One gram of soil was added to 50 ml sterile medium containing (per litre) 10 g proteose
peptone, 10 g casein hydrolysate, 1.5 g anhydrous MgSO4, 1.5 g K2HPO4 and 10 ml
glycerol (PAF medium) in a 250-ml flask. The flask and its contents were incubated in a
shaker (200 r.p.m.) at a temperature 300
C. After 24 h, a 1-ml aliquot was removed from
the growing culture, transferred to 50 ml of sterile PAF medium in a 250-ml flask and
incubated at 200 r.p.m. in a shaker for 24 h, at 300
C, the same temperature as the first
incubation.