COLLECTION, CULTIVATION, OBSERVATION 719 



tion, which mixture is passed through a sieve and then filtered 

 through a thick absorbent cotton. Balantidium which shows posi- 

 tive geotropism, is freed of faecal debris by passage downward 

 through cotton in V-tube. The ciliates are introduced into the cul- 

 ture tubes. Incubation at 37°C. Subcultures are made every 7-22 

 days. Nelson found that autoclaved medium is unsuitable until a 

 living bacterial population has been established. Balantidium can 

 also be cultivated in the media given for the intestinal amoebae. 



Microscopical examination 



Protozoa should be studied as far as possible in life. Permanent 

 preparations while indispensable in revealing many intracellular 

 structures, cannot replace fresh preparations. The microscopic slides 

 of standard size, 3" by 1", should be of white glass and preferably 

 thin. The so-called No. 1 slides measure about 0.75 mm. in thickness. 

 For darkfield illumination thin slides are essential. No. 1 coverglasses 

 should be used for both fresh and permanent preparations. They are 

 about 130-1 70m thick. The most convenient size of the coverglass is 

 about 7/8 square inch which many prefer to circular ones. 



The slides and coverglasses must be thoroughly cleaned before 

 being used. Immerse them in concentrated mineral acids (nitric acid 

 is best fitted) for 10 minutes. Pour off the acid, wash the slides and 

 coverglasses for about 10 minutes in running water, rinse in distilled 

 water, and keep them in 95% alcohol. When needed they are dried 

 one by one with clean cheese cloth. Handle slides and covers with a 

 pair of forceps. If thumb and fingers are used, hold them edgewise. 



A. Fresh preparations 



In making fresh preparations with large Protozoa care must be ex- 

 ercised to avoid pressure of the coverglass on the organisms as this 

 will cause deformities. If small bits of detritus or debris are included 

 in the preparation, the coverglass will be supported by them and the 

 organisms will not be subjected to any pressure. Although ordinary 

 slides are used most frequently, it is sometimes advisable to use a 

 depression slide especially for prolonged observation. To make a 

 preparation with this slide, a small drop of water containing speci- 

 mens is placed in the center of a coverglass, and is covered by a small 

 circular coverglass (about 1 cm. in diameter), which in turn is cov- 

 ered by a depression slide with a thin coat of vaseline along the edge 

 of the depression, so as to make an air-tight compartment. In turning 

 over the whole, care must be taken to prevent the smaller circular 



