136 Phylum Protozoa 



cool to room temperature, distribute it in dishes* holding 200 cc. each, 

 and place 3 or 4 grains of the prepared wheat in each dish of water. 

 The dishes are now stacked and allowed to stand 2 or 3 days, during 

 which time enough air is dissolved to make the medium ready for in- 

 oculation. If it is desired to immediately inoculate the dishes the water 

 may be artificially aerated. 



In general this first inoculation is made with material (bacterial glea, 

 etc.) from successful cultures and after considerable growth has set up 

 around the wheat grains (in 3 or 4 days) we subculture into the dish 

 the particular organism desired from the best of the previous cultures. 



Initial cultures of an organism are made in the same way except that 

 inoculations are made from the medium in which the organism was 

 collected. 



We have made no attempt to control the pH of our cultures within 

 any narrow limits but the range has been between 7.05 and 7.40. Our 

 spring water generally has a pH value of about 7.05, a two-weeks cul- 

 ture about 7.40 and a two-months culture about 7.12. There is some 

 slight seasonal variation in the water which we use. 



We have cultured very successfully in this medium the following groups 

 of fresh-water inverteb r ates: 



Protozoa — Several species of Amoeba, Actinophrys, various Difflugia, 

 Chilomonas, Peranema and many other small flagellates, Paramecium, 

 Vorticella, several hypotrichs, and many other Infusoria. 



Plathelminthes — Catenula, some 15 species of Stenostomum, Micros- 

 tomum, and small triclads. 



Nemathelminthes — Several species of freshwater nematodes. 



Rotifera — Some 20 species. 



Annelida — Several species of oligochaetes. 



Bryozoa — Plumatella. 



Arthropoda — Copepoda, Cladocera, Ostracoda, Hydracarina, mos- 

 quito and midge larvae. 



We especially recommend this method as a means of maintaining 

 constant supplies of Amoeba for class use. We have for 5 years main- 

 tained cultures in the original dishes by simply pouring out the water 

 from each dish every two months and adding fresh water (boiled, cooled, 

 aerated) and 3 or 4 grains of boiled wheat. A sufficient number of 

 Amoebae stick to the bottom of the dish when the water is poured off 

 to seed the culture. 



* The ice-box dishes (Hazel Atlas Glass Company) which we use for most of our 

 general culture work may be purchased for 10 cents each at any io-cent store. They have 

 a capacity of 400 cc; they may be stacked; and they may be obtained in either clear or 

 green glass. These dishes are, for general purposes, quite as satisfactory as the much more 

 expensive pyrex dishes sold for such purposes. 



