PRESENT STATUS OF INVESTIGATION OF BEE DISEASES. 29 
the drop of fluid, while the specimens can be kept permanently and compared 
with one another. 
In order to study the growth of the spores, I used a cultivation on the agar- 
agar cultivating material which had been kept at the temperature of the body 
for fourteen days, and which consisted almost entirely of spores, though a few 
bacilli were present. As the result of several experiments, I have got a series 
of preparations which have been taken at various times (15 minutes, 30 min- 
utes, 40 minutes, 1 hour, 14 hours, 1 hour 50 minutes, 2 hours, 2 hours 20 
minutes, 2 hours 50 minutes, 2 hours 55 minutes, 3 hours 20 minutes, + hours 
20 minutes, 5 hours, 5 hours 35 minutes, 5 hours 40 minutes, and 7 hours 50 
minutes), and the course of events is shown in Plate X.¢ The bacilli 
stain with various anilin dyes—best, I think, with methyl-violet; but the 
spores resemble the spores of other bacteria in not taking on the stain. The 
cover glasses on which the organisms are dried are passed three times through 
the gas flame and floated on the surface of a fairly strong watery solution of 
methyl-violet for one or two hours. They are then washed in water, and 
afterwards laid in weak acetic acid (1 per cent) till no more stain comes out. 
They are again washed in water, allowed to dry at the ordinary temperature, 
and mounted in Canada balsam. A spore-bearing cultivation shows the bacilli 
stained violet and the spores unstained, with the exception of their outline, 
which is of a faint violet color. In most cases no trace of the rod in which 
the spore was formed can be seen. The first change which is observed on 
cultivation is that in many cases the outline of the rod in which the spore was 
formed becomes faintly visible. This can be seen in 15 minutes, and is, I 
think, simply due to swelling by the fluid, as it is also evident to some extent 
in the case of spores soaked in water for the same length of time. In from 
half an hour to an hour it is evident that the bacilli which were present in the 
original material are beginning to multiply, and a considerable number of rods 
are now seen containing spores. It is evident that these spores are newly 
formed, as extremely few bacilli containing spores were seen in the original 
material, whereas in the preparations taken from a half hour to an hour a 
considerable number are present. That some of the rods, instead of growing 
by fission, at once proceed to form spores is probably to be explained in this 
way. When the cultivation was removed from the incubator, some bacilli were 
growing by fission, some were forming spores, and some had passed into a state 
ready to form spores. The first go on growing by fission, the last complete 
their spore-formation, which was arrested by removal from the warm tempera- 
ture. That actively growing rods would not have formed spores so early 
is evidenced by the facts observed in the second series of observations on the 
formation of spores. The next thing that is observed is that several of the 
spores take on the stain, and are as intensely violet as the adult bacilli. The 
number of the spores which take on the stain in this way goes on increasing 
as time passes, till in about four hours almost all the spores stain violet. In 
three hours the first indication of sprouting of these spores becomes evident. 
The stained part of the spore loses its oval shape, becomes elongated, and is 
soon seen to burst through the spore-capsule at one part. It then presents the 
appearance of a short rod, with a pale envelope embracing one end. This rod 
gradually leaves the spore-capsule and then goes on multiplying as a full- 
grown bacillus. In specimens taken from four to five hours all stages of 
growth can be seen, and the remains of the ruptured spore-capsules are evident. 
The bacilli appear to grow mainly by fission, but I have seen appearances 
a@Not reproduced here. 
