37§ Phylum Arthropoda 



the larvae in a constant temperature water bath which has capacity for 

 6 pans. In the winter months this is heated to 70 F. by contained elec- 

 trical heating units. In the summer it is cooled to 70 F. by the coils of 

 a Frigidaire refrigerating plant. 



The infusions are made, and the larvae subsequently kept when the in- 

 fusion is ripe, in white enameled cream pans, about 1 2 inches in diameter 

 and 2% inches deep. 



Early successful culture of the larvae on a large scale is attributable to 

 the employment of pans of hay infusion for their rearing, the rich auto- 

 genous plankton of which, especially when supplemented by a supply of 

 yeast, affords an abundance of food and results in the production of large 

 vigorous pupae and imagines. While successful, hay infusions have not 

 been satisfactory; their qualities vary widely depending on the hay em- 

 ployed; they are from a biological standpoint very complex; and they 

 may only be employed for larval nutrition after a lengthy process of 

 fermentation. It was observed that pans were satisfactory for larval 

 nutrition after the reaction had become alkaline and if a dense growth of 

 Paramecium and flagellates occurred. Considerable experimentation 

 was carried on in an effort to reproduce these conditions in a simpler man- 

 ner. After trying extracts from various vegetables and seeds, as well as 

 synthetic media, wheat infusions were found to serve as a satisfactory 

 base for cultures and now hay infusions are abandoned. 



Wheat infusions are prepared as follows: One or two ounces of sound 

 wheat grains are placed in a beaker which is partly filled with tap water. 

 The beaker is then placed over a flame and the water boiled for a few 

 minutes. When boiled, about 250 grains are placed in an enamel pan 

 with 2 liters of tap water. The pan is placed where it will receive very 

 diffused light in the laboratory. After 2 or 3 days when a visible bacterial 

 growth is present it is heavily inoculated from a previously prepared 

 plankton culture in wheat infusion. After 4 or 5 days' further incubation 

 at room temperature nebulous masses of flagellates in descending con- 

 vection currents are clearly visible and the pan is ready for the introduc- 

 tion of larvae. At the time when a series of pans is prepared a separate 

 culture is made to serve for the inoculation of the next series. The 

 original mixed culture of Paramecium and flagellates was secured from 

 a satisfactory ripened pan of hay infusion. Alkalinity is maintained by 

 adding 1 gram of powdered calcium carbonate to each pan and neutrali- 

 zation of acid is probably facilitated by the gentle stirring of the settled 

 CaC0 3 from the bottom once a day. After being placed in service a 

 culture will give from 2 to 3 weeks' service before being discarded. 

 Water loss by evaporation should be replaced. If a slimy envelope de- 

 velopes about 4th stage larvae, the affected larvae may be placed in a 

 1% solution of NaCl for 30-45 minutes and then returned to their pan. 



