22 Studies in Bacteriosis 



and bright, but in 44 (and seven days later in 48) cases a colourless 

 gelatinous substance was observed around the piece of tissue lying at 

 the bottom of the tube. On staining, this appeared as a zoogloea of 

 bacteria of similar shape to P. Proteamaculans, but smaller in size. This 

 did not develop further and was interpreted as a swollen mass of the 

 gum-like substance containing dead bacteria. This phenomenon of the 

 swelling of the gum has been observed in the case of dead or dying 

 leaves kept under moist conditions; small beads of the yellow gum were 

 seen on the surface of the diseased spots, and these could be removed, 

 spread on a slide and stained. They showed the same appearance of small 

 embedded bacteria as described above. In order to test the viability of 

 these organisms small masses of the gum of the size of a pin's head and 

 containing some millions of the bacteria were ground with sterile sand 

 and water, and the water then plated on agar and incubated. Four 

 experiments of this kind were made; in three cases no growth was ob- 

 tained, but in the fourth numerous colonies developed. The experiments 

 differed in that the gum used in the three with negative result was so 

 dry as to be quite brittle, w^hile in the fourth it was noticeably much 

 softer in consistency. The presence of living organisms in the gum exuded 

 from a dead leaf would seem to discount the hypothesis of active bac- 

 teriolysis taking place within this medium. 



The foregoing experiments emphasised the difficulty of isolating the 

 parasite, but failed to determine the percentage of spots containing 

 viable bacteria. The number of cases where P. Proteamaculans appeared 

 was very few, but it was impossible to say how far the sterilisation 

 of the leaf surface had contributed to this result. Experiments with 

 unsterihsed leaves were equally unsuccessful through the difficulty in 

 recognising the parasite in the presence of the numerous other organisms 

 which developed. One of these, a small Sarcina, very closely resembled 

 diplo-forms of the Pseudomonas. 



Experiments were next made to determine the fate of bacteria in 

 spots produced by artificial infection. The surface of a fresh leaf of a 

 young seedling of Protea cynaroides was first washed with 60 per cent, 

 alcohol, scratched in several places with a sterile needle and the scratches 

 covered with uniform drops of a bouillon culture of P. Proteamaculans. 

 On the sixth day the leaf was washed with sterile water to remove 

 organisms from the surface. The first spot was cut out on this day and 

 the others at intervals of every second or third day, and the number of 

 bacteria counted. The spots were cut out with a sterile cork-borer, ground 

 in 5 c.c. of sterile water with 1 gm. of sterile sand, suitable dilution plates 



