Dichloran rose bengal chloramphenicol agar drbc

The growth of mesophilic, fastidious anaerobic bacteria, LAB, Enterobacteriaceae and yeasts were enumerated using Plate Count Agar and Blood Agar (PCA; Oxoid Ltd., ThermoFisher Scientific, Waltham, MA, USA. BA; Blood agar base and defibrinated horse blood; Oxoid and ThermoFisher Scientific), deMan, Rogosa and Sharpe agar (MRS; Oxoid), Violet Red Bile agar with Glucose (VRBG; Oxoid) and Dichloran Rose Bengal Chloramphenicol agar (DRBC; Oxoid), respectively. PCA plates were incubated aerobically for three days at 20 °C. MRS and BA plates were incubated anaerobically for five days at 20 °C and three days at 20 °C, respectively. Anaerobic incubation was performed using the AnaeroGen Atmosphere Generation System (Oxoid). VRBG and DRBC plates were incubated aerobically for two days at 30 °C and 5 days at 25 °C, respectively.
The samples collected from the mid-level of the containers of days 0, 3, 7, 28, 91 (or day 63 from Producer 6) were subjected to microbial plating. (For the Producers 4, 5 and 6 only the end samples were collected and thus plated the first year). Samples of frozen fermented fish brine were thawed and 50 µL of non-diluted or appropriate 10-fold dilutions (using peptone water) were spread on agar plates using an automated spiral plater (Don Whitley Scientific Limited, Shipley, UK).

On days 0, 7, 14, and 21, two cucamelons from each treatment group were removed and stomached as described above. One milliliter of the cucamelon homogenate was serially diluted in PBS, spread on plate count agar (PCA; 5 g L⁻¹ enzymatic digest of casein, 2.5 g L⁻¹ yeast extract, 1 g L⁻¹ glucose, 15 g L⁻¹ agar-agar), and incubated at 25°C for 3 days to enumerate the total mesophilic aerobic bacteria. One milliliter of the cucamelon homogenate was also serially diluted in PBS, spread onto dichloran rose bengal chloramphenicol agar (DRBC; Oxoid), and incubated at 25°C for 5 days to enumerate yeasts and molds.

Viable microbial counts were performed at time of flowering (45 DAT) and at harvest (90 DAT), to assess the impact of the treatment with microbial consortia on the cultivable microbial community. Soil rhizosphere samples, 9 replicates for each treatment, were collected as previously reported (Romano et al., 2020b (link)). Ten grams of rhizosphere composite samples (n=3) were suspended in 90 mL of quarter-strength Ringer’s solution (Oxoid, Milan, Italy). After shaking for 30 minutes, a dilution series was prepared in quarter strength Ringer’s solution, and aliquots were used to inoculate different solid and liquid media. Total heterotrophic aerobic bacteria were enumerated on Plate Count Agar (PCA; Oxoid, Milan, Italy) plates and incubated for 2 days at 28°C; whereas fungi were counted on Dichloran Rose Bengal Chloramphenicol Agar (DRBC, Oxoid) plates and incubated for 7 days at 28°C. To determine target microbial groups based on inoculum characteristics, free-living (N₂)-fixing aerobic bacteria were counted on the Augier medium (Romano et al., 2020a (link)), detecting a brown patina on surface of the liquid medium of positive tube after 15 days of incubation at 28°C; selective count of Trichoderma was performed as described by Caruso et al. (2020) (link).
All tests were carried out in triplicate. Microbiological data were expressed as CFU or MPN g^-1 of soil.