Annex White book.pdf

liquor, 25 g of saffron in sterilized condition should be weighed in Stomacherbag and 225 ml of peptone regulative solution should be added.The preparation is placed in the Stomacher bag for 1 minute in orderto be homogenized. We receive then mother liquor in a relation of 10-1(1:10). Further on, we take 1 ml of mother liquor in a sterilized pipetteand dilute it up to 10 ml in peptone regulative solution. We homogenizeby receiving a decimal solution of 10-2 (1:100).Escherichia coli tallyingEscherichia coli determination takes place by tallying the concentrationsdeveloped on a cultivation plate (dry matter) where a knownsample amount has been inoculated at a determined amount of timeand at incubation temperature (44,5oC for 24 hours).The testing method is as follows: we dissolve the cultivation mediumTergitol B.C.I.G. in a warm water bath. We cool it at 45oC andkeep this temperature constant. We take 1 ml of each solution that hasbeen already prepared (10-1, 10-2) and place it in different Petri plates.We stir lightly with circular moves in order to homogenize the sample.We leave the sample to solidify at room temperature and let each plateincubate on reverse position at 44,5 o C for 24 hours. This procedure isapplied twice. After 24 hours we count all blue colored concentrationsthat have been developed on the plates.Sulfite reducing clostridia tallyingThis determination takes place by tallying the concentrations developedin a tube that contains an adequate cultivation medium andspecified sample amount. The procedure is applied after the sample hasremained for a certain amount of time at specified temperature and inanaerobic conditions.The testing procedure is as follows: we take 4 tubes of cultivationmedium agar - agar SPS and dissolve it completely in warm water bath,lowering the temperature to 45oC (the solutions remain at this constanttemperature until use). After homogenization of solutions 10-1 and 10-2 we take a sample fraction of about 5 ml with a sterilized pipette andplace it in sterilized flame proof glass tubes. We heat the tubes in a waterbath at 80-85 o C for 5 minutes. We inoculate the sample in every agaragarSPS tube (1 ml of the 1:10 solution and 1 ml of the 1:100 solution)and homogenize the solution. We cover the tube surface with sterilizedVaseline and leave it to solidify at room temperature. The tubes areincubated at 46 o C for 48 hours in anaerobic conditions. After 48 hourswe count all black concentrations developed in the tubes.The amount of sulfite reducing clostridia contained in 1 g sampleis calculated by multiplying the concentrations developed with solutionmedium applied in each case.170