186 The Philippine Journal of Science i9i8 



I make use of what is called the round-bottom-vial method. Vials 

 of from 5 to 8 cubic centimeters' capacity may be used. They 

 should be filled to about two-thirds of their capacity with the fix- 

 ing fluid, and material from a rich part of the culture should be 

 transferred to them with a pipette. The organisms are instantly 

 killed and fixed. When they have settled to the bottom of the 

 vial, the supernatant fluid should be decanted or cautiously drawn 

 off with a pipette, leaving the fixed organisms in a mass in the 

 bottom of the vial. Alcohol of 70 per cent strength should be 

 added, and when the organisms have again settled, this should be 

 withdrawn and 95 per cent alcohol substituted. The organisms 

 are allowed to settle once more. Keep the vial corked, especially 

 in wet weather, to avoid absorption of water by the alcohol. 



Slides are prepared by lightly smearing a little Mayer's albu- 

 men fixative over an area at the center of the slide about the size 

 of a 5-centavo piece. This is made up by adding an equal quan- 

 tity of glycerin to the white of egg, which has been previously 

 well beaten. Add 1 gram of sodium salicylate to each 50 cubic 

 centimeters of the mixture as a preservative. Mix and filter 

 carefully before using. 



A small quantity of the sediment from the bottom of the vial, 

 together with a minimum amount of the alcohol, is then drawn 

 up into a capillarj^ pipette and forcibly spurted on the surface 

 of the albumen film. The slide is then immersed in 95 per cent 

 alcohol contained in a Coplin staining jar. From the time the 

 protozoa are transferred to the slide until the preparation is 

 finally sealed under the cover glass, the slide should never be 

 allowed to dry. If it dries, the preparation is ruined. From the 

 95 per cent alcohol the slide is transferred to 70 per cent alcohol 

 containing sufficient iodine to color it a port wine shade. It 

 should remain there for ten minutes or possibly longer in order 

 to remove the excess of mercury from the organisms. The 

 iodine should be then washed out in 70 per cent clear alcohol and 

 the slide passed down through 50 per cent alcohol to water by 

 three- to five-minute stages. The preparation is then stained. 



After staining, the slide is washed in tap water, is carried 

 into 50 per cent alcohol where it should remain for three to five 

 minutes, and finally is put into 70 per cent alcohol. Differentia- 

 tion, when required, is carried out at this stage in the acidified 

 70 per cent alcohol before mentioned, the excess of acid after- 

 ward being thoroughly washed out in clear 70 per cent alcohol 

 for at least fifteen minutes. After this the slide is passed 

 through 95 per cent alcohol, 100 per cent alcohol, and xylol, five 

 minutes . in each change, and finally mounted in xylol Canada 



