Anaerocult system

B. subtilis and E. faecium strains were cultivated on Luria-Bertani (LB) agar at 37°C under aerobic conditions whereas the oral Streptococcus strains (Table 1) were cultivated in anaerobic conditions on Todd Hewitt (TH) agar at 37°C overnight using the Anaerocult® System (Merck, Germany).

Microbiological evaluation was performed following the protocols established by the Applied and Environmental Microbiology Laboratory of Animal Health and Anatomy Department of the Veterinary Faculty (UAB). Samples were grown in blood agar (Columbia agar supplemented with sheep blood), Man–Rogosa–Sharpe agar (MRS, tryptic soy agar (TSA), McConkey agar (MK), Baird–Parker agar (BP) supplemented with egg yolk tellurite emulsion, Sabouraud agar supplemented with 0.5 gr/L chloramphenicol, and tryptone sulfite neomycin (TSN) agar. All culture media were purchased from Liofilchem Srl (Italy), with the exception of Columbia agar, which was purchased from Bio-Rad Laboratories (USA). Oxygen and temperature conditions for the different culture media are shown in Table 1.
Samples were processed within one hour of sampling. Swabs were streaked on Petri dishes following a continuous streak method. Then, plates were incubated for 24 h at the different stated conditions and an initial counting of colonies was performed. Those plates with negative growth were incubated during 24 more hours and the counting was repeated at the end of this second period of incubation. Plates in anaerobic conditions were incubated for 48 h with an Anaerocult system (Merck KGaA, 64271 Darmstadt, Germany), whereas Sabouraud agar plates were incubated for 7 days before being discarded.

Thirty grams of silage were suspended in 270 ml of ¼-strength Ringer solution (2.25 g l⁻¹ NaCl, 0.105 g l⁻¹ KCl, 0.06 g l⁻¹ CaCl₂, 0.05 g l⁻¹ NaHCO₃) (Merck, Darmstadt, Germany) and homogenized in a mixer for one minute. From this suspension, total bacterial counts were analyzed on plate-count agar (5.0 g l⁻¹ enzymatic digest of casein, 2.5 g l⁻¹ yeast extract, 1.0 g l⁻¹ glucose, 15 g l⁻¹ agar, pH 7.0) (Merck, Darmstadt) after aerobic incubation at 30 °C for 2 days. Lactic acid bacteria (LAB) were quantified from this suspension on de Man, Rogosa and Sharpe (MRS)-agar (10 g l⁻¹ enzymatic digest of casein, 10 g l⁻¹ meat extract, 4 g l⁻¹ yeast extract, 20 g l⁻¹ glucose, 2 g l⁻¹ K₂HPO₄, 1.08 g l⁻¹ tween 80, 2 g l⁻¹ triammonium citrate, 5 g l⁻¹ sodium acetate, 0.2 g l⁻¹ MgSO₄ × 7 H₂O, 0.05 g l⁻¹ MnSO₄ × 4 H₂O, 14 g l⁻¹ agar, pH 5.7) (Merck, Darmstadt). MRS agar plates were incubated anaerobically using an Anaerocult system (Merck, Darmstadt). Yeasts and molds were detected using yeast extract glucose chloramphenicol (YGC)-agar (5.0 g l⁻¹ yeast extract, 20.0 g l⁻¹ glucose, 0.1 g l⁻¹ chloramphenicol, 14.9 g l⁻¹ agar, pH 6.6) (Merck, Darmstadt). These agar plates were incubated at 25 °C for 3 days.