BY R. GKEIG-SillTH. 77 



the heap covered •with a white mould, and a portion taken from well inside the 

 heap contained budding yeast-like cells. These were living, for they did not 

 become stained by watery eosin. It is possible that this preliminary mixed fer- 

 mentation at a comparatively low temperature brings about the production of 

 certain nutrients which are necessary for the life of high temperature bacteria 

 in the main fermentation. However this is a matter for future investigation. 



A sample of bai'k was taken from a stack when the top tier was in active 

 fermentation. Digging down about six inches, the temperature was found to be 

 180° r. and a sterile bottle was tilled with the hot bark, taken to the laboratory 

 and subjected to investigation. 



It may be mentioned here that a sample of the tempered bark contained 

 50.5% of water driven ofS at 100° and 51.7% at 130° C. while the stack-bark 

 lost 59.7% at 100°, and 61.9% at 130°. 



The microscopic examination of a watery suspension of the bark showed 

 stout rods of various lengths, and threads. It was thought that the latter might 

 be actinomyces-forms but as an Actinomyces colony was only once found upon 

 the plates, it is probable that they were bacterial threads. During the investi- 

 gation, it was noted that the bacteria formed threads of various lengths very 

 easily. They occurred in the older cultures and may be looked upon as de- 

 generate or involution forms. 



A high temperature organism was eventually obtained but before this oc- 

 curred some observations were made which led up to the successful issue. As 

 plate cultivation at 60° is not an easy method of isolating bacteria, an attempt 

 was made at 37°. Unfortunately the colonies that developed would not grow at 

 60°. Had the incubation of the plates been prolonged, punctiform colonies 

 would have appeared ; one race of the thermophilic organisms was subsequently 

 obtained in this way. A preliminary incubation of the bark with water was 

 found to be necessary and it was shown, later, that an alkaline liquid was very 

 much better. Colonies were obtained by stroking agar-slopes with suspensions 

 of the incubated bark and also by smearing plates which in order to minimise the 

 drying and condensation that occurs at 60° were wrapped in butter-paper and 

 put into damp chambers or larger Petri-dishes . With regard to the tubes, the 

 plugs were pushed down and the mouths loosely closed with ordinary wooden 

 corks. Rubber caps perish quickly at 60°. 



The fii-st trials with ordinary nutrient agar were negative; no growths were 

 obtained and this led to the preparation of a special saccharose agar medium. 

 Later work, however, showed that the ordinary nutrient agar was quite good for 

 gi'owing the high temperature' organism, and the negative preliminary tests must 

 have been due to the ignorance of the necessity for a preliminary incubation of 

 the bark with water or with dilute alkali. 



The agar-medium suggested by the early test consisted of saccharose 1 %, 

 peptone 0.3 %, meat-extract 0.2 %, potassium citrate 0.3 %, magnesium sulphate 

 crystals 0.2%, and calcium chloride 0.05%, made neutral to phenolphthalein . 

 It proved a very suitable mediimi but, as it had a tendency to soften and the 

 slopes slid down in their tubes when incubated at 60°, the second batch was made 

 with 2.5% of agar. The first active growths were obtained on this medium 

 from a portion of the stack-bark that had been covered with water and incubated 

 at 60°. The suspension was smeared over an agar slope and in 20 hours at 60° 

 a luxuriant growth had spread over the agar which was broken up witli numerous 



