Tryptone soya broth

Escherichia coli NCIMB 10243 was grown overnight in Tryptone Soya Broth (Oxoid CM129) in an orbital shaker at 35 C. This was used to inoculate water samples containing biocides (2 mL of culture added to 18 mL sample). Final concentrations of the biocides were 200 ppm. Samples were taken from the biocide/bacteria solutions at various time-points and both ATP assays and plate counts performed. 

Inoculate a sample of the bacterium from an agar slope into a medium of 9 mL Tryptone Soya Broth or Nutrient Broth and incubate overnight in an orbital shaker at 37°C. 

NOTE The type of broth used is often sterile tryptone soya broth that may be presented in double strength to allow for dilution with buffer, saline, or water to simulate the process. Any suitable liquid culture medium may however be used but the ability of the broth to support growth should be demonstrated. 

Media are prepared to defined formulations using requisite amounts of appropriate chemicals. Many of the more commonly used media, such as nutrient broth, tryptone soya broth, malt agar, and potato dextrose agar, are available in pre-mixed dehydrated form from various specialist suppliers. Any components of media that are heat-sensitive must be added to the remainder of the medium after it has been heat-sterilized by autoclaving this is normally done by syringing in these particular components as concentrated aqueous solutions through disposable sterile filter units available from various specialist suppliers. 

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. 

Fig. 2. Thermal survival characteristics of S. senftenberg 755W at 60 °C in O, the centre of 3-cm cubes of beef , ground beef A, phosphate buffer (0067 M), pH 70), All menstrua were pre-equilibrated to 60 C and held in 10 g quantities in sterile, polyethylene stomacher bags. Aliquots of washed cell suspensions (previously grown at 37 °C in tryptone soya broth) were added by sterile syringe. Survivors were enumerated on tryptone soya agar.

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. 

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