At twenty-four hours, cells treated with 0.075% EHG appeared equally or more permeable to the dye than cells treated with 0.675% phenoxyethanol. This longer-exposure observation needs to be read separately from the thirty-minute culture-count result.
For the DNA-leakage assay, treated cells were removed by centrifugation before DNA in the supernatant was measured. DNA from lysed cells supplied a reference for the total cellular DNA pool. The assay therefore allowed the appearance of intracellular material outside the cells to be assessed separately.
The study found that EHG could damage membrane integrity even at low concentrations. A cell with an affected membrane may still retain the ability to form a colony. Membrane changes can consequently coexist with an unchanged culturable count.
Comparing DNA leakage with survival
Figure 2 provides a concrete comparison. After 30 minutes, DNA leakage from cells exposed to phenoxyethanol at a lethal concentration of 1.35% could resemble leakage after exposure to 0.15% EHG without cell death. Similar leakage thus occurred alongside different survival outcomes.
This comparison clarifies the relationship between the measurements. DNA leakage indicates a change in the barrier retaining intracellular material; culture counts show whether treated cells can still form colonies. Interpreting the relationship requires attention to the concentration and exposure time used in each assay.
The authors linked EHG’s potentiating effect to impaired membrane integrity. EHG affected the membrane, and joint treatment with phenoxyethanol produced rapid killing in the study. The individual and combined culture counts in Figure 1 supply evidence of the combined effect, while the leakage results in Figure 2 provide evidence about the membrane response.
