1. Soak two samples of 1 kg seed per seed lot in SR buffer (2 ml per gram of seed) in sterile containers for 20 h at 5°C.
2. Stir each suspension thoroughly with a sterile glass rod and then remove a 20 ml sample.
3. Make three serial tenfold dilutions in SR and plate 0.1 ml of each dilution (three duplicates of each).
4. Incubate plates at 28°C for 4–5 days and then count the number of pathogens and saprophytes.
solution 1: proteose peptone no. 3 (Difco) (10 g), CaCl2 (0.25 g), tyrosine (1 g), agar (15 g), distilled water (500 ml);
solution 2: skim milk (10 g), distilled water (500 ml);
solution 3: Tween 80 (10 ml). Autoclave solutions separately and mix when still hot. Cool to 50°C and add the following antibiotics under sterile conditions: cephalexin 80 mg/l, cycloheximide 200 mg/l and vancomycin 10 mg/l.
Goszczynska, T. and Serfontein, J. J. 1998. Milk-Tween agar, a semiselective medium for isolation and differentiation of Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. phaseolicola and Xanthomonas axonopodis pv. phaseoli. Journal of Microbiological Methods. 32(1): 65-72.