Reinforced clostridial agar

The ceca with contents were stored at 4°C for determination of cecal microbiota. The cecal digesta was processed within 24 h (5 (link)). Each cecal digesta homogenate was serially diluted from 10-1 to 10-8. Coliforms were cultured using HiCrome E. coli HiVeg Agar (Product code: MV1295; Hi-Media Laboratories, Mumbai, India), anaerobic bacteria were cultured using Wilkins Chalgren Anaerobic Agar (Product code: M832; Hi-Media Laboratories), Staphylococcus aureus was cultured using HiCrome Staph Agar (Product code: M1837; Hi-Media Laboratories), Lactobacillus spp. were cultured using Lactobacillus MRS Agar (Product code: M641; Hi-Media Laboratories), Pseudomonas aeruginosa was cultured using Cetrimide Agar (Product code: M024; Hi-Media Laboratories), and the Clostridium were cultured in Reinforced Clostridial Agar (Product code: M154; Hi-Media Laboratories). Diluted digesta samples were streaked onto the agar plates and incubated at 37°C for 48 h, while the clostridia plates were incubated anaerobically at similar temperature. Visible colonies were enumerated using a colony counter and the results were expressed as log₁₀ CFU/g of cecal digesta.

The counting of fecal select bacterial populations was performed by vortexing 1 g of homogenized fresh feces in 9 mL of normal saline (0.9% NaCl solution). Bacterial populations were counted using the serial dilution method (10¹ to 10⁸) in duplicate by plating in the following media: reinforced clostridial agar (Himedia, Mumbai, India) for clostridia, deMan, Rogosa, and Sharpe agar (Himedia) for lactobacilli^{41 (link)}, MacConkey agar (Himedia) for coliforms⁴² , and Bifidobacteria agar (Himedia) for Bifidobacteria. Lactobacilli and coliforms were cultured anaerobically in an anaerobic jar on relevant agar plates for 24 h at 37 °C, while Bifidobacteria and Clostridia were incubated on the plate for 24 to 48 h. Each plate's bacterial colony was counted and expressed as log₁₀ CFU/g of fresh feces. The ratio of lactobacilli to coliforms was determined by dividing the colony-forming units (CFU) of lactobacilli by the CFU of coliforms.