Pleuro-Pnewmonia of Cattle. 169 



opalescence in the culture medium. This opalescence was ex- 

 tremely slight, and could be best recognised by holding the tubes 

 in such a manner that varying degrees of light fell upon them, 

 at the same time comparing each tube with one or more control 

 tubes of the same medium, which had been incubated, but which 

 had not been inoculated. In order to recognise the presence of 

 this slightly opalescent culture in Martin's broth serum medium 

 it is absolutely essential that each lot of tubes inoculated and 

 incubated should be efficiently controlled by incubating tubes 

 of the same broth serum medium, which have not been inoculated, 

 and comparing them from time to time. 



Sub-cultures made into Martin's broth plus ox ' serum, and 

 incubated at 37°C. showed in 3 to 4 days the same opalescent 

 -appearance noted in the primary cultures. This opalescence 

 could still be obtained after several generations of subcultures. 



Examination of stained films, made with this opalescent broth, 

 under the microscope . with a magnification of 1000 diameters, 

 failed to reveal any recognisable micro-organisms, the material 

 in the film staining as a homogenous mass. 



In order to make sure that the opalescence in the broth was 

 .due to the growth of micro-organisms, sub-cultures from the 

 broth tubes were made on to Martin's-broth-agar with sterile ox 

 serum added after it had been sloped. On this solid medium 

 very fine colonies developed in four days, at first only recog- 

 nisable by means of a lens, but by the eighth day they assumed 

 the size of a pin's point. More or less colourless at first, and 

 appearing like drops of dew on the surface of the medium, they 

 later on became slightly opaque. These colonies appeared on the 

 surface of the agar, but were firmly embedded into it, and were 

 dislodged with difficulty. In most cases they had to be dug out 

 -of the agar, so firmly were they attached. Several colonies were 

 removed, and stained for microscopic examination en bloc. They 

 stained readily with the basic stains, but were decolourised by 

 'Gram's method. Although the shape of the colony could be 

 distinguished under the microscope, individual organisms could 

 not be seen with a magnification of 1000 diameters. 



Sub-cultures from the Martin's broth tubes were made into 

 Martin's broth tubes containing 1 per cent, of various sugars 

 — saccharose, glucose, maltose, lactose, and the alcohol deriva- 

 tives — mannite and dulcite. In the mannite and dulcite tubes no 

 -growth took place. This experiment was subsequently repeated 

 with other cultures of the organism inoculated into mannite broth 



