Mrs agar plates

In order to analyze different bacterial species from the gut microbiome of Drosophila, both white[-] and Oregon-R male flies (9 individuals) were surface sterilized by washing with 10% bleach, 70% ethanol and PBS before homogenization and plating on MRS and ACE agar plates. MRS agar plates (Oxoid, Thermo Scientific) contain (in 1000 mL dH₂O): Agar (15 g), casein peptone, tryptic digest (10 g), meat extract (10 g), yeast extract (5 g), glucose (20 g), Tween 80 (1 g), K₂HPO₄ (2 g), Na-acetate (5 g), (NH₄)₂ citrate (2 g), MgSO₄ x 7 H₂O (0.2 g), MnSO₄ x H₂O (0.05 g), pH 6.2–6.5. ACE agar plates (Blum et al., 2013 (link)) contain: (in 1000 mL dH₂O): Agar (15 g), yeast extract (8 g), casein peptone (15 g), glucose (10 g), after autoclaving: acetic acid (3 mL), ethanol (p.a.) (5 mL) and Cycloheximid (100 mg). The plates were incubated at 28°C for three to five days and single colonies were picked and isolated on new agar plates for three rounds to obtain pure cultures. These were then stored in glycerol stocks for later DNA extraction and analysis.

Dry matter (DM) was determined in triplicate by oven drying for 48 h at 60°C. A mixture 40 g of silages in 360 ml of distilled water was stomached for 3 min (Bagmixer, Interscience, France), the filtered aqueous extract was used for estimation of lactic acid content, ethanol and acetic acid. Lactic acid content was determined by the spectrophotometric method of Barker and Summerson (1941) . Ethanol and acetic acid were determined with a gas chromatograph equipped with a semi capillary FFAP (nitroterephthalic acid-modified polyethylene glycol) column (Hewlett Packard, Waldborn, Germany), over a temperature range of 40–230°C. Microbiological evaluation included enumeration of LAB on Rogosa and MRS agar plates (Oxoid, Basingstoke, United Kingdom), yeasts and molds were enumerated on malt extract agar plates (Difco, Detroit, MI, United States) acidified to pH 4.0 with lactic acid. The plates were incubated at 30°C for 3 days.

The rectal swabs were immersed in maximum recovery diluent (Oxoid) to prepare serial dilutions that were spread onto MRS agar plates (Oxoid) for the isolation of LAB. The serial dilutions were also spread onto PCA plates (Oxoid) to estimate the percentage of LAB when compared to total microaerophilic viable bacteria. Plates were incubated at 37 °C for 48–60 h under microaerophilic conditions. A total of 50 different colonies were then selected from MRS-agar plates containing approximately 20–100 colonies from each of the 26 collected faecal samples (a total of 1300 colonies), and cultivated in Thermo Scientific Nunc MicroWell™ 96-well plates with MRS broth at 37 °C for 48–60 h under microaerophilic conditions.

Stock strains (B. infantis 52486, B. longum SP 07/3, L.GG, and LcS) were stored frozen at −80 °C in Microbank^® mixed vials according to the manufacturer's instructions (ProLab Diagnostics). After defrosting, the strains were grown in MRS agar plates (Oxoid Ltd., UK) at 37 °C under anaerobic conditions for 3 days. A single colony from each strain was then transferred to a hungate tube containing 10 ml MRS broth (Oxoid Ltd., UK) with 0.05% l-cysteine hydrochloride (Sigma) and incubated for a further 24 h under the same conditions. Following this, 100 μl of the liquid culture was removed, added to a new MSR broth tube and grown at 37 °C in a shaking incubator (Cooled Orbital Incubator, Gallenkamp, Loughborough, UK). Bacteria were harvested in the exponential phase and transferred to centrifuge tubes. After washing twice at 400 × g for 10 min, the bacteria were resuspended in 1 ml RPMI 1640 medium (Lonza, UK) and diluted to a concentration of 1 × 10⁷ cfu/ml.