52 THE ROYAL SOCIETY OF CANADA 



twenty cells or so all the cells will be filled with homogeneous proto- 

 plasm. After two or three days, the colonies become too large to be 

 seen entire under the oil immersion lens. At this stage, the cells 

 become granular and some of them disintegrate as before described, 

 and in the case of Aj and Aj, the organisms produce their large cap- 

 sules. 



Pigment Production. 



The brown and black pigment characteristic of azotobacter cultures 

 is produced apparently only when there is a lack of suitable available 

 nutrient material, when the organisms in the area where the pigment is 

 produced have ceased to multiply, and when the culture is aerated. In 

 plates that are thickly seeded, the maximum growth of the colonies under 

 such conditions is soon attained and pigment production at once begins. 

 In plates that are thinly seeded, the maximum growth of the colonies 

 is longer in being attained and pigment production is correspondingly 

 deferred. If the medium is unevenly spread over the plate, the pigment 

 production occurs early with the colonies that are situated where the 

 medium is scant and where the medium is plentiful the pigment produc- 

 tion by the colonies does not occur until later. If streak cultures are 

 made on tubes of Ashby's agar and growth is allowed to take place 

 until pigment production is just commencing, and then some of the tubes 

 are sealed up with wax and others left unsealed, pigment production 

 will cease in the tubes that are sealed and continue rapidly in the 

 tubes that are not sealed. The pigment is produced and retained 

 within the bacterial cells. It does not occur in the capsules nor in the 

 medium. If a preparation mounted in water is made from a pigmented 

 colony, the mature organisms seen in clusters under the oil immersion 

 lens present a smoky brown or black appearance, something after the 

 appearance of the mature spores of Aspergillus niger. 



Staining Reactions. 



Of the stains tried the following four gave the best results: — 



1. Saturated alcoholic solution of Gentian Violet. This stains the 

 young homogeneous cells evenly violet; with older cells that are granular, 

 the granules of the No. 1 type are not stained and the granules of the 

 No. 2 type which are not always in evidence are stained. The most 

 striking reaction of this stain is with the capsules. The capsules of 

 young cells are negative and the organism of the centre is positive, 

 later the organism becomes slowly negative and the capsule strongly 

 positive. See PI. II, Figs. 4, 5, and 6; PI. Ill, Fig. 2. 



