GENUS BACTERIUM 219- 



very small isolated gray specks would appear in the film of 

 medium on the surface at the circumference of the tubes, and 

 a gray circular cloud appeared immediately below the surface 

 and very soon spread to the top, especially if some of the fluid 

 parts of the medium had been expressed in the cooling process. 

 Not infrequently, also, the growth appeared to begin in the 

 surface liquid. Such growths became distinctly visible after 

 two days' incubation. Distinct colonies only appeared in the 

 deeper part of the medium, that is to say, at the site of and 

 below the grayish ring, after a week, and sometimes longer. 

 The distance below the surface at which these colonies ap- 

 peared varied considerably in different cases. A few isolated 

 ones were occasionally found half an inch and even more be- 

 low the surface, that is to say, in what might be called the 

 anaerobic zones of the tubes. 



"We were able eventually to convince ourselves that these 

 occasional colonies were not impurities, although the appear- 

 ance of a larger number of colonies in this situation may be 

 taken as proof positive that the culture is impure. 



' * The majority of the sub-surface colonies appeared in the 

 zone comprised between % an d % inch below the surface. The- 

 number varied from one or two to hundreds ; usually they were 

 very numerous. In some cases we found a lower zone of col- 

 onies about y of an inch from the surface, and an upper layer 

 of small gray isolated colonies so near the top that they raised 

 the surface of the medium and finally burst through the thin 

 solid film and spread over the top. 



' ' On most of the tubes which grew at all, as has been pre- 

 viously mentioned, there was a visible growth after two days' 

 incubation, but in a number of cases we have seen the tubes re- 

 quire ten days ' incubation before a growth became visible. The 

 latter appeared almost invariably at about 14 i ncn below the 

 surface of the medium. 



"The colonies vary in size from a pin point to the head 

 of a large pin. The smaller ones are grayish in color, but a& 

 they increase in size, the center becomes of a distinct rusty 



