15 



and preserves the natural transparency and pigmentation of the tissue, making it 

 valuable for gross anatomy and museum purposes. As a fixer, an aqueous solu- 

 tion of 2 to 4% strength may be employed, or it may be used, which is preferable, 

 in conjunction with other chemicals, as picric acid, in picro-formalin, or chromic 

 acid and acetic acid. 



Formalin is a 36 to 40% solution of formaldehyde (gas) in water. A small 

 amount of formic acid is also present. A 10% solution of formalin, that is a 4% 

 solution of the formaldehyde is a satisfactory strength for most histological 

 purposes. 



Fix 12 to 24 hours, remove to 67% alcohol for a day, 82% alcohol one to 

 several days. Stain as you wish. 



30. Osmic acid. A very useful as well as expensive reagent and somewhat 

 difficult to use. It is generally employed as a fixer in conjunction with other 

 reagents, as in the mixtures ( 18 and 19). When used alone as a fixer weak 

 solutions are generally best A to 1%. It penetrates slowly and it "over-fixes" 

 cells very easily, obscuring detail and giving the parts a homogeneous, glassy 

 appearance. Over-fixed cells cannot be stained, or with great difficulty. More 

 or less blackening of the protoplasm also occurs. It may be used chiefly to demon- 

 strate fat, which is blackened by it, and the zymogen of pepsin and trypsin, which 

 it preserves and browns slightly. 



Fix small (about 2 mm. thick or less) pieces of tissue in 1% osmic acid for 6 to 

 12 hours, wash well in water (running or changed frequently) for 12 to 24 hours, 

 and place in 67% and 82% alcohols. It is somewhat difficult to prevent pure 

 osmic acid of this strength from over-fixing the tissue, and cell detail is generally 

 lost, though the form of cells is well preserved. 



31. Nitric acid. A 10% solution of nitric acid is serviceable in fixing the 

 blastoderm of the chick. 



32. Muller's fluid. Formula: Potassium dichromate, 2.5 grams; sodium 

 sulphate, 1 gram; water, 100 c. c. Make up from stock solutions. This fluid is 

 more of a hardener than a fixer; it should be avoided (as likewise Erlicki's fluid 

 and potassium dichromate) when the preservation of nuclear structure is desired. 

 Staining after its use is sometimes difficult. It is, however, occasionally useful 

 for general work, although such formulas as Zenker's fluid or Kelly's fluid are 

 generally to be preferred. 



Place the object in an abundance of the fluid and harden for from 1 to 8 

 weeks, changing the fluid at first each day. In general, 10 to 14 days will be 

 sufficient. Wash in running water for 24 to 48 hours or longer, remove to 67% 

 alcohol for 1 to 2 days, 82% alcohol several days. Keep in the dark while in the 

 alcohols, and change to fresh when the fluid is colored yellow. Tissue hardened 

 in Muller's fluid cuts well, and it is useful in preparing sections of large organs, or 

 organs with much connective tissue. Its chief usefulness is, however, in the study 

 of the nervous system ( 199-). 



33. Erlicki's fluid. Formula: Potassium dichromate, 2.5 grams; copper 

 sulphate, 1 gram; water, 100 c. c. Make up from stock solutions. This is quite 

 similar to Muller's fluid in its action and results, save that its action is more rapid 

 and stronger. Therefore, it had better be employed with smaller objects, and 

 allowed to act only 2 to 14 days. Otherwise, employ like Muller's fluid. 



