Milk samples were hand-mixed and opened in a biosafety level II cabinet. Bacteriological examination of milk samples was performed as recommended previously [59 ,60 (link)]. Briefly, 10 μL of milk were streaked by the quadrant streaking method over Blood Agar Base (bioMérieux, Warsaw, Poland), Mac Conkey Agar (BTL, Warsaw, Poland), Mannitol salt agar (Oxoid Ltd., Basingstoke, UK), and Edwards Medium (Oxoid Ltd., Basingstoke, UK) plates. Plates were incubated at 37 °C, and then read after 24 and 48 h. The bacteria were tentatively identified according to their cultural and morphological appearance, and Gram’s reaction [61 ]. Detailed identification of isolated bacteria was performed using standard biochemical tests and API tests (bioMérieux, Warsaw, Poland) [62 ,63 (link)].
Blood agar base
Manufactured by bioMérieux
Sourced in Poland
Blood Agar Base is a microbiological culture medium used for the isolation and identification of a variety of microorganisms, including both aerobic and anaerobic bacteria. It provides the necessary nutrients and growth factors to support the cultivation of a wide range of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Mannitol salt agar, by Thermo Fisher Scientific (2 mentions)
Edwards medium, by Thermo Fisher Scientific (2 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Api tests, by bioMérieux (1 mentions)
Nutrient broth, by HiMedia (1 mentions)
Tryptone bile x glucuronide agar, by Merck Group (1 mentions)
Endo agar, by HiMedia (1 mentions)
Api coryne system, by bioMérieux (1 mentions)
4 protocols using blood agar base
1
Milk Bacterial Profiling Protocol
2
Microbial Culture Conditions Optimization
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Microorganisms were cultured at 22°C ± 1°C for 24–48 h on Difco Bacto agar (Difco, USA), nutrient broth (HiMedia, India), and blood agar base (BioMerieux, France) media.
3
Comprehensive Bacterial Identification Protocol
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Microorganisms’ morphological, cultural, and chemical properties were studied using conventional methods with differential diagnostic media and test systems. The bacterial phenotypes were studied using conventional methods according to the classification system from Bergeys manual 1984–1989 [26 ]. Microorganisms were cultured at 37°C ± 1°C for 24 or 48 h on the following media: Blood Agar Base (Biomerieux, France), Nutrient Broth for the general cultivation of less fastidious microorganisms (HiMedia, India), Endo Agar (HiMedia), Tryptone Bile X-glucuronide agar (Merck, Germany), and Chromogenic E. coli/coliform agar (Merck). The ENTERO-Rapid 24 kit (PLIVA-Lachema, Czech Republic) was used to differentiate microorganisms. Serological identification was conducted at the Federal Budgetary Institution of Science “State Scientific Center for Applied Microbiology and Biotechnology” https://obolensk.org/en/about-eng (Russian Federation) using the “E-coli serum” and “Anti-adhesive serums”, prepared at this Institution and on “Armavir Biofactory” (Russian Federation).
4
Milk Bacteriology: Comprehensive Identification Protocol
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Bacteriological examination of milk samples was performed according to Sztachańska et al. [39 (link)]. Briefly, 10 μl of milk were cultivated on Blood Agar Base (BioMérieux Poland), MacConkey Agar (BTL, Poland), Mannitol salt agar (Oxoid Ltd., England), and Edwards Medium (Oxoid Ltd., England). Plates were incubated at 37°C and read at 24 and 48 h later. Colonies were identified by their colony morphology and Gram staining. Detailed identification of isolated bacteria was performed using standard biochemical tests and API-Coryne system (bioMérieux Poland).
For C. pseudotuberculosis diagnosis, milk samples were inoculated onto brain heart infusion (BHI) agar supplemented with 5% defibrinated sheep blood and chocolate agar. The plates were incubated aerobically for approximately 48 h at 37°C. Colonies that morphologically resembled C. pseudotuberculosis were Gram stained. Gram-positive colonies were further tested for urease activity, synergistic hemolytic activity with Christie, Atkins, and Munch-Peterson factor from Rhodococcus equi and carbohydrate fermentation (glucose, lactose, and sucrose). Strains that were positive for urease and glucose fermentation and negative for lactose and sucrose fermentation were identified as C. pseudotuberculosis [40 (link)].
For C. pseudotuberculosis diagnosis, milk samples were inoculated onto brain heart infusion (BHI) agar supplemented with 5% defibrinated sheep blood and chocolate agar. The plates were incubated aerobically for approximately 48 h at 37°C. Colonies that morphologically resembled C. pseudotuberculosis were Gram stained. Gram-positive colonies were further tested for urease activity, synergistic hemolytic activity with Christie, Atkins, and Munch-Peterson factor from Rhodococcus equi and carbohydrate fermentation (glucose, lactose, and sucrose). Strains that were positive for urease and glucose fermentation and negative for lactose and sucrose fermentation were identified as C. pseudotuberculosis [40 (link)].
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