Mycoplasma broth

Acetone, chloroform, ethyl acetate, ethanol (all were AR grade from Merck, South Africa), DPPH, (Sigma, Germany), Muller Hinton broth, Muller Hinton agar (Oxoid) ADC Middlebrook supplement, Middlebrook 7H9 broth (Fluka), glycerol, Iodo-nitro-terazolium chloride (Fluka), Mycoplasma agar, Mycoplasma broth, Yeast malt broth and Yeast Malt agar (Oxoid).

Briefly, 10‐fold dilutions were performed on a sample of each challenge material (both pre‐ and post‐administration samples), by adding 200 μL culture to 1.8 mL of Mycoplasma broth (Oxoid) and titrating to 10⁻⁷. A 200 μL aliquot of each dilution (10⁻⁴–10⁻⁷) was inoculated onto complete Mycoplasma agar (Oxoid). The plates were incubated in 5% CO₂ in a humidity chamber at +37°C (±2°C) for 3–6 days, after which time the resultant colonies were counted at each dilution for each of the duplicate plates, and the CFU/mL of each challenge inoculum was calculated as follows. The concentration of the challenge was calculated as follows. For each dilution of the challenge, duplicate colony counts were recorded and the mean number of bacterial colonies was calculated. Where possible, the mean count from the lowest dilution which gives the most reliable value (ideally between 30 and 300 colonies) was used to calculate the challenge concentration (CFU/mL) according to the formula: CFU/mL = mean × dilution factor × 5.

All samples were pre-enriched into broth media; brain heart infusion (BHI) broth (Oxoid, Basingstoke, Hampshire, England, UK) or Mycoplasma broth (Oxoid, Basingstoke, Hampshire, England, UK) in case of Mycoplasma detection. A loopful was streaked into different microbiological media including 5% sheep blood agar media (Columbia Blood Agar base; Oxoid, Basingstoke, Hampshire, England, UK), MacConkey agar (Thermo Fisher Scientific, GmbH, Germany), Mannitol Salt Agar (HiMedia Laboratories, Mumbai), and Mycoplasma agar media (Oxoid, Basingstoke, Hampshire, England, UK). The inoculated plates were incubated for 48 h at 37°C in aerobic or anaerobic conditions except for Mycoplasma species where the incubation period extended up to 14 days under microaerophilic condition. Pure cultures were identified biochemically by conventional methods [15 -17 (link)].