FIXING AND HARDENING REAGENTS. 31 



turbid. The hardening process is completed in alcohol. It is well 

 adapted for embryological work. 



3. Picro-Nitric Acid (P. Mayi'r). Some substitute nitric acid for 

 the sulphuric acid, but for our purposes there is no advantage in 

 this. Small pieces of tissue not over J cm. in diameter prefer- 

 ably those that contain little connective tissue are hardened for 1-3 

 hours. They are washed with alcohol until most of the yellow 

 colour disappears. Sections can be stained with haematoxylin. 



4. Nitric Acid (Altmami). Use a 3 per cent, watery solution of 

 pure nitric acid. This has a sp. gr. of 1.02. Use as small pieces 

 as possible, and leave them in the mixture just until they are fixed, 

 i.e. t from a quarter to half an hour. Strong solutions, if they act. 

 too long, dissolve the chrornatin. The tissues are then hardened 

 successively in 70, 80, and 90 per cent, alcohol. It is particularly 

 useful for preserving the nuclei of cells, mitotic figures, embryological 

 tissues, and the retina. 



5. Pernyi's Fluid : 



Nitric acid (10 per cent.) , 40 cc. 



Chromic acid (0.5 per cent.) . . . 30 ,, 



Alcohol . . . . . 30 



It is a light greenish-blue liquid, and is specially useful for 

 hardening ova and embryos. Time, 46 hours for a small embryo 

 and 4-12 hours for the tissues of vertebrates. The tissue is trans- 

 ferred direct to 75 per cent, alcohol without previous washing in 

 water. Borax carmine may be added to it, and then this mixture 

 hardens and stains at the same time (Garbini). 



6. Chromo-Formic Acid (flabl's Fluid). To 200 cc. of J per 

 cent, chromic acid add four to five drops formic acid. It must be 

 freshly prepared, and fresh tissues small pieces are placed in it 

 for 12-24 hours. The tissues are thoroughly washed in water, and 

 hardened in alcohol of gradually increasing strength. Sections can 

 be stained in hsematoxylin and safranin. It is specially useful for the 

 study of mitosis and nuclei generally. It has this advantage, that 

 tissues hardened in it do not afterwards darken. 



7. Chromo-Acetic Acid (Fhmming). As chromic acid by itself 

 is apt to cause shrinking of some of the more delicate textures, it 

 has been proposed to mix it with a substance which has an opposite 

 effect, such as acetic acid. The following combination is specially 

 recommended by Flemming for fixing the achromatic spindle in 

 cells : 



Chromic acid .... $-|* grain. 



Glacial acetic acid . . . rs cc - 



Water .... 100 ,, 



The tissues must be small in size, not above 3-5 mm. in diameter. 



