STAINS AND STAINING 29 



In a successful preparation of this type, chromosomes and lignified cell walls 

 are stained bright red, the contents of the cells having purplish resting nuclei 

 against a bright orange cytoplasm. There is a very vivid, but somewhat vari- 

 able, differentiation of the remaining structures. Parasitic fungi are particularly 

 well shown in bright green whether they are penetrating cytoplasm or the 



lignified portion of the cell wall. 



Stains for Special Purposes 



The number of stains for special purposes is legion. They belong properly 

 in the field of the specialist in the particular tissue which they demonstrate. 

 There are, however, a few which are sufficiently interesting and simple to 

 justify their inclusion in a beginner's handbook. 



Fats. Most fats are normally dissolved from tissues in the course of embed- 

 ding in wax. If, however, frozen sections are made by the technique described 

 on page 71, it is possible to demonstrate differentially the fat globules by soaking 

 the sections in an alcoholic solution of a fat soluble, but water insoluble, dye. 

 The classic method is to use a saturated solution of sudan IV in 70 per cent 

 alcohol. This stains fat globules red. A blue color may be obtained by using 

 a saturated solution of oil blue N in 60 per cent isopropyl alcohol. Sections 

 stained by this method cannot be dehydrated and should be mounted in 

 Farrants' medium (see p. 35). 



Skeletons. It is often useful, in the study of embryos or very small verte- 

 brates, to be able to stain the skeleton differentially. 



Specimens, such as fish fry, which have bony skeletons should be preserved 

 in 70 per cent alcohol made slightly alkaline by saturating it with borax. When 

 they are thoroughly hardened, a 0.5 per cent solution of alizarin red S in abso- 

 lute alcohol is added in the proportion of 1 ml. of stain for each 100 ml. of 

 preservative. The alizarin forms a red lake with the calcium in the bones. When 

 the bones are red enough, the surplus stain is washed out of the other tissues 

 with alkaline alcohol. The embryos are then dehydrated and mounted in balsam. 



Cartilaginous skeletons cannot be directly stained, as are bones, but may be 

 indirectly stained in toluidine blue. The embryos are fixed in any fixative not 

 containing picric acid— mercuric mixtures are preferred by most people— and 

 thoroughly washed. They are soaked for 24 hours in: 



Van Wijhe's Stain: 



70% Alcohol 100 ml. 



Hydrochloric acid 0.1 ml. 



Toluidine blue 0.1 Gm. 



Then they are differentiated in 0.1 per cent hydrochloric acid in 70 per cent 



alcohol until no more color comes away. Dehydration and mounting in balsam 



will show the cartilage alone stained clear blue. 



