454 CONTROL OF CULTURES 



watch glasses are enclosed in cellophane bags, the ends of which are 

 folded over, and the whole sterilized in the autoclave. After cooling, the 

 bags are carefully opened and 5 ml. of sterile wash fluid is placed in each 

 watch glass by means of sterile serological pipettes. The protozoan to 

 be sterilized is placed in the first bath by means of a micro-pipette 

 inserted through the open end of the bag. There are three obvious ad- 

 vantages in this modification, aside from simplicity of apparatus. The 

 opening of the bag is at the side of the dish and at some distance from 

 it. The top of the dish and therefore the fluid is never exposed to the 

 air from above. The same situation is here repeated as obtains when 

 making tube inoculations in ordinary bacteriological technique, where 

 the tube is always held at a slant. This system is less dangerous than 

 one in which the top of a Petri dish must be removed, and obviates 

 the necessity for a hood or drape. The second advantage is that the ob- 

 server may follow the movements of the protozoan at all times and may 

 then readily draw it up in the transfer pipette. This is usually impossible 

 or difficult when using a Petri dish with the cover in place, as the water 

 of condensation reduces the visibility markedly. Water does not condense 

 on the cellophane, and observations are therefore not hampered. The 

 third advantage is simply one of choice of containers. The Syracuse 

 watch glasses holding 5 ml. of fluid raise enormously the all-important 

 dilution factor. 



When it is possible to obtain large numbers of Protozoa in heavy con- 

 centrations, sterilization may be accomplished by centrifugation. This 

 method, although unsuccessful in the hands of Hargitt and Fray ( 1917) , 

 has been used to advantage recently (Kidder and Stuart, 1939). It is 

 recommended for use with those species of Protozoa which are so small 

 as to make them difficult to follow under the powers of a dissecting 

 microscope. By choosing a washing fluid favorable for the species to be 

 used and carrying the number of washes far enough, it is usually possible 

 to recover large numbers of sterile Protozoa after the final wash. The 

 method which was finally adopted for the sterilization of Colpoda 

 steinii (see Burt, 1940, for species designation) is quoted below: 



After excystment had occurred the ciliates were concentrated by slow 

 centrifugation and the concentrate removed to a single, sterile, cotton-plugged 

 centrifuge tube. This ciliate concentrate was diluted with 10 ml. of sterile 

 distilled water and recentrifuged at a speed which would just throw down 



