Sheep blood agar plates

Approximately 0.01 ml of milk from each sample was cultured on 5% sheep Blood Agar Plates and MacConkey Agar Plates (Oxoid, UK) [19 ]. The agar plates were initially incubated at 37°C for 18-24 h. If no growth appeared after 24 h, incubation was extended for another 24 h before final judgment was taken. From growth-positive plates, isolates were sub-cultured on nutrient agar (HiMedia, India) plates at 37°C for 24 h to obtain pure cultures. Microorganisms were identified based on colony morphology, hemolysis characteristics on blood agar, and Gram-staining [19 ]. Gram-positive bacteria were then confirmed based on the results of the catalase test, oxidase test, DNase test, growth on mannitol salt agar, and Microbact 12S (Thermo Fisher Scientific, USA). Gram-negative bacteria were confirmed based on lactose fermentation on MacConkey Agar, Kligler Iron Agar, catalase test, oxidase test, and IMViC test (HiMedia Laboratories, India).

Photobacterium damselae subsp. damselae (Pdd) RM-71 was isolated from diseased turbot during an outbreak in Galicia (Spain) (Fouz et al., 1992 ). A RM-71 rifampin-resistant derivative (AR57) was created (Rivas et al., 2011 (link)). AR57 and its Rif^R derivatives were routinely grown at 25°C on tryptic soy agar (TSA) (SIGMA 22091), or in trypic soy broth (TSB) (SIGMA 22092), with final NaCl concentrations of 1.5%. Sheep blood agar plates (Oxoid), containing tryptone, peptone, yeast extract, NaCl, pH 7.3 were used for conjugative mating. E. coli strains were routinely grown at 37°C in Luria-Bertani (LB) broth (ROTH) and LB agar [LB broth with 1.5% agarose (Agar-Agar Merck Millipore)], supplemented with antibiotics when appropriate. Antibiotics were used at the following final concentrations: kanamycin at 50 μg ml^-1, ampicillin sodium salt at 100 μg ml^-1, and rifampin at 50 μg ml^-1.

The LAB isolates were cultured in MRS medium for 18–24 h at 37°C. Streak plate methods were performed on sheep blood agar plates (Oxoid, Germany) to analyze hemolytic activity. And then the plates were incubated at 37°C. The appearance of clear zones around the bacteria colonies was indicated as α-, β-, or γ-hemolysis.

The collected samples were serially diluted with de Man, Rogosa and Sharpe (MRS) broth (Oxoid) and plated on MRS agar plates containing 0.5% (w/v) CaCO₃ (BDH, Poole, UK). These were then incubated under aerobic and anaerobic conditions at 37 °C for 48 h. For anaerobic incubations, liquid or solid MRS medium was supplemented with 0.3 g L⁻¹ L-cysteine-HCl (Merck, Darmstadt, Germany) giving rise to MRS-Cys. After incubation, individual colonies surrounded by clear acidification halos were randomly selected, purified on MRS agar with or without cysteine (depending on their aerobic or anaerobic isolation), and examined according to colony morphology, Gram staining, catalase reaction, and hemolytic activity using sheep blood agar plates (Oxoid). Catalase negative, γ-hemolytic, Gram-positive rods were deemed to be LAB and were stored at −80 °C in MRS broth supplemented with 15% (v/v) glycerol (Merck).

Enzymatic activities were evaluated out by placing 5 µL of a TSB-1 culture of each strain at OD₆₀₀ ≈ 0.5 on the surface of TSA-1 plates supplemented with the appropriate substrate. All assays were repeated three times to ensure reproducibility of results. TSA-1 plates supplemented with 1% egg yolk were used to determine phospholipase activity [35 (link)]. TSA-1 plates with 1% Tween 20 were used to confirm lipolytic activity, as Tween 20 is easily hydrolyzed by esterase because it contains esters of lower chain fatty acids [36 (link)]. Both lipolytic activities (phospholipase and esterase) were evaluated after 24 h incubation at 25 °C. The gelatinase activity test was carried out on TSA-1 plates supplemented with 0.4% gelatin, and the results were recorded after 48 h incubation at 25 °C by covering the surface of the agar plate with a 12.5% (w/v) HgCl₂ solution. For the hemolysis test, 5 µL of an inoculum in TSB-1 at OD₆₀₀ ≈ 0.5 was placed on sheep blood agar plates (Oxoid, Hampshire, UK) and incubated at 25 °C for 48 h.

Hemolysis assays on agar plates were conducted by picking a colony of each isolate previously grown on TSA-1, and inoculating it on sheep blood agar plates (Oxoid). For swimming motility assays, single isolated colonies of a 18 h culture agar plate for each strain were picked with a sterile plastic tip and stabbed into motility agar, which was prepared with TSB-1 broth supplemented with 0.25% bacteriological agar. For hemolysis and motility assays, pictures were taken at 24 h post-inoculation of the plates. Experiments were repeated three times to ensure that the hemolytic haloes and motility radius of the strains were reproducible.

Hemolysis assays on agar plates were conducted by picking a colony of each strain previously grown on TSA-1 and inoculating it on sheep blood agar plates (Oxoid) followed by growth at 25°C. For the phospholipase/lecithinase activity assay, 3 μl of overnight cultures in TSB-1 were spotted onto TSA-1 plates supplemented with 3% egg yolk extract (Oxoid), and results were evaluated after 24 h of culture at 25°C. Hydrolysis of lecithin by the phospholipase yields water-insoluble diglycerides that cause the appearance of an opaque precipitate. The gelatinase activity assay was carried out by spotting 3 μl of a TSB-1 overnight culture onto TSA-1 plates supplemented with 1% gelatin (Oxoid), and results were developed after 48 h of incubation at 25°C by covering the agar plate surface with a 12.5% (wt/vol) HgCl₂ solution. Hydrolysis of gelatin by the gelatinase enzyme causes the appearance of a translucent halo around the bacterial colony upon addition of HgCl₂.