208 
Amoebae 
On all of these plates colonies of moulds and bacteria from the air 
made their appearance after 1-6 days. The colonies were examined 
under the low pow'er of the microscope in the inverted plates and, in 
14 out of 86 plates, amoebae were found present, in association with one 
or more of the bacterial colonies. The details of the experiments are 
tabulated on pp. 210, 211. 
Subcultures of these amoebae were readily obtained by taking a 
loopful of the mixed growth of amoebae, either vegetative forms or 
cysts, and bacteria, and stroking it on the surface of a fresh plate of 
Musgrave’s medium. The subcultures were generally kept in a moist 
atmosphere at a temperature of 22°-28° C. for the first day or two: 
the optimum temperature for their multiplication probably lies within 
these limits. Generally, after from 12 hours to two days, there is an 
abundant crop of motile amoebae along the needle-stroke of inoculation; 
as a rule, after 5-6 days many of the amoebae are found to have become 
encysted. 
In order to obtain a culture which would certainly represent only 
one species of amoeba, the following method was adopted ; A loopful 
of growth is inoculated at one point of the plate; thence a radiating 
stroke is produced on the surface of the medium with a fine platinum 
needle. On examining this stroke under the low power several amoebae 
are generally found at the end of it. From this point a second stroke 
is made, and so on, until finally one is selected which contains only 
a single amoeba towards its extremity: this single amoeba is then cut 
off from the rest of the growth by a stroke of carbolised vaseline painted 
on the surface of the medium after the method of Walker (1908). Pure 
cultures thus obtained were most easily pr’eserved from contamination 
by subculturing on test-tube “ slopes ” of Musgrave’s agar. 
With a Zeiss A A lens and a No. 12 eyepiece the main processes of 
the life-cycle may be roughly followed on the inverted Petri plates, but, 
for examination under the higher powers of the microscope, the following 
method of subculture was employed : a slab of Musgrave’s agar, half 
an inch square, is cut out of a fresh plate of the medium with a sterile 
knife and placed on a sterile slide. A loopful is then taken from an 
old culture containing cysts and stroked on the surface of the agar 
square; a sterile cover glass is placed on top and its edges are sealed 
with melted paraffin wax. An air space should be left between the 
edges of the agar slab and the paraffin wall. Thus a microscopic moist 
chamber is provided, sealed against external contamination. Such a 
preparation may be kept in a warm microscope chamber at a tempera- 
