F. T. Brooks and A. Sharples 73 



the cotton wool into the reservoir of water where it becomes 

 aggregated. 



The cultures started originally from the pink incrustation were now 

 used to inoculate sterilised blocks of Hevea wood. The cultures so 

 obtained were identical with those derived from the Necator spores so 

 that, apart from other evidence, this alone indicated the connection 

 between the two forms. 



The cultures on wood blocks were placed under different conditions 

 to ascertain whether these influenced the development of the coloura- 

 tion. Some were placed in the laboratory in diffuse light, others in 

 direct sunlight, and others in darkness. Kant (10) states that his agar 

 cultures placed in darkness remained white though after illumination 

 for a short time a pink colouration appeared. In our experiments the 

 cultures grown in darkness behaved like those in the light except that 

 the pink colour did not develop so rapidly. 



In older cultures the mycelium often turns a dirty brown colour as 

 though some impurity had entered. However, small portions of this 

 mycelium seen by transmitted light show the characteristic colouration. 

 The hyphae in the old brown cultures are closely aggregated, corrugated, 

 septate, much vacuolate, with numerous clamp connections. The 

 hyphae in young cultures are septate, vacuolate, but not corrugated, 

 whilst clamp connections are less numerous. 



In one culture placed in bright light and in another kept in diffuse 

 light an aggregation of hyphae took place along the upper edge of the 

 blocks to form a bright pink mass. The aggregation continued until 

 a solid mass half an inch high and half an inch in diameter was formed, 

 attached to the block by a thinner base. It resembled a number of 

 closely attached Necator pustules (Fig. 11). Examined microscopically 

 the mass was found to consist of short cells somewhat irregular in size 

 forming a kind of pseudo-stroma. 



These Necator-\ike masses are often formed in the cotton wool at 

 the base of the culture tubes and sometimes upon the surface film of 

 mycelium in the reservoir. Their spore-like nature is indicated when 

 small pieces are used to start new cultures upon wood blocks. In every 

 case the mycelium develops copiously within 24 hours, while, as stated 

 above, 50 per cent, of attempts to start new cultures with portions of 

 the usual form of mycelium result in failure. Under natural conditions 

 it is probable that the cells forming these pustular masses would become 

 detached from one another in a manner similar to those forming typical 

 Necator pustules. 



