132 BETTS ! THE FUNGI OF THE BEF-HIVE. 
Petri dishes or flasks) containing various nutritive media. In some 
cases, pure cultures were at once obtained; more usually, several 
sub-cultures had to be made before the species were separated. Drop 
cultures were made use of occasionally, chiefly in order to watch the 
germination of spores. Since December, 1911, the poured plate 
method has been almost exclusively employed in making cultures 
direct from the combs. 
All the ryit test-tube cultures were reliable, the tubes being 
sterilized as described below. The Petri dishes and flasks were un- 
fortunately not sterilized with adequate thoroughness.’ In the few 
cases, however, where my cultures of a species were originally derived 
from a culture made in a flask, Petri dish, or drop-culture cell (and 
consequently not absolutely reliable), subsequent work with poured 
plates (as well as an examination of combs) has verified the presence 
of the fungus on the combs, thus confirming the 1911 results. 
Throughout the course of the work on which this paper is 
based, the methods of sterilization were as follows. All glass-ware 
(except drop-culture cells) was sterilized in an oven, test-tubes and 
flasks being previously plugged with cotton wool. After being filled 
with a suitable quantity of the nutritive medium, all tubes, whether 
intended for sloped tubes or for poured plate work, were -sterilized 
by boiling in water for at least twenty minutes on three successive 
days (time being reckoned from beginning of ebullition of the 
water). Forceps, wires, etc., were sterilized in a spirit-lamp flame. 
The usual precautions were observed when removing and 
replacing the cotton wool plugs of culture-vessels. 
The following media were used :— 
Pollen-decoction. This was made by boiling in water pieces of 
comb in which the bees had stored pollen, and straining repeatedly 
till the wax, cocoons, and bulk of the pollen-grains were removed. 
It was made up with 1 to 2 grammes of bar agar-agar per 1co ccm. 
of decoction, or with gelatine,” and was usually left acid. This 
medium was used a good deal; but has latterly been for the most 
part abandoned in favour of the honey media. 
Honey, diluted with 3 or 4 times its volume of water, was found 
to be an excellent medium for most of the fungi studied. It was 
generally made up with gelatine, but agar was also used. An 
1The medium was boiled, and poured into the dishes and flasks, which were then 
used for cultures without further sterilization. They were nearly always kept for some 
days before use. : 
2The flasks used during 1912 were also treated in this manner. 
8In all cases where gelatine was used, the proportions were: 10 grammes gelatine 
(Gold Label) to roo ccm. of liquid. 
