LABORATORY AND GREENHOUSE STUDIES. 133 



Plate la, numerous pink colonies. 

 Plate 1, numerous pink colonies. 

 Plate 1'-, Four tiny pink colonies. 

 Plate la'-, Four tiny pink colonies. 



Thus, some of these plates suggest the presence of Bacillus coli. 

 Four tubes were made from the diseased material, marked thus: No. 1, 

 No. 1 a, No. 1 b, and No. 1 c. From these dilutions were made, 

 marked the same, and from them second dilutions were made, 

 marked No. P, No. la-, No. lb-, and No. Ic-. Plates from these 

 tubes have already been described for one day's growth. In two 

 days they appeared as follows, in the order of their dilution: 



No. 1: Well sown with round pink colonies, mostly typical of Bacillus coli. 



No. 1^: Five round pink colonies, unlike Bacillus coli. 



No. la: Many luxuriant pink, irregular, smooth colonies, unlike Bacillus coli. 



No. la^: Six colonies, unlike Bacillus coli. 



No. lb: Well sown with round, pink colonies, typical of Bacillus coli. 



No. lb-: Several colonies; none like Bacillus coli. 



No. Ic: Well sown with small, round pink Bacillus coli colonies. 



No. 1 c^: Four colonies, unlike Bacillus coli. 



These notes were made on the plates after transfers, so that some 

 colonies which might have been Bacillus coli were destroyed by the 

 needle. Transfers from these plates to litmus milk were incubated 

 at 37° C. All the tubes became reddened and coagulated in 48 hours. 

 Transfers were made from the litmus-milk cultures into nitrate 

 bouillon and incubated at 37° C. for 48 hours. At the end of that 

 time they were tested and all showed reduction of nitrates to nitrites. 

 Transfers were then made from the litmus milk to fermentation tubes 

 containing neutral red and dextrose, and these were incubated at 

 37° C. After 48 hours every one of the 10 tubes showed the tj^^ical 

 greenish-yellow color reaction in the closed arm of the fermentation 

 tube characteristic of Bacillus coli. Transfers were made to gelatin 

 and incubated at 37° C. for 48 hours. They were then placed in an 

 ice box to permit hardening. After 10 hours all of these tubes were 

 found to be perfectly firm, thus showing that no liquefaction of the 

 gelatin had taken place. No further tests were made, as it was 

 believed that sufficient had been shown to indicate that Bacillus 

 coli was in the tissues into which it had been injected in inoculation 

 No. 1. 



Inoculation No. 2, of May 7: The outer sheath was very slightly 

 water soaked about the inoculation hole. The under leaves were 

 rotted only a slight distance; more than in the checks, but scarcely 

 enough to plate out. 



Inoculation No. 3, of May 7: The outer sheath was bro\Mi rotted 

 2 millimeters about the inoculation hole on the inner side. Above the 

 hole were numerous brown spots, apparently stomatal infections 



228 



