Marketed samples of Indigo carmine, Sunset yellow, Erythrosine and Ponceau 4R dye and their aluminum lakes. Materials used in the preparation of the lakes including aluminum hydroxide powder (batch nos. 103,374,AD3 and paste P61). Nutrient agar, Vogel-Johnson’s medium, Mac Conkeys medium, Cetrimide medium and agar media of these and mannitol salt agar were from M/s Hi media, India. Other stains and chemicals employed were of highest purity.
Sampling, sample handling and sample condition-
All the samples obtained were powders, excepting aluminum hydroxide, which was supplied also as a paste. The samples were received in tightly closed glass bottles.
The surface of the sample container was disinfected with an aqueous mixture of 80 % alcohol v/v and 1 % v/v. The surface was dried with sterile gauze before opening and removing the contents in laminar air flow cabinet.
(I) Preparation of stock inoculums -
One-gram test sample was aseptically inoculated in 25 ml of sterile nutrient broth. The inoculated medium was kept undisturbed for about 15 minutes, to ensure complete transfer of microbes from sample to the medium. The clear supernatant liquid was then used a sample for performing the tests.
(ii) Determination of MPN by serial dilution method-
One ml of stock inoculums prepared as mentioned under (i) was diluted with 9.0 ml nutrient broth to give a dilution of 1:10. this was further serially diluted with sterile nutrient broth to give 1:100, 1:1000 and 1: 10000
The labeled tubes were inoculated at 37 + 1 deg.C for 24- 48 hours and 5-7 days after which they were observed for the presence or absence of growth. The tube in which the growth was not clear from appearance was sub-cultured into fresh sterile nutrient broth tubes (0.1 ml was inoculated into 10 ml sterile nutrient broth). Negative and positive (E.coli ATCC 11775) control was maintained to test the effectiveness of sterilization procedures and growth promoting property of the nutrient media respectively.
(iii) Determination of bacterial count by plate count technique-
The stock inoculums prepared under (i) was serially diluted with sterile physiological saline to give 1:10,1:100,1:1000,1:10000 ,1:1,00,000 ,1: 10,00,000 dilutions. Depending on the MPN count values determined above in (1) 1.0 ml of the last two dilutions of the sample showing growth were pour plated using 15.0 ml of sterile nutrient agar. The plates were then incubated at 37 + 1 deg.C for 24 hours and 5-7 days and the MPN values were determined.
(iv) Detection of fungi-
One ml of the stock inoculums mentioned under (i) was added to 90 ml of Saborauds broth. Positive control (Candida albicans ATCC) and negative controls were maintained side by side. The tubes were incubated at room temperature (RT) for a period of 7-15 days.
In the next post, we shall discuss further on the experimental and isolation procedures of the microbes. To be continued in Part 3