The aim of the test is to evaluate the efficacy of the tested cosmetic preservative to reduce the microbial load of a strain following the Minimun Inhibitory Concentration and Minimum Bactericidal protocols using appropriate microbiology techniques.


Used microrganisms

The microorganisms used to evaluate the antimicrobial activity of the formulation have been previously agreed with the customer and are listed below:

Staphylococcus aureus (ATCC 6538);
Staphylococcus epidermidis (ATCC 12228);

Escherichia coli (ATCC 8739);
Pseudomonas aeruginosa (ATCC 9027);

Candida albicans (ATCC 10231);
Aspergillus brasiliensis (ATCC 16404).

Growth media

The growth medium used for the MIC analisys is:
– TSB (Tryptone Soy Broth, Acumedia Lab Neogen Culture Media, COD: LAB004)

The growth media used for the preparation of the MBC subcultures are:

–  TSA (Tryptone Soy Agar, Condalab, COD: 1068);

–  SDA (Sabourad Dextrose Agar, Condalab, COD: 1024).

–  PDA (Potato Dextrose Agar, Condalab, COD: 1022).

Determination of microbial load

The ability of the tested cosmetic preservative to reduce the microbial load of the strains was evaluated by the Mininum Inhibitory Concentration (MIC) in 96-wells plates.

Preparation of the sample and inoculation

Serial dilutions of the tested preservative were prepared on a 96-wells plates (14% – 12.5% – 11% – 9.5% – 8.0% – 6.5% – 5.0% – 3.5% – 2.0% – 0.5%) diluting in specific medium broth for the selected strains.
Samples (180 μl) of each dilutions or medium broth (for the inoculum control) were put on 96-wells plates; then 20 μl of the microbial strain were added (final concentration 10^6 CFU/ml for bacteria and 10^5 CFU/ml for C. albicans and A. brasiliensis). Plates were incubated for 24 h at 37°C and for 48 h at 25°C for bacteria and yeasts/moulds, respectively.

The same protocol was followed for each strain.

Evaluation of bactericidal activity

Once the MIC was determined in a 96-well plate, 10 μl of the control, the MIC and two concentrations (increasing and decreasing with respect to the MIC) were plated in duplicate, in particular TSA plates incubated for 24 h at 37 °C for bacteria, SDA plates incubated for 48/72 h at 25 °C for C. albicans and PDA plates incubated for 72/120 h at 25 °C for A. brasiliensis. After incubation time, the colonies on the plate were enumerated in order to calculate the reduction of the microbial load obtained from each of the tested concentrations compared to the control.

The same protocol was followed for all selected strains.