Salmonella shigella agar

The composite cecal material sample (1 g) of each hen was diluted with 9 mL of 0.9% saline solution and mixed using a vortex. Viable counts of bacteria in the cecal samples were then estimated by plating serial 10-fold dilutions (in 1% peptone solution) on lactobacilli de Man, Rogosa, and Sharpe (Lactobacilli MRS), MacConkey, and Salmonella shigella agar plates (Difco Laboratories, Becton Dickinson, Franklin Lakes, NJ, USA) in order to isolate Lactobacillus, Escherichia coli, and Salmonella, respectively. The Lactobacilli MRS plates were then incubated for 48 h at 37°C under anaerobic conditions, and the MacConkey and Salmonella shigella plates were incubated for 24 h at 37°C under aerobic conditions. Lactobacillus, E. coli, and Salmonella colonies were counted immediately after removal from the incubator.

Re-isolation numbers for Salmonella Gallinarum was determined for liver, spleen, and feces collected from each sacrificed chicken at 5 dpi. In addition, feces were retrieved from sterilized paper that was used to line the chicken's housing unit. For non-selective pre-enrichment processing, 0.1 g of tissue and feces from the cecum of each sample were aseptically collected and added to 10 mL of sterile Buffered Peptone Water (Sigma-Aldrich), homogenized completely, and incubated at 37℃ for 24 h. Then, the homogenate was transferred from 1 mL of the pre-enrichment broth to 10 mL Tetrathionate Broth (TTB; Difco Laboratories) and incubated at 37℃ for 24 h for selective enrichment processing. The TTB was serially diluted 10-fold in PBS, and 50 µL of each dilution was spread onto a Salmonella Shigella agar (Difco Laboratories) plate and incubated at 37℃ for 24 h. The resulting characteristic black-colored colonies were counted and expressed as CFU/0.1 g tissue, but only for those plates with counts of 30 to 300 colonies per plate.

On d 100, cecal contents from 8 birds per treatment were gathered into individual, sterile culture tubes and immediately transported to the laboratory for immediate examination. In the analytical procedure, a gram from each cecal sample was amalgamated with 9 mL of sterile peptone broth (Becton, Dickinson and Company, Sparks, MD) and subjected to a 60-s vortexing. To ascertain the viable bacterial counts present, 10-fold serial dilutions (spanning from 10⁻¹ to 10⁻⁸) were methodically plated on distinct agar mediums: Lactobacilli MRS agar (Difco Laboratories, Detroit, MI) for lactic acid bacteria, MacConkey agar (Difco Laboratories, Detroit, MI) for coliform bacteria, and Salmonella-Shigella agar (Difco Laboratories, Detroit, MI) for Salmonella. Postplating, Lactobacilli agar plates were incubated anaerobically at 37°C for a 24-h duration, while both MacConkey and Salmonella-Shigella agar plates were aerobically incubated at the same temperature and time frame. Postincubation, bacterial colonies specific to Lactobacillus, E. coli, and Salmonella were enumerated, with the results articulated in the logarithm of colony-forming units per gram (log₁₀ CFU/g).

One gram of collected sample from the cecum was diluted with 9 mL of sterile peptone water and mixed for 1 min on a vortex stirrer. Samples were serially blended from 10⁻¹ to 10⁻⁶, and were injected by 50 μL in 3 selective agar media as follows; Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) for Lactobacillus spp., coliform bacteria for MacConkey agar (Difco Laboratories, Detroit, MI, USA) and Salmonella-Shigella (SS) agar (Difco Laboratories, Detroit, MI, USA) for Salmonella. Plates were then incubated aerobically at 37°C for 24 h (MacConkey agar and SS agar) or anaerobically at 37°C for 24 h (Lactobacilli MRS agar). The viable colonies of the respective bacteria were counted and expressed as the log 10 of colony-forming units (cfu) g⁻¹ of cecal content.

Fresh intestinal samples from each treatment were taken at the end of the experiment. A total of 15 broilers per treatment were slaughtered and sampled after a 12-h feed withdrawal. Their large and small intestines were used to enumerate Lactobacillus, E. coli and Salmonella. Small intestinal digesta were collected from a 4–5 cm segment between the front and rear parts of the Meckel's diverticulum. Large intestinal digesta were collected from a 2–3 cm front part of the cloaca. To enumerate intestinal Salmonella, approximately 200 mg of sample was diluted 10 fold by blending them with anaerobically sterilized phosphorus buffered saline (PBS, 0.1 M, pH 7.0) and homogenized. Afterwards, a 0.1 ml sample was serially diluted 10³∼10⁷ and spread onto sterilized flat Rogosa agar (Difco Laboratories, Detroit, MI, USA), Salmonella-Shigella (SS) agar (Difco Laboratories, Detroit, MI, USA), and MacConkey's agar (Difco Laboratories, Detroit, MI, USA) for Lactobaicllus, Salmonella, and Escherichia coli culture. Lactobacillus medium agar plates were then incubated anaerobically at 37°C for 48 h. Salmonella and E. coli medium agar plates were incubated at 37°C for 24 h under aerobic conditions. Colonies on each flat medium were counted using a colony counter. Results were transformed as colony-forming units (CFU) at log₁₀ per gram.