MICROSCOPY 67 



away spores from their attachments and, in general, is rather unsatisfactory 

 for filamentous fung-i, although it is very satisfactory as a mounting medium 

 for most 3^east and yeastlike organisms. Some of the various formulae of 

 laetophenol give good results, as does also lactic acid alone. The latter has 

 the disadvantage of preventing the use of many dyes in staining. 



So far as I am aware, laetophenol was developed in French laboratories, 

 the formula of Amann (1896) being phenol crystals 20 gm., lactic acid 20 gm., 

 glycerol 40 gm., and distilled water 20 gm. These are dissolved with gentle 

 warming and then is added anilin blue (a mixture of the tri-sulphonates of 

 tri-phenyl pararosanilin [C.I. 706] and of di-phenyl rosanilin) otherwise known 

 as cotton blue (C. B. Poirier). Other compounds, such as methyl blue, are 

 also called cotton blue, but are said to be distinctly inferior for this purpose. 

 Sartory (1924) recommends 0.5% dye to his laetophenol. Linder (1929) ad- 

 vocates the same formula while Henrici (1930) adds only 0.05% of the dye. 

 I have found that the dye added directly to the laetophenol gives a blue back- 

 ground to the preparation. Consequently, I use a 1% aqueous solution of 

 cotton blue, place a drop on a clean slide, inoculate with the fungus, lower a 

 cover glass on the mount, and then allow a drop of laetophenol to be drawn 

 in under as shown previously for glycerol. By this method, the excess dye is 

 pushed to the edge of the cover slip and the laetophenol forms a clear back- 

 ground for the blue fungus. 



Weston (1929) recommends the addition of a small quantity of nigrosin, 

 water soluble, either aqueous, or the picric acid solution described by Curtis 

 and Colley (1915) in order to stain nuclei as well. Since the dye varies in 

 different samples, at present Weston has found no other way than to add some 

 of the dye, try it, and then add more dye or more laetophenol until satisfac- 

 tory results are obtained. Sartory also recommends a mixture of Sudan III 



1 part, and lactic acid 1000 parts by weight. This is ground in a mortar with 

 slow additions of small amounts of the lactic acid. The mixture is then heated 

 in a flask on a water-bath until it is completely dissolved, cooled and filtered. 

 One part of anilin blue is added, also 1-2 drops of tincture of iodine for each 

 10 c.c. of solution. This triple stain colors fatty bodies, amyloid compounds, 

 and the fungus protoplasm. Before adding the cover slip the mount should 

 be heated gently until vapors are given off. 



Spore Stains. — Maneval (1924) suggests the following stain for yeast 

 spores. Spread a film of cells in a drop of water on a slide and dry in air, 

 fix by passing through a flame 12-15 times; stain with hot carbolfuchsin 1-3 

 minutes; wash with water; destain with 5% sulphuric acid 2-3 seconds; wash 

 with water and stain with methylene blue 3 seconds, Avash with water. In 

 1929, he suggested the following procedure : stain with carbolfuchsin or 

 carbol-methylene blue; destain with 5% acetic acid; treat with 5% tannin for 



2 minutes; wash and counterstain with methylene blue (after carbolfuchsin) 

 or with safranin (after carbol-methylene blue). Old spores should be heated 

 in a small amount of sterile water for 10 minutes on a hot water-bath before 



