i6o RESEARCH IN PROTOZOOLOGY 



The protozoa were grown in large numbers together with many 

 bacteria. The cultures were then diluted with sterile medium and 

 the individual trichomonads were picked up by means of a Barber 

 pipette, taking as few bacteria as possible. These were then intro- 

 duced into a culture of fibroblasts, epithelium, mesothelium and 

 sympathetic nerve fibers, grown from the stomach or intestine of 

 a chick embryo seven or eight days old. 



Since the above experiments were performed Cleveland has re- 

 ported the obtaining of cultures of TricJiomonas free from bac- 

 teria. These would be admirable for introducing into the tissue 

 cultures. By using these the effect of the protozoa alone could be 

 observed. In the writer's work so far reported only the mechanical 

 effect of the protozoa could be studied, as a few bacteria were 

 always present and the effect of their products on the tissue cells 

 could not be distinguished from the effect of the protozoan 

 products. 



There are two general methods of growing tissues outside of 

 the body ; one the Harrison-Carrel method in which plasma and 

 embryonic juices are used as a nutrient medium ; the other the 

 Lewis method where Locke-Lewis is used. The Lewis method 

 is better fitted for these problems as it offers a liquid medium for 

 the protozoa, whereas the plasma soon coagulates and becomes too 

 dense for the protozoa to move about in. 



For the Locke-Lewis (Lewis, 1924) chicken bouillon is very 

 carefully made up and adjusted to pH 6.4-6.6. The Locke solution 

 has the following formula: NaCl 0.9%, CaCL 0.025%, KCl 

 0.042%, NaHCOs 0.02%. In making the Locke-Lewis one uses 

 eighty-five per cent of Locke solution, fifteen per cent of chicken 

 bouillon and 0.25 to one per cent of dextrose. This is sterilized in 

 the autoclave for fifteen minutes at fifteen pounds pressure. 



Chick embryos from six to nine days old are used for the ex- 

 periments. All the instruments, Petri dishes and coverslips are 

 sterile. The eggs are opened at the broad end and the embryo re- 

 moved with forceps or a spatula and placed in a small Petri dish 

 containing Locke-Lewis which has been slightly warmed. Here the 

 embryonic membranes are removed, the chick opened and the 

 organs to be used in the experiments are taken out and placed 

 in other small Petri dishes containing warmed Locke-Lewis in 

 which they are cut into small pieces about 0.5 mm. long. With 

 a sterile pipette two or three of these pieces with a small drop 



