OSMIC ACID 



180 



OSSIFICATION 



Osmic Acid. This is the tetroxide of 

 osmium and has no acid properties. 

 It comes in sealed glass tubes usually 

 each containing 1 gm. To make the 2% 

 aq. sol . of osmic acid generally employed, 

 wash the label off the tube with soap 

 and water. After washing repeatedly 

 in aq. dest. rinse in absolute alcohol and 

 dry. Carefully clean the inside of a 

 glass stoppered bottle and of a graduate 

 in the same way. With clean forceps put 

 the tube in the bottle. If it is not easily 

 broken by vigorous shaking it will be 

 necessary to take it out, file one side, 

 break and return to the bottle. Finally 

 add 50 cc. of aq. dest, measured in the 

 graduate. The osmic acid slowly dis- 

 solves forming a clear light yellow solu- 

 tion. Do not hasten solution by heat. 

 Keep in dark or subdued light. To use 

 a bottle made of colored glass or the out- 

 side of which has been blackened is a 

 bad practice because it hides the con- 

 dition of the solution from the person 

 using it. If there is a blackening of the 

 solution its potency is probably reduced. 

 An indicator of concentration, dis- 

 covered by Tschngaeff, has been im- 

 proved by Palmer (R., J. Roy. Micr. 

 Soc, 1930, 50, 221-226). _ 



The fumes of osmic acid are very in- 

 j urious to the eyes . They are a good fixa- 

 tive for well separated cells as in smears. 

 They blacken the chromaffin cells of the 

 adrenal charged with epinephrine or its 

 precursor (Cramer, W., Fever, Heat 

 Regulation, Climate and Thyroid- 

 Adrenal Apparatus. London: Long- 

 mans, Green & Co., 1928, 153 pp.) 

 Alone, a solution of osmic acid is a fair 

 fixative for mitochondria and by pro- 

 longed action may reveal the Golgi 

 apparatus. See critique by Owens and 

 Bensley (II. S. and R. R., Am. J. Anat., 

 1929, 44, 79-109). But osmic acid 

 penetrates very badly indeed and is best 

 employed in mixtures with other chem- 

 icals as in the fixatives of Altmann, 

 Mann, Bensley, Flamming and others. 

 Its chief value is that it blackens many 

 but not all fatty droplets. However it 

 also blackens some materials which are 

 not fatty. Osmic acid plays an impor- 

 tant part in the Marchi method for 

 nerve fiber degeneration. 



Osmic Acid Method for fat. When reduced 

 to osmium dioxide in the presence of 

 some fats it blackens them as may be 

 seen by the examination of tissues fixed 

 in fluids containing osmic acid (Alt- 

 mann's, Flemming's etc.) but unless 

 rigidly controlled other substances may 

 be blackened as well or not all of the fats 

 may be shown. See remarks by Owens, 

 H. B. and Benslev, R. R., Anat. Rec, 

 1929, 44, 79-109. It is best to proceed 

 as advised by Mallory (p. 119). Place 



frozen sections of tissue fixed in 10% 

 formalin for 24 hrs. in aq. dest. 1% osmic 

 acid 24 hrs. (or Flemming's or Marchi 's 

 solution). Wash thoroughly in running 

 water 6-12 hrs. Abs. ale. for several 

 hours in order to get secondary stain- 

 ing of palmitic and stearic compounds as 

 well as of oleic. Wash in water and 

 mount in glycerin jelly (glycerin alone 

 will do). Fat is black against a yellow- 

 ish brown background. Non-fatty sub- 

 stances like tannic acid and eleidin of 

 epidermis are also blackened. 



For nerve fibers (Dr. J. L. O'Leary, 

 personal communication). Use fresh or 

 10% formalin fixed material. Tie a 

 stretch of freshly isolated nerve to short 

 length of glass rod and immerse in 2% 

 aq. osmic acid. Leave for 24 hrs. Wash 

 4-6 hrs. in running water. Dehydrate 

 in ascending alcohols and doubly imbed 

 by the Peterfi method as follows : Pour 

 1% celloidin in methyl benzoate (which 

 takes about 1 month to dissolve) into a 

 dish. Add absolute alcohol and the tis- 

 sue. The latter gradually sinks into the 

 celloidin. Transfer to 2-3% celloidin 

 in methyl benzoate. Leave 2-4 days. 

 Drop tissue directly into benzol. After 

 a few hours in benzol begin infiltration 

 in paraffin at 40°C. This takes 12-24 

 hrs. Change paraffin several times and 

 imbed. 



Ossicles, see Ear. 



Ossification. Demonstration of in embryos 

 and fetuses up to IS weeks by staining 

 with alizarin red S (Richmond, G. W. 

 and Bennett, L., Stain Techn., 1938, 

 13, 77-79). Eviscerate. Fix in 95% 

 alcohol 2 weeks or more. Rinse in tap 

 water and put in 1% aq. KoCO.i for 

 month or longer. Clear soft parts and 

 make bones clearly visible by placing in 

 1% aq. KOH for 10 days or more. (Spec- 

 imens fixed in formalin instead of alco- 

 hol require about 1 month in 10% KOH) 

 If tissues become too soft harden in 

 equal parts glycerin, 95% alcohol and 

 water 12-24 hrs. and continue KOH if 

 necessary. In last few days reduce 

 KOH to 0.5%. Wash in running tap 

 water 12 hrs. Immerse in 0.1% aq. 

 alizarin red S to which few drops 1% 

 aq. KOH has been added for 30-60 min. 

 Wash for 30 min. in running tap water. 

 Remove deep purple color from soft 

 parts by immersing in 20% aq. glycerin 

 containing 1% KOH. For small speci- 

 mens reduce KOH to 0.5%. This de- 

 colorization may require 1-2 weeks be- 

 fore ossified skeleton remains deep red 

 in transparent background. Dehydrate 

 by passing slowly through 95_% ale, 

 glycerin and aq. dest. in following pro- 

 portions 10 : 20 : 70—20 : 20 : 60—30 : 30 : 40— 

 40:40:20—50:50:0. Seal in specimen 



