Chapter III —49— Morphology 



discussion. The presence of fine grains in preparations treated with 

 dilute methylene blue was looked upon as substantial evidence of the 

 presence of a nucleus (321). Others considered these granules, how- 

 ever, as merely fatty bodies or of a metachromatic nature. Krassilni- 

 Kov submitted evidence that these granules consist of chromatin sub- 

 stance, which plays the role of a nucleus. Young hyphae contain 

 single grains which are larger in older cultures and could be distin- 

 guished only with difficulty from the rest of the protoplasm. 



By use of the Feulgen reaction, von Plotho (340) demonstrated 

 that the reacting substance is distributed in the protoplasm of the actino- 

 mycetes in all stages of its development. He reached the conclusion 

 that nuclear substance is present in the cell plasma. This substance 

 can become concentrated into special nuclear bodies, especially in the 

 mature spores. The presence of thymonucleic acid bears evidence of 

 this fact. These results are not in agreement with those obtained by 

 RippEL (361), who believed that the bodies stained by the Feulgen re- 

 action are fats in nature. 



Jensen described (186) the staining reactions of Micromonosfora 

 as follows: The hyphae and spores stain easily with all the usual bac- 

 terial stains, such as carbol fuchsin, aqueous fuchsin, methylene blue, 

 gentian violet. Delafield's haematoxylin gives fine and clear prepara- 

 tions, especially when material is fixed with sublimate alcohol. The 

 spores stain more intensely than the hyphae. All the strains are gram- 

 positive, but never acid-fast. Whether nuclei are present in the spores 

 and mycelium was difficult to decide because of the minuteness of the 

 objects. Preparations were stained by the method of Schumacher for 

 demonstrating nuclear material. The preparation was dried on a slide 

 and treated for 2 to 4 hours with 25 per cent hydrochloric acid, washed 

 first with water, then for 10 seconds with dilute Na2C03 solution, and 

 finally stained for 30 seconds with carbol thionine. The presence of 

 deeply stained minute granules was demonstrated in old spores, in 

 germinating spores, and in young mycelium. 



The nuclear method of staining bacteria was applied successfully to 

 the staining of sporulating actinomycetes (222). It consists in fixing the 

 cells with osmic acid in N HCl, and staining with the Giemsa stain. 

 The acid is usually applied for 6 to 20 minutes at 55 °C., and the stain, 

 diluted 1 to 30, 5 to 30 minutes. The preparations are dehydrated with 

 acetone and x)'lose and mounted in Canada balsam or in the weak stain- 

 ing solution. To show the cell boundaries, the osmic acid preparations 

 are placed for 30 minutes in a 5 per cent aqueous solution of tannic acid, 

 rinsed in water, stained for 2 to 4 minutes in crystal violet 1 : 10,000, and 

 mounted in the stain or in water. 



General Morphology:— 



^Colony formation— Growth of an actinomyces on a solid or in a 

 liquid medium results in the formation of a mass of growth usually 



