xin, D, 5 Haughwout: Protozoa of Manila and Vicinity: I 181 



timothy hay in 100 cubic centimeters of tap water for ten min- 

 utes, allowing it to stand uncovered for from twelve to twenty- 

 four hours to allow for the growth of bacteria. Care must be 

 taken that the water does not contain hypochloride of lime or 

 any of the algicides. Pond or well water may be substituted. 

 Distilled water is poor in oxygen, and in fact that constitutes 

 a strong objection to boiled infusions, which likewise tend to 

 become overgrovrai with bacteria that exert a deleterious effect 

 on the protozoa. An interesting and suggestive paper on this 

 subject has been recently written by La Rue.^ He makes his 

 medium of sterile timothy hay and filtered fap water. The hay 

 is made into small compact bundles, which are tightly wrapped 

 in several layers of cheese cloth. The bundles are sterilized at 

 from 15 to 17 pounds' pressure in the autoclave for fifteen min- 

 utes or more and then dried. This sterilization is intended 

 mainly to kill encysted protozoa. It does not kill all bacteria. 

 The tap water is filtered through filter paper into sterile con- 

 tainers, which should have the eff'ect of removing many of the 

 larger protozoa. It is my experience, however, that most of 

 the smaller forms pass readily through filter paper. La Rue 

 makes up his medium in the proportion of 10 grams of the 

 sterile hay to 2 liters of the filtered tap water. These propor- 

 tions, the author says, may be considerably varied. The original 

 level of the fluid should be marked and maintained by the addi- 

 tion of filtered tap water from time to time. One or 2 grams 

 of sterile hay may be added each week. 



A refinement in the methods of cultivating free-living pro- 

 tozoa has been introduced by Hargitt and Fray ^ in their method 

 for sterilizing Paramecium and cultivating it with known strains 

 of bacteria. Their paper is of great interest and significance 

 in connection with experimental work, but the method is too 

 difficult of application and, indeed, is not intended by the authors 

 to apply to the maintenance of ordinary laboratory cultures. 

 The principles brought out, however, are of interest to all work- 

 ers who are dealing with the problems of the cultivation of 

 protozoa. 



Cultures of protozoa have been classified by Williams as being 

 of three types : 



Mixed cultures. — Those that contain the "omnium gatherum" 



' La Rue, G. B., Notes on the culturing of microscopic organisms for 

 the zoological laboratory, Trans. Am. Micros. Soc. (1917), 36, 163. 



° Hargitt, G. T., and Fray, W. W., The growth of Paramecium in pure 

 cultures of bacteria, Journ. Exp. Zool. (1917), 22, 421. 



