Sydney G. Paine and Emily M. Berridge 21 



washed rapidly with 65 per cent, alcohol, and a portion containing one 

 of the diseased areas cut from the leaf and ground under aseptic con- 

 ditions with sterile sand and 5 c.c. of sterile water. After a lapse of a 

 minute or so for the settling of the sand, 1 c.c. of the liquid was further 

 diluted with 4 c.c. of water, and 1 c.c. of this dilution was plated in 

 bouillon agar. In the successful experiments the number of colonies of 

 P. Proteamaculans was never great, and since these represented roughly 

 one twenty-fifth of the total number in the leaf tissue taken, it must be 

 concluded that the number of viable organisms present was much less 

 than might have been expected considering the extent of the disorgani- 

 sation of the tissue. The method may perhaps appear rather drastic and 

 liable to loss of bacteria by adsorption on the sand and detritus and 

 by their actual destruction in the grinding process. A certain amount 

 of loss does occur, but as shown later (footnote, p. 23), it is not more 

 than about 20 per cent, of the organisms present. It seems then 

 that some factor militating against the bacteria comes into action after 

 a lesion of a certain size has been produced. 



In this case a large percentage of the spots might be expected to con- 

 tain no viable organisms. To determine this percentage 100 "spots" 

 were cut aseptically from leaves and dropped each into a separate tube 

 containing 5 c.c. of bouillon; in order to aid the escape of bacteria each 

 area of the diseased tissue was usually cut through the middle. After 

 incubation for three days the broth in all cases remained perfectly clear 

 and after streaking the broth from 75 tubes across the surface of nutrient 

 agar plates, no growth of bacteria was obtained. The experiment was 

 repeated with 64 diseased spots and again after three days none showed 

 the presence of P. Proteamaculans, although 17 tubes showed turbidity 

 by other organisms. Five of these examined after seven days' incubation 

 still showed no development of P. Proteamaculans; two tubes, however, 

 which had been put aside were found to contain this organism when 

 examined ten weeks later. It seemed possible that the reddish-brown 

 pigment which had diffused from the tissue and coloured the broth 

 quite strongly in certain cases, or perhaps some other diffusible toxin 

 from the leaf, had a retarding influence upon the rate of development 

 of the organism. Six of the most strongly coloured tubes were therefore 

 inoculated from a pure culture of P. Proteamaculans, and although 

 growth was not very vigorous at first, all six developed the organism and 

 were strongly turbid after a week or so. Marked retardation of growth 

 was therefore not due to this cause. A third set of 65 tubes was pre- 

 pared as before. At the end of nine days the broth in all cases was clear 



