243 
THE DEVELOPMENT OF PIROPLASMA CANIS 
IN CULTURE. 
By GEORGE H. F. NUTTALL and G. S. GRAHAM-SMITH. 
Plate XIX and 1 Text Figure. 
In the course of their investigations upon different species of Piro- 
plasma numerous observers have kept infected blood in the ice-chest, or 
at room temperature, for various periods of time prior to the inoculation 
of animals. On the evidence afforded by microscopic examination alone 
some observers have concluded that the parasites are capable of con¬ 
siderable multiplication in defibrinated blood kept in vitro. Under 
these conditions many corpuscles undergo haemolysis and in consequence 
the parasites appear to be more numerous, We believe that this source 
of error explains the conclusions arrived at by Lignieres and others with 
regard to the multiplication of P. bovis in extravascular blood (see p. 253). 
It is only within the last few years that systematic attempts have 
been made to cultivate the different species of Piroplasma. We ha,ve 
previously referred (1905, Journ. of Hygiene, v. 245) to the negative 
results which followed the attempts of Nocard and Motas (1902, 
pp. 274—275) to cultivate P. canis, although they established the 
fact that the parasites remained alive and virulent in blood which 
had been preserved in the dark and cold for 25 days. Kinoshita (1907, 
p. Ill) has kept the parasite alive for 31 days on ice. In an earlier 
paper we have described the forms of parasites observed in defibrinated 
dog’s blood up to 48 hours after its removal from the animal. 
Kleine’s observations. 
Kleine (1906, pp. 10—15) states that most of his attempts to culti¬ 
vate P. canis in vitro proved negative until he adopted Robert Koch’s 
suggestion and studied the early changes which take place in cultures 
made by diluting defibrinated piroplasma blood with salt solution. 
Kleine infected young dogs by the intraperitoneal injection of about 
Parasitology i 16 
