To make an initial dilution (10−1), 10 mL camel's milk was homogenized with 90 mL of saline water (8.5 g/L). The suspension was utilized for making appropriate serial dilutions up to 10−8 by incorporating 1 mL into 9 mL of sterile saline water in sterile tubes. One milliliter of these dilutions was pour-plated in M17 agar, de Man Rogosa (MRS) agar [11] and kanamycin aesculin azide agar (Merck, Germany). After incubation at 37°C for 48 hours under anaerobic condition (using anaerocult A gas packs; Merck), individual different colonies were phenotypically selected. Colony enumeration was conducted after incubation and was recorded as CFU per liter of milk.
Kanamycin aesculin azide agar
Manufactured by Merck Group
Sourced in Germany
Kanamycin aesculin azide agar is a selective and differential culture medium used for the isolation and identification of enterococci. It contains kanamycin and sodium azide as selective agents, and aesculin as a differential indicator. The medium allows the growth of enterococci, which hydrolyze aesculin and produce a black or brown-black coloration in the medium.
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2 protocols using kanamycin aesculin azide agar
1
Enumeration of Camel Milk Microbiome
2
Characterizing NSLAB Dynamics in Ripening Cheese
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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 (Figure2B ).
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
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