91.KORTHOF Korthof basal medium: · Dissolve the following components in 900 ml sterile distilled water (use a 3 litre flask): – 0.80 g Proteose Peptone No. 3 (Difco); 1.40 g NaCl; 0.02 g NaHCO3; 0.04 g KCl; – 0.18 g KH2PO4; 0.96 g Na2HPO4.2H20; 0.001 g Vitamin B3 (nicotinamide). · Mix the above and add sterile distilled water up to 1 litre. · Autoclave for 20 min at 121 °C. · Cool overnight at room temperature. · Pass through filter paper (Whatman No. 1, or equivalent). · Check for the pH (the final pH of the medium should be in the range 7.2–7.4). · Autoclave for 30 min at 116 °C. Rabbit serum · Collect rabbit serum (commercial rabbit serum can be used). Because of variation from one animal to another, the use of pooled rabbit serum (obtained from animals on an antibiotic-free diet) is recommended. · Check with the MAT for the presence of antibodies against leptospires. The MAT should be negative. · Inactivate the serum for 120 min at 56 °C in a water bath. · Centrifuge at high speed (22 000 g for 30 min) to remove cellular debris. · Filter-sterilize through a 0.22 μm Millipore filter. · Dispense the rabbit serum in 30 ml volumes in tubes; store at –70 °C until use. Lysed blood cells · Collect 5 ml of rabbit blood (from a seronegative animal on an antibiotic-free diet) in a 250-ml flask containing glass beads. · Mix by gently shaking for 10–15 min to remove fibrin. · Add 10 ml of sterile distilled water, mix and leave at 4 °C overnight. · Centrifuge at high speed (22 000 g for 30 min) to remove cellular debris. · Filter-sterilize the supernatant through a 0.22 μm Millipore filter. · Dispense the supernatant in 2 ml volumes in tubes and store at –70 °C until use. Vitamin B12 · Dissolve 100 mg vitamin B12 in 10 ml sterile distilled water pH 4.5 (use sterile distilled water and adjust its pH to 4.5 with 1N HCl). · Filter-sterilize through a 0.22 μm Millipore filter. Preparation of the final medium · Add the following to the Korthof basal medium: 60 ml sterile, heat-inactivated rabbit serum; 2 ml sterile, heatinactivated lysed blood cells; 0.1 ml sterile vitamin B12 stock solution. · Filter-sterilize through a 0.22 μm Millipore filter. · Dispense the Korthof medium in 4–5 ml volumes in tubes. · Tyndalize three times for 60 min at 56 °C. · Store the medium at 4 °C until use. |
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