PROTOZOA 569 



place another drop on a fresh fdm and repeat till a single cyst is 



obtained on the film. The method is simple, and with practice 



one can make half a dozen such cultures in an hour. Place the 



slide film surface downwards above water in a Petri dish (this is 



conveniently done by resting it on two corks), and cover the dish. 



Examine day by day, till numerous amoebic are found, and then 



allow them to encyst. From this culture cysts may be inoculated 



into an ordinary test-tube slope, and so cultures obtained of the 



one species of amoeba feeding on any bacteria that happen to be 



carried over in the inoculum with it — -sub-cultures being made as 



often as desired. Most soil amccbae appear to feed upon almost 



any of the common bacteria, but there is a considerable element 



of luck as to whether its favourite food will be carried over at every 



sub-culture. It is better, therefore, to determine for which of the 



many organisms present the amoeba, being cultivated, shows 



preference and then to try to obtain it in pure culture. 



1115. To obtain a Culture of One Species only of AmcEba feeding 



upon Bacteria of only a Single Species — a ' ' so-called ' ' Pure 



Mixed Culture. To a tube containing a culture in which the 



amoebae have mostly encysted, add hydrochloric acid solution 



strong enough to kill all organisms except the encysted amceba?. 



(Some soil cysts are said to tolerate 2 per cent, hydrochloric acid 



for twenty-four hours, but others may be killed in much less 



time by acid only one-tenth that strength, and preliminary 



experiments will be necessary. Dobell Parasitology, xix, 1927, 



N 

 p. 288) finds that E. histolytica cysts can survive in — HCl {i.e., 



0-36 per cent.) for as long as three hours at ordinary temperature, 



N . 



whereas — kills all active amoebae in ten to thirty minutes.) Then 

 20 



pour off the acid, neutralise with sodium bicarbonate, and 

 thoroughly wash the cysts on the slope with sterile water. With 

 a platinum loop scrape off the cysts and inoculate into a fresh 

 tube of medium, adding a loopful of a dilute emulsion of the 

 bacterium with which it is desired to cultivate the amoebae. 



Of course, precautions against infection must be observed 

 throughout and cultures are generally kept in tubes plugged with 

 cotton-wool. It is, however, possible to lessen the risk of con- 

 tamination of plate cultures by inverting them into a little mercuric 

 chloride solution in the lids. 



II. CULTIVATION OF BLOOD PROTOZOA 



1116. In all tlicsc processes it is most essential to avoid all 

 bacterial contamination, consequently aseptic conditions nmst 

 prevail throughout. For further information consult text-books 

 on Bacteriology or Wenyon's Protozoology. 



