Tryptose sulphite cycloserine agar

Experimental diets and excreta samples were analyzed in triplicate for dry matter (DM, method 930.15;
[16 ]), crude protein (CP, method 990.03;
[16 ]), ash (method 942.05;
[16 ]), calcium, and phosphorus (method 985.01;
[16 ]). Gross energy was measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL), and chromium concentrations of experimental diets and excreta samples were determined with an automated spectrophotometer (Jasco V-650, Jasco Corp., Tokyo, Japan) according to the procedure of Fenton and Fenton
[17 (link)]. The ATTD (%) of nutrients was calculated by the following formula:

Where
N_f = nutrient concentration in feces (%)
N_d = nutrient concentration in diet (%)
C_f = chromium concentration in feces (%)
C_d = chromium concentration in diet (%)
The microbiological assay of excreta was carried out by the procedure suggested by Choi et al.
[18 (link)]. The microbial groups enumerated were total anaerobic bacteria (TAB; plate count agar, Difco Laboratories, Detroit, MI, USA), Bifidobacterium spp. (MRS agar), coliforms (violet red bile agar, Difco Laboratories, Detroit, MI, USA), and Clostridium spp. (Tryptose sulphite cycloserine agar, Oxoid, Hampshire, UK). The anaerobic conditions during the TAB and Clostridium spp. assays were created by using the gas-pak anaerobic system (BBL, No. 260678, Difco, Detroit, MI, USA).

The excreta samples from the small intestine and the cecum per replicate were pooled in sterilized plastic bottles and mixed. The samples (1 g) were put together in anaerobic dilution solution (9 mL) by keeping them under CO₂. The serial dilutions (10⁻⁵ to 10⁻⁸) were made by using the anaerobic dilution solution and diluted solution and were plated at 1 mL onto agar plates in triplicate. The total anaerobic bacteria were determined on plate count agar (Difco Laboratories, Detroit, MI, USA) and incubated at 37°C for 24 hours under anaerobic condition. The lactobacillus spp. was determined on MRS agar (with 0.20 g/L NaN₃ + 0.50 g/L L-cystine hydrochloride monohydrate) and incubated at 39°C for 48 hours under anaerobic condition. The Clostridium spp. was determined on tryptose sulphite cycloserine agar (Oxoid, Hampshire, UK) and incubated at 37°C for 24 hours under an anaerobic condition. The Escherichia coli (E. coli) was determined on MacConkey agar and incubated at 37°C for 24 hours under anaerobic condition. The anaerobic condition was made in a gas-pack anaerobic system (BBL, No. 260678; Difco, USA) [33 (link)].