20 THE ROYAL SOCIETY OF CANADA 



After three days this culture had assumed a characteristic appearance. 

 The growth was white, semi-transparent, rather dry, very adherent 

 to the medium and had a pecuhar cracked and wrinkled surface 

 (Culture I)^ A finely granular deposit had formed in the medium 

 in the neighbourhood of the growth which was found to consist of 

 minute spherular nodules of calcium carbonate. This will be referred 

 to in a later paragraph. A film prepared from the culture at this 

 stage^ showed a predominance of similar forms to those seen in the 

 young growth but the organisms were frequently thicker and ran up 

 to 4-5/1. in length. Both their form and the staining were less regular 

 than in the young culture. 



All the cultures kept at air temperature showed growth after six 

 days. The character of the culture from the surface sample was 

 similar to that obtained at 28°C after 3 days and similar organisms 

 were present (Culture VI). The 20 fathom sample gave a faint 

 dotted growth which did not develop sufficiently to assume any definite 

 character. There was a slight deposition of calcium carbonate in the 

 medium. A film showed round ended bacilli about -Sfi. thick and 

 ranging from 1 to 2 -5//. long mixed with a longer and more slender 

 organism (about -25^. thick by 3 or 3-5 ^. long) (Culture XXII). 

 The culture from the 100 fathom sample grew more freely. The 

 growth was almost transparent and colourless and in the early stages 

 moist and spreading. After a few weeks it became drier and more 

 adhesive to the medium and a considerable deposition of calcium 

 carbonate occurred in the medium. A film prepared from this culture 

 showed a small coccus or very short bacillus (width about -4^., 

 length -4 to -S/z.) (Culture VII). 



Each of the three samples of water was also plated on the same 

 medium using Ice. of water and lOcc. of medium. The plates were 

 kept at 28°C. No colonies developed in the case of the 20 fathom or 

 the 100 fathom sample. It was subsequently found that this was due 

 to the temperature of incubation being too high, no satisfactory 



1 The numbers of the cultures refer to the summary of cultural characteristics 

 at the end of the paper. 



2 The method used for staining films throughout this work was as follows: The 

 film having been fixed by flaming in the usual manner was treated with 2% acetic 

 acid for two minutes. As much as possible of the acid was then poured off the 

 coverglass and freshly prepared Anilin Gentian Violet applied for about two minutes. 

 This method was found to give much better results than either Carbol Fuchsin, 

 Lofflers Methylene Blue or Aniline Gentian Violet used direct. In many cases these 

 failed to stain the organisms at all. The organisms frequently seemed to be sur- 

 rounded by a substance which prevented the penetration of the stain and which 

 the acetic acid treatment removed. 



