244 
Cultivation of Piroplasma 
10 c.c of defibrinated piroplasma blood. When the parasites became 
numerous in the blood of the infected dogs (they usually died on the 
third or fourth day) he bled the animals to death under chloroform, 
defibrinated their blood and added 0’5 c.c. of blood to 0’5 c.c. of salt 
solution. A series of 20 such cultures in test tubes were made at one 
time and kept at 27° C. 
After the lapse of 18 hours Kleine decanted the clear supernatant 
fluid from the tubes and upon examining the deposit found that it con¬ 
tained bodies corresponding in form to those described by Koch (1905, 
1906) in piroplasma infected ticks. Many of the parasites occurred in 
the form of clubs with about six rays protruding from the broader 
portion, whilst the tapering extremity also bore two or more radial 
processes. The parasites appeared peculiarly rigid, but, on closer 
examination in drop culture, amoeboid movements could be detected, 
the parasites becoming slowly rounded or elongated, whilst the length 
and number of the radial processes changed. These changes were 
already observable after eight hours, but were most marked after 18 
hours. 
After 48 hours some parasites had attained a length of 14 p, and 
a width of 4 p, the rays at the broad end measuring 9 fM, those at the 
opposite end measuring up to 19 /x in length. These large parasites 
occurred together with parasites of normal size. When stained by 
Giemsa’s method the large forms with radii showed a large chromatin 
mass at the end of the club, and usually a secondary chromatin mass at 
the tapering extremity. Kleine states that the rays appear blue, unless 
intensely stained when they take on a red colour. When the parasites 
were numerous in the blood, masses of radiate bodies occurred, the 
tapering extremities of the parasites converging to a common centre. 
When the parasites were very numerous the development of these large 
forms appeared to be inhibited. At times Kleine observed “ fused 
forms ” such as Koch has described in ticks, sausage shaped bodies 
with radii protruding from the extremities and containing large masses 
of chromatin at the ends of the sausage and secondary masses of 
chromatin situated about midway along their length. 
After two days at 27° C. the parasites appeared on the whole 
larger than normal. In forms from early cultures both chromatin 
masses stain in the same way, but in those derived from cultures two 
days old the chromatin mass within the clubs retains the peculiar black- 
red appearance, whilst the larger chromatin mass surrounded by radii 
appeal’s more lightly stained. After 2—3 days the parasites cease to 
