May 15, 191S Wilt of Gipsy-Moth Caterpillars 117 



of these boxes can be conveniently placed in an autoclave at one time, 

 and the heat of the steam effectively penetrates through the pasteboard, 

 thus assuring sterihzation. After sterilization, a vacuum is produced 

 and the boxes are taken out in just as dry and solid condition as when 

 first put into the autoclave. Every box was autoclaved before using, 

 for, even if new, they may become infected in some way during transit 

 from the factory to the laboratory. After the boxes were removed from 

 the autoclave, they were placed in rooms which had never contained wilt 

 material, and one caterpillar was introduced into each box. In order 

 to have conditions as uniform as possible, all caterpillars belonging to a 

 single experiment were kept in the same room, being fed with red-oak 

 leaves during the entire period. Red-oak leaves remain fresh much 

 longer after being picked than those of either black oak or white oak and 

 were chosen both for this reason and because red oak is one of the most 

 favored food plants of the gipsy moth. To preclude the possibility of 

 introducing the disease by means of the food, the red-oak leaves were 

 shipped daily from a locality not infested by the gipsy moth. 



SERIES I 



First-stage caterpillars, after having successfully passed their physical 

 examination, were placed in their boxes on June 1 7 and were infected on 

 June 18. Wilted caterpillars that had been collected a few hours before 

 were ground up in a mortar with just enough sterile water added to facili- 

 tate the grinding. The material was then strained through cheesecloth, 

 after which it amounted to 40 c. c, and was divided into two lots of 20 

 c. c. each. One lot was diluted with 25 times its volume, while the other 

 was diluted with 50 times its volume of sterile water, in order to keep 

 the pores of the Berkefeld filters free. A concentrated, unfiltered emul- 

 sion of diseased material is so full of polyhedral bodies, cellular debris, 

 hairs, and pigment granules that it very soon plugs up a filter. Concen- 

 trated material, furthermore, is rather thick and slimy, and a film soon 

 becomes deposited on the outside of the candle, thus withholding the 

 virus. Both lots were then thoroughly shaken and filtered through paper 

 filters by means of suction. This filtrate was passed through Berkefeld 

 filters, grade N, and used for the infection experiments. 



The filtrates were always controlled culturally and in most cases 

 remained sterile, in so far as bacteria were concerned. One must, of 

 course, be careful to autoclave the Berkefeld filters and all receptacles 

 before using them. One or two cases where bacteria were obtained from 

 tubes inoculated with Berkefeld filtrates could be traced to faulty 

 technique. After shaking the filtrate well, 2 or 3 c. c. were always 

 centrifuged and the bottom sediment examined microscopically for poly- 

 hedra; but these were never found, and nothing could be observed, even 

 90271°— 15 2 



