Isolation and Characterization of Anaerobic Gut Bacteria

The chickens were sacrificed under chloroform anesthesia by cervical dislocation. Whole caeca with their contents originating from 18 random healthy chickens or hens 4 to 40 weeks of age were removed during necropsy, chilled on ice and transported to an anaerobic chamber for further processing within one hour. The caeca were opened in an anaerobic chamber (10% CO₂, 5% H₂ and 85% N₂ atmosphere; Concept 400, Baker Ruskinn, USA) and 0.5 g of content was squeezed into 4.5 ml pre-reduced PRAS dilution blank (0.1 g magnesium sulfate heptahydrate, 0.2 g monobasic potassium phosphate, 0.2 g potassium chloride, 1.15 g dibasic sodium phosphate, 3.0 g sodium chloride, 1.0 g sodium thioglycolate, 0.5 g L-cysteine, 1000 ml distilled water; final pH 7.5 +/− 0.2 at 25 °C) and mixed thoroughly. All samples were serially diluted in pre-reduced PRAS dilution blank and plated on Wilkins-Chalgren anaerobe agar (WCHA) (Oxoid) supplemented with 30% of rumen fluid. The rumen fluid was collected from cows by an oral probe, filtered through cheesecloth, centrifuged at 8000 g for 30 min and sterilised by filtration through a 0.22 μm filter. Aliquots of rumen fluid were stored at − 20 °C. WCHA was additionally supplemented with 5 mg/l hemin, 1 mg/l cellobiose, 0.5 g/l soluble starch, 1 mg/ml maltose, 0.2 ml vitamin K1 solution (0.1 ml of filter sterilized vitamin K1 in 20 ml 95% ethanol) and 0.5 mg/ml L-cysteine. Approx. 10 well-separated colonies of different morphology were selected from each agar plate after a five-day incubation at 37 °C and purified by subculture on WCHA. All isolates were stored at − 80 °C in pre-reduced PRAS dilution blank containing glycerol at 20% concentration and equal volume of sterile sheep blood. Sensitivity of pure anaerobe cultures to air oxygen exposure was tested exactly as described elsewhere [6 (link)]. Briefly, bacterial cultures were serially diluted in anaerobic chamber and plated on 4 copies of WCHA. One copy of WCHA was left in the anaerobic chamber to determine initial counts of each anaerobe. The remaining 3 copies of WCHA plates were placed into a standard aerobic 37 °C incubator and after 1, 8 and 24 h, a single copy of agar plate was returned back to the anaerobic chamber to check for growth restoration.