706 GENERAL TECHNIQUES FOR CLASSES OF PLANTS 



forty-five minutes. Remove excess stain with water and 

 differentiate in 95 per cent, alcohol, acidulated with a few drops 

 of hydrochloric acid. Clear in carbol-turpentine or carbol-xylol, 

 wash with xylol and mount in balsam. 



To stain " germinating spores on the epidermis of infected 

 plants, prepare a leaf by placing a drop of spore suspension on the 

 surface and keep it in suitable cultural conditions. Cut out test 

 areas, fix them in equal parts of glacial acetic acid and 95 per 

 cent, alcohol for twenty -four hours, wash in 50 to 70 per cent, 

 alcohol, stain fifteen to thirty minutes in Pianese Illb, wash for 

 two minutes in water, hastily run through acid alcohol, dehydrate 

 and clear as above. 



KoBEL {Mitteil. Naturf. Ges. Bern., 1920, p. 44) stains the 

 mycelium and haustoria of Peronosporacese and Uredinales m a 

 solution of 0-1 grm. anilin blue in 50 c.c. of lactic acid and 

 100 c.c. of water. Stain sections for five minutes, rinse, and warm 

 in a few drops of lactic acid. The mycelium is stained intensely 

 blue while the host tissue remains nearly unchanged. The stam 

 fades. 



Cotton blue has frequently been advocated as a stain for fungal 

 hyphiE. The stain may be dissolved in glycerin, lactic acid or 

 lacto-phenol (Amann's medium) and the material either mounted 

 in the coloured medium or after staining transferred to glycerm, 

 lacto-phenol or glycerin jelly. When a solution of the stain is 

 used as mounting medium it should be quite dilute. Stainmg is 

 accelerated by gentle warming over a spirit flame. 



Bright (J. Boy. Micr. Soc, Lond., 1925, p. 141) examines 

 mildewed cotton material by staining in picro-nigrosin or cotton 

 blue, mounting in balsam, and examining the preparations under 

 a low power using the powerful light source of a pointolite. The 

 cotton fibres are rendered invisible and the fungal mycelia stand 

 out clearly. 



Chesters {Ann. Bot., xlviii, 1934, p. 820) has described three 



methods for the use of cotton blue : — 



1. For phycomycetes, sterilise a clean slide placed in the 

 centre of a Petri dish and then fill the latter with about 30 c.c. 

 of clear agar medium. This produces a thin film over the slide. 

 Inoculate and incubate. Fix the mycelium in the agar for 

 twenty-four hours at incubator temperature with 1 per cent, 

 chromo-acetic acid. Remove slide with its attached agar film 

 and wash thoroughly. Place slide in 10 per cent, glycerin and 

 allow it to concentrate to the strength of Amann's medium. 

 Stain six to twenty-four hours in 0-5 per cent, solution of cotton 

 blue in Amann's medium and differentiate in several changes 

 of this medium until no further colour is removed from the hyphae. 

 Wash out in 70 per cent, alcohol and carefully dehydrate to 

 absolute alcohol. Pass through mixtures of alcohol and xylol 



