Methods selleckchem Selection criteria for subjects and sample collection Subjects from two healthy Indian joint-middle class families with similar eating habits comprising of three successive generations staying under one roof and with no history of gastrointestinal diseases, no genetic disorders and no antibiotics consumed in the past six months were selected. Age of individuals in Family S was S1 (26 years), S2 (8 months), and S3 (56 years) and in family T was T1 (14 years), T2 (42 years),
and T3 (62 years). Stool samples were collected in a sterile N2 flushed bottles on the same day from each individual within a family and within 2 hours were transported to laboratory. Samples of family S were processed for isolation of strict anaerobes and VE-822 chemical structure remaining samples from both the families were frozen at −70°C for further molecular analysis. All the experiments were carried out with approval from Institutional (NCCS, Pune) Ethical Committee. A written informed consent was obtained from the subjects, in case of children written consent was obtained from their parents. Isolation of strict anaerobes Three samples from family S were processed for isolation study. Each sample was serially diluted in pre-reduced sterile phosphate buffer (pH 7.0) BMN 673 manufacturer 0.3 g, K2HPO4, 0.18 g, KH2 PO4 , 0.45 g, NaCl, 0.46 g, (NH 4) 2SO4 ,
0.05 g, CaCl2 , 0.09 g, Mg2 SO4 ; H2O, 0.001 g, resazurin,
0.5 g, L- cysteine HCl; H2O and observed under phase contrast microscope (Nikon Eclipse 80i, Japan) in order to obtain morphological details and density of bacteria (cells ml-1). Serial dilutions were carried and 0.1 ml of each dilution from 10-5 to 10-8 of the fresh sample were placed on the pre-reduced medium agar plates in an PAK5 anaerobic chamber (Anaerobic system 1029, Forma Scientific Inc., USA) with gas phase of N2:H2:CO2 (85:10:5). The plates were incubated at 37°C in built-in incubator in the anaerobic chamber. Two non-selective media namely Peptone Yeast Extract Glucose (PYG), Brain Heart Infusion (BHI) (OXOID LTD., England) and one selective medium namely Bile Esculin (BE) were used for the isolation. Enrichments were set up for all fecal samples in PYG, BHI and BE medium to culture bacteria present in low numbers in the feces. One gram of fecal sample was suspended in 9 ml pre-reduced sterile broth. After consecutive transfers to enrich different bacteria, the enrichment cultures were serially diluted up to 10-8. The last four dilutions were placed on the pre-reduced respective medium agar plates under anaerobic conditions and were kept for incubation at 37°C. Direct isolation and enrichment plates were incubated for 5 days and well grown morphologically different colonies were picked after every 24 h during the 5 days incubation.
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