Antimicrobial Test
The antimicrobial activity was tested against two different bacterial strains: Staphylococcus aureus ATCC 29213 for gram positive and a clinical isolated Escherichia coli for gram negative. Each strain was grown aerobically in Nutrient Broth (NB; Oxoid, UK) at 37 °C for 24 h. A preliminary assay was prepared in order to assess (i) the non-toxicity of the liquid culture media used for dilutions (maximum recovery diluent, MRD, Oxoid, UK) of bacterial suspension, (ii) the non-toxicity of the neutraliser (D/E Neutralising Broth; Liofilchem, Italy) and (iii) the non-toxicity of the different gels after neutralisation. In order to do so, bacterial suspensions and the different diluents were left in contact at 37 °C for the test period. After this, decimal dilutions were performed, samples were inoculated in triplicate on Nutrient Agar (NA; Oxoid, UK) and incubated at 37 °C for 24 h. Counting was performed and the total viable cell count was calculated.
Vitality reduction activity was tested according to the BS EN 1040:2005 (European Committee for Standardization [ECS], 2005) as modified by Grispoldi et al. (8), using the tested neutraliser to stop the antibacterial activity of the gels at any given time. For the assay, sterile tubes were prepared with solutions of bacterial suspension and the hand sanitisers were diluted to different v/v concentrations (33%, 50%, 66% and 75%). The solution was left in contact with the diluted ABHRs for 8 min. Then, the appropriate volume of neutraliser was added. After 5 min, the mixture decimal dilutions were prepared. Samples were inoculated in triplicate on NA and incubated at 37 °C for 24 h. Counting was performed and the total viable cell count was calculated. Only the plates showing a number of colonies included in a 1.5 102–3.0 103 (maximum deviation of 10%) were used to perform the result calculation (9).