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Technical Report CH – 691/2016 EVALUATION OF ANTIMICROBIAL ACTIVITY 1. Preparation of bacterial suspension Bacterial strain ( Salmonella typhimurium ATCC 14028) was maintained as described in EN 12353. In order to prepare the working culture of the test organism, the bacterial strains were inoculated by streaking on TSA dishes and incubated at 37°C for 18 hours. This procedure was repeated three times, and the third subculture was used for the study. For the preparation of the bacterial test suspension, 10 mL of diluent was taken and placed in a 100 mL flask with 5 g of glass beads. Then a loopful of cells was transferred into the diluent. The cells were suspended in the diluent by immersing the loop and rubbing it against the side of the flask to dislodge the cells. The flask was then shaken for 3 minutes using a mechanical shaker. The bacterial suspension obtained was then transferred into a new sterile tube and the number of the cells was adjusted to obtain a concentration from 1.5 x 10 8 cfu/mL to 5 x 10 8 cfu/mL using diluent. The estimated number of bacteria was determined microscopically using a Thoma cell. This suspension was maintained at 20°C and used within 2 hours. The adjusted bacterial suspensions were diluted in order to obtain 10 -6 and 10 -7 serial decimal dilutions using diluent. An aliquot of 1 mL from each dilution obtained as described above was inoculated in duplicate into two separate Petri dishes using the pour plate technique. Approximately 15-20 mL of melted TSA cooled to 45°C was then added. The bacterial strains were incubated at 37°C for 24 hours. After incubation, the number of the colonies of each test strain grown on the Petri dishes was determined. Only For Petri dishes with a number of colonies between 14 and 330 were considered for the colony count. The concentration S 0 of the test suspension inoculated on the paper carrier was calculated using the following equation: where: 0,050 0,1 c = is the sum of the Vc-value taken into account = is the number of Vc value taken into account in the lower dilution = is the number of Vc value taken into account in the higher dilution d = is the dilution factor corresponding to the lower dilution page 8 of 16
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Technical Report CH – 691/2016 2. Culture media and reagents 2.1 General The reagents were of analytical grade and/or appropriate for microbiological purposes. 2.2 Water The water was free from substance that are toxic or inhibiting to bacteria and fungi. It was freshly glass distilled and not demineralised water. 2.3 Tryptone Soya Agar (TSA) A commercially available Tryptone Soya Agar medium, sterilized in the autoclave, was used for the maintenance of bacterial strains and performance of viable count. 2.4 Neutralizer The neutralizer used for this study was prepared as follows. Lecithin Polysorbate 80 L-Histidine Water 3 g/L 30 g/L 1 g/L 1000 g/L It was sterilized by passing through a filter with a maximum effective pore size of 0.45 µm. 2.5 Diluent A commercially available Maximum recovery diluent, sterilized in the autoclave, was used for serial decimal dilutions. 2.6 Preparation of the test article solution for the test procedure and validation of neutralization method The test items were used as supplied, without dilution. 2.7 Preparation of the paper carriers Paper carriers were prepared with 2x2 cm paper squares. The carriers were sterilized in the autoclave. page 9 of 16
