1903.] with especial Reference to its Nitrogenous Metabolism. 459 



The algal material also grew exceedingly well when stroked upon the 

 following ammoniacal medium : — 2 • grammes NH 4 C1, 1 • gramme 

 Na 2 C0 3 , 0*5 gramme KotIP0 4 , 15*0 grammes agar-agar, in 1 litre 

 water. The growth was, however, contaminated with numerous 

 bacteria. 



This evident ready assimilation of ammonia pointed to the possibility 

 of the organism's playing some part in the "purification " of sewage 

 or further natural " purification " of sewage effluents, and its nitrogenous 

 metabolism was studied with this view. 



Mode of Preparation of a Pure Culture. 



The first step necessary towards a study of the chemical physiology 

 of the alga was the preparation of a pure culture. As the separation 

 of algse in pure culture is not of very common occurrence, few as 

 yet having been obtained, it may be permissible to describe in detail 

 the method adopted. The following methods were tried without 

 success. Sterilisation, as regards bacteria, was attempted by exposure 

 of liquid cultures to sunlight, and hydrogen peroxide was 'also tried 

 as a means of killing the adherent bacteria, but the alga appeared 

 to be susceptible also. More purely bacteriological methods were 

 employed, viz. : — by stroking in great dilution over the surface of 

 potatoes, and by " pouring " plates of ammonium agar, or ammonium 

 gelatine, and incubating in sunlight, at a low temperature. All these 

 methods failed for one reason or another, but the organism was 

 finally separated in the following way. It seemed advisable to employ 

 an ammonium-containing medium, and ammonium agar was therefore 

 selected. Plates of this medium were poured and allowed to set. An 

 ammoniacal solution was also made and sterilised, having the following 

 composition : — • 5 gramme NaoC0 3 , • 5 gramme K 2 HP0 4 , • 1 

 gramme NH 4 C1, 1 litre tap-water or distilled water. This solution 

 will in future be alluded to as " solution A." 



A few drops were allowed to drop upon the surface of the poured 

 plates, then a small portion of the impure material was added to the 

 first plate, and the whole was brushed over the surface. The same 

 brush was then used to brush the liquid over the second plate, and 

 so on, to six plates or more, without the addition of fresh algal 

 material, so that a considerable dilution was obtained. The plates were 

 kept in the light, in as much sunshine as was possible, protected 

 from dust, and in a damp atmosphere. In seven to fourteen days, green 

 growth was generally visible, and after three or four weeks, definite 

 algse-colonies were to be distinguished among the colonies of bacteria. 



In this way pure cultures of Chlorella pyrenoidosa were obtained from 



(1) Material of Ducat Filter Bed (Hendon). 



(2) Sludge wash-water (Leeds). 



