Lactobacillus selective lbs agar

For bacteriological analysis, cheeses were aseptically sampled at Day 1, 14, 28, and Month 3 of ripening. The samples were placed in a sterile stomacher bag, diluted 1:10 with sterile 2% trisodium citrate and homogenized using a stomacher (Iul Instruments, Barcelona, Spain) for 5 min. Independent duplicate samples were taken at each time point and serial dilutions were prepared as required. Starter cells were enumerated on LM17 agar (Merck, Darmstadt, Germany) after incubation at 30°C for 3 days. Total NSLAB (lactobacilli) were enumerated on Lactobacillus selective (LBS) agar (BD, Oxford, UK) after 5 days incubation at 30°C. Coliforms and enterococci were enumerated on violet red bile agar (BD) and Kanamycin aesculin azide agar (Merck) after 24 h incubation at 30°C.
To confirm that the majority of NSLAB belonged to the inoculated adjuncts, Pulsed-Field Gel Electrophoresis (PFGE) was performed as described by Stefanovic et al. (2017a (link)). Isolates from Month 3 samples were analyzed, and the PFGE patterns were compared with the patterns of the three adjuncts (Figure 2B).

Ileal and AC contents were homogenized in PBS by vortex and aseptically plated in triplicate using an Eddy Jet spiral plater (Neu-Tec, Farmingdale, NY, USA) on Lactobacillus selective (LBS) agar (BD, Franklin Lakes, NJ, USA). The agar plates were incubated for 48 h at 37°C anaerobically (5% CO₂, 5% H₂ and 90% N₂). Bacteria were counted using the spiral plater manufacturer’s methods and expressed as colony forming units (CFU) per gram of homogenized sample. Ten colonies per piglet per sample were randomly selected, cultivated in MRS, resuspended in 12.5% glycerol and stored at -80°C.