250 Development of a Standardised Agar Mediimi 



colony. It seems, therefore, that the retention of the water cannot be 

 due merely to the closely packed bacteria separating it from the under- 

 lying agar. 



Two explanations of these facts suggest themselves. Either sub- 

 stances that hinder the access of the water to the agar may cover the 

 surface in neighbourhood of the bacterial growth, or else the organ- 

 isms may produce a local change in the agar gel such that its capacity 

 to absorb water is reduced. The latter hypothesis can be tested by grow- 

 ing the organism on the lower surface of a film of agar and measuring 

 the rate of absorption of water drops by the agar immediately above 

 the growth and elsewhere. Under these conditions, any substances pro- 

 duced by the organism on the surface of growth will no longer He between 

 the agar and the water drops, whereas a change in the absorption 

 capacity of the agar would reveal itself, and, if found to occur, the depth 

 to which the gel is affected could be observed by varying the thickness of 

 the agar film. This test was applied by the following experiment. A very 

 small droplet of 12 per cent, gelatine was placed in the centre of each of 

 six sterile petri dishes, and each droplet was inoculated with spores of 

 B. dendroides and allowed to set. Nutrient agar medium, melted and 

 cooled to 42° C, was then poured into each dish. The gelatine kept the 

 spores adhering to the glass so that all subsequent growth of the organism 

 took place beneath the agar film. By varying the quantity of agar in 

 the dish, the thickness of the film was varied. Quantities of from 5 to 

 20 c.c. were used, giving films of from about 1 to 3 mm. in thickness. 

 After about 48 hours incubation there was good growth along the bottom 

 of each plating. Drops of distilled water, 0-02 c.c. in volame, were placed 

 on the agar surface in each dish, both above the bacterial growth, and 

 outside the colony area, and their rates of absorption by the agar were 

 measured. On all the platings, the water was absorbed at an equal rate 

 above the bacterial growth and outside this area. It appears, therefore, 

 that there is no alteration in the water absorbing capacity of the agar 

 gel in the neighbourhood of the colony. We must therefore conclude 

 that the retention of moisture on the surface about the bacterial growth 

 is due to some secretion, probably of a mucilaginous nature which hinders 

 access of the water to the underlying agar. 



The rods, that are at first produced by germination of the spores, 

 are non-motile, but soon peritrichous flagella are developed. These have 

 mean length of about lO/x, and are undulating. In the young colony the 

 cells bear 8 to 15 flagella, but this number appears to be reduced to about 

 half in the older growths. 



