24 BULLETIN 828, U. S. DEPARTMENT OF AGRICULTURE. 



tate reaching all parts of the insect body, and the solution was also constantly shaken 

 during the exposure. 



Approximately 35 striped beetles were dissected (June 29) after a 7-day feeding on 

 wilted cucumber plants in pots, and inoculations were made to the leaves of as many 

 cucumber seedlings by mixing the intestinal contents of each beetle in a drop of tap 

 water and pricking the mixture into the leaves. These plants were kept in beetle- 

 free cage with 20 or more uninoculated control plants. Two of the inoculated plants 

 developed typical cases of wilt starting from the points of inoculation with intestinal 

 contents. 



Finally, on July 5, 1918, the last experiment was repeated, using sixty striped and 

 thirty 12-spotted cucumber beetles and fifteen squash lady birds. All had been 

 previously fed for 10 days on wilted cucumber plants in pots and had been treated for 

 five minutes before dissection with mercuric chlorid and alcohol, as in the preceding 

 test. Five plants out of the 60 inoculated with intestines of striped beetles promptly 

 developed wilt. All these cases showed the typical stringy slime from the cut ends 

 of the vascular bundles, and cultures made from one of these plants gave Bacillus 

 tracheiphilus. Inoculations in this experiment with intestines of the other two insect 

 species gave negative results. 



These dissection experiments prove definitely that both striped 

 and 12-spotted cucumber beetles may harbor the wilt organism in 

 their intestinal tracts. In fact, a much larger percentage of infec- 

 tions took place from intestinal contents than from mouth parts, 

 although this may have been due to the fact that the mouth parts 

 were harder to break up and mix thoroughly with the water on the 

 leaves. They also show that only a small proportion of the beetles 

 gave infection from the intestines even after feeding seven days or 

 more on wilted plants. Thus, in the two tests with 12-spotted 

 beetles, the intestines gave infection in about 50 and 8 per cent of the 

 individuals, respectively. In the four tests with striped beetles the 

 proportion varied in round numbers between 6 and 12 per cent. 

 There was very little difference in the proportion of wilt carriers 

 among individuals taken while feeding directly on wilted plants and 

 those not feeding at all when taken for the test. 



BACTERIAL ISOLATIONS FROM BEETLES. 



One attempt was made (Dec. 10, 1917) at direct isolation of Bacillus 

 tracJieiphilus from the intestinal tracts of wilt-fed striped cucumber 

 beetles. 



Ten individuals were fed for two weeks upon cucumber plants wilted from pure- 

 culture inoculation. They were then dipped for several seconds into 95 per cent 

 alcohol, for 60 seconds in 1 to 1,000 mercuric chlorid, quickly washed in sterile water, 

 and then given five more washings of several minutes each in sterile water. The in- 

 testinal tracts were then carefully dissected out as described under ' ' Dissection experi- 

 ments," without allowing the body contents to come in contact with the surface of 

 the beetle. The intestines in each case were crushed in sterile distilled water with a 

 sterile glass rod and allowed to stand for 20 minutes, when four series of dilutions in beef 

 bouillon were made. From these tubes one series of poured plates was made at once 

 and another the next morning after the bouillon had become slightly cloudy. From 

 most of the plates developing colonies, pure cultures of one type of bacteria (A) 



