Oxoid agar

Charcoal agar. 1.0% Oxoid agar, 0.5% (w/v) activated charcoal, adjust pH to 6.5 with HCl or NaOH. 

Yeast extract and the various broth and agar media are available commercially from Oxoid Ltd. or Difco Labs (Appendix 3). 

Dissolve 40 g blood agar base (Oxoid, Basingstoke) in 1 I of distilled water by standing in a boiling water bath. 

These included nutrient agar (Oxoid), Mueller-Hinton Agar (Oxoid), Desoxy-cholate Citrate Agar (Oxoid), and MacConkey Agar (Oxoid). 

This medium was prepared by adding sterile defribrinated sheep blood (10%) to sterile molten nutrient agar (Oxoid) at 55 °C. 

Erwinia herbicola NCIMB 12126 was obtained from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK), HEPES buffer sachets and magnesium acetate were obtained from Sigma (Poole, UK), adenylate kinase assay kits were obtained from Acolyte Biomedica (Salisbury, UK), sterile tissue culture grade distilled water was obtained from Gibco (Paisley, UK), L-broth and tryptone soya agar plates were obtained from Oxoid (Basingstoke, UK). 

The plate counts at To were estimated by diluting the neat broth culture 1 in 10 with Neutralised Peptone Water (NPW) containing per L 1.0 g Bacteriological Peptone (Oxoid, L37), 8.8 g Sodium Chloride (May Baker), 3.0 g Amisol 910 (Degussa) and 30.0 g Tween 80 (BDH, 560234H) and then decimally with Phosphate Buffered Saline (Oxoid). For biocide treated samples 1 mL was diluted in 9 mL of NPW, mixed and allowed to stand for 5 min (for neutralisation of the biocide). The solution in NPW was diluted decimally (0.1 mL in 0.9 mL) in PBS. Appropriate dilutions (0.1 mL) were plated out on Tryptone Soya Agar Plates (bioMerieux) and incubated at 37 °C for 24 h. 

Enrichment broths have consisted of brain heart infusion (BHI) with cysteine and yeast extracts (Songer et al., 2009) and Oxoid C. difficile medium without agar (Rodriguez-Palacios et al., 2009 Weese et al., 2010 Table 3.1). These enrichment media may be supplemented with cefoxitin ( 16 pg/ml) and cycloserine ( 500 gg/ml) or moxalactam (32 pg/ml) and norfloxacin (12 gg/ml). Alcohol shock and subculture on commercial solid media described above follow incubation under anaerobic conditions for 2-12 days. 

Ingredients of Oxoid selective agar (except agar), 0.1% sodium taurocholate... 

AnaeroGen Compact transparent pouches (AnOOlOC) from Oxoid (Cambridge, U.K.) with atmospheric generation system for anaerobic growth on agar plates. 

Total viable psychrotrophic counts (TVC) and counts of H2S-producing bacteria were evaluated on iron agar (lA) as described by Gram et al. (1987) with the exception that 1% NaCl was used instead of 0.5% with no overlay. Surface-placing was used for all counts. Plates were incubated at 15°C for 4-5 days. Bacteria forming black colonies on lA produce H2S fi-om sodium thiosulphate and/or cysteine. In all experiments cooled Maximum Recovery Diluent (MRD, Oxoid) was used for dilutions. All samples were analyzed in duplicate and results presented as an average. 

Both organisms were grown in TSB medium, consisting of Tryptone Soya Broth (Oxoid Ltd. 3%, w/v) supplemented with inorganic ions and vitamins as described by Abbas-Ali and Coleman (1977). Batches of medium (50ml), contained in 250 ml conical flasks, were inoculated with bacteria from Tryptone Soya Agar (Oxoid Ltd.) slopes by means of a platinum loop. The cultures were incubated in a "Gyratory" incubator-shaker (model G25, New Brunswick Scientific Co., New Brunswick, N.J., U.S.A.) at 37 C in the case of S. aureus and 25 C for A. salmonicida. 

By plate-diffusion The assay medium contains Oxoid peptone 10 g, Lab-Lemco 15 g and agar powder 15 g made up to 1 litre with water and adjusted to pH 7-2. For seeding the medium use about 0 4ml of a sensitive strain of Staphylococcus aureus (the Heat ley penicillin assay strain is suitable) grown for twenty-four hours in nutrient broth to each 100 ml of agar medium. 

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