Solid medium for plating: SP-agar: It contained g/l: sucrose, 300; casein-peptone, 10; KH,PO,,
0.5; MgSO, -7H,O, 0.5; FeSO,. 7H20,0.007;
ZnSO,. 7 H,O, 0.006; agar-agar, 18; and tap water to
volume. Before sterilization the pH of the medium was adjusted to 6.2 with ammonia (German pa-
tent, 1973). After sterilization 1.25 ml of 0.5% solution of fluorescein or 2',7'-dichlorofluorescein in
ethanol were added to 260 ml of the medium. This was poured into steril Petri dishes and when the
agar had solidified, placed in an incubator (24 "C)and left for some days before inoculating the
media.

The seed stage medium was modified after Hung. pat. (1967).It contained g/l: sucrose, 100; succi-
nic acid, 10; proflo, 10; Ca(NO,),, 1; KH,PO,, 0.25; MgSO,. 7 H20, 0.25; KCl, 0.12; FeSO,. 7 H20,
0.007; ZnSO, . 7 H,O, 0.006 in 1 1 of dest. H,O.
The production stage medium contained g/l: sucrose, 100;succinic acid, 10; Ca(NO,),, 1 ; KH,PO,,
0.3; MgSO, 7 H,O, 0.25; KCl, 0.12; NaCl, 20; FeSO, 7 H,O, 0.007; and ZnSO, 7 H,O, 0.006 i n
1 1 of dest. H,O. p H 5.2 in both media was adjusted with ammonia before sterilization.
Analytical methods : Growth of submerged cultures was determined by mycelial dry weight.
50 ml of broth were filtered, mycelium washed with water and dried a t 85 "C t o constant weight.
Alkaloids: Culture filtrate suitably diluted (2 ml) was mixed with VAN URK
reagent (4 ml) being prepared as described by AGURELL (1966) and the blue color determined spectrophotometrically
with reference t o the standard solution of ergotamino tartrate. Mycelial alkaloids were extracted from washed and homogenized mycelium with a mixture of acetone and 4% tartatric acid (1 : 1).
The collected extract was properly diluted with water and used for alkaloid determination as des-
cribed above. The same procedure as for mycelium was also used for the alkaloid determination i n
a single colony

the whole ordeal is an exercise in patience anyway

Those beautiful vials of alkaloids make it all worthwhile, good work.