GENERAL TECHNIQUES FOR CLASSES OF PLANTS 707 



to pure xylol and infiltrate with xylol-balsam. The method can 

 also be used for the early stages in formation of some perithecia 

 and pycnidia, 



2, For fungal hypha; in woody tissues : stain thin sections in 

 slightly warm 0-5 per cent, cotton blue in Amann's medium for 

 five to fifteen minutes. Wash out excess stain with Amann's 

 medium and remove this with 70 per cent, alcohol. Counterstain 

 in safranin ten minutes and remove most of excess stain in 70 per 

 cent, alcohol ; dehydrate, clear in xylol and mount in balsam. 

 Fungal hyphae are stained blue, xylem red. 



3. Examination of hypha^ upon cut surfaces of infected tissue 

 (especially timber) may be carried out using a Leitz Ultropak 

 vertical illuminator, after a pre-treatment of the surface with 

 cotton blue. Smooth the surface with a sharp razor and immerse 

 it in a slightly warmed 0-5 to 1-0 per cent, solution of cotton 

 blue in Amann's medium for about ten minutes and wash away 

 excess stain. Place block on slide and cover prepared surface 

 with a coverslip. 



Lepik {Phytopath., xviii, 1928, p. 869) obtains differential 

 staining of Peronosporaceae as follows : Remove paraffin from 

 sections with xylol or turpentine ; pass through absolute and 

 90 per cent, alcohols into a mixture (solution No. 1) of 10 grm. 

 phenol, 10 c.c. cone, lactic acid, 20 c.c. cone, glycerin and 20 c.c. 

 alcohol for ten to fifteen minutes. Then stain two hours in a 

 mixture of 0-02 grm. cotton blue 4B (thought to be identical with 

 oxanin blue 4BX or dianyl blue G) and 0-1 grm. safranin in 100 c.c. 

 of solution No. 1. Differentiate in solution No. 1 and wash in 

 absolute alcohol. Treat twenty to thirty minutes in a weak solu- 

 tion of safranin in clove oil and differentiate in clove oil. Clear in 

 xylol and mount in balsam. Mycelium blue, host tissue red. 



Ferrari {Soc. Internag. Microbiol. Boll. Sez. Italiana, iii, 1931, 

 p. 26) recommends Cuccati's picro-carmine, which gives a rather 

 intense rose colour. He also uses ruthenium red, which colours 

 the cells red-violet. He finds that while a 10 to 20 per cent, 

 aqueous solution of caustic potash bleaches the host cells, the 

 mycelial cells are changed to a yellowish-red. 



Ferrari {Atti 1st. Bat., " Giovanni Briosi " e Lab. Critogam. 

 Italiano R. Univ. Pavia, ii, 1930, p. 81). Immerse sections of 

 host tissue in alcohol for ten to twenty minutes and then stain 

 one to three minutes in a solution of 0-1 grm. ruthenium red in 

 15 c.c. distilled water. In obstinate eases stain the tissue twelve 

 to twenty-four hours or heat the stain to boiling. Differentiate 

 in a ten to twenty per cent, solution of caustic potash. The 

 mycelium stains reddish-yellow, while the host tissue is destained. 

 The stain nuist be kc])t in the dark. 



Dickson {Science, Hi, 1920, ]). 03) stains in a 2 per cent, solu- 

 tion of Magdala red in 85 per cent, alcohol for five to ten minutes, 



23—2 



