OSMIC ACID METHOD 



248 



OVARY 



Bensley (H. 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, Flemming 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 Bensley, 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 18 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. KjCOj 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 

 jar in the final mixture of alcohol and 

 glycerin. 



A rather similar technique leading up 

 to dehydration in absolute alcohol, 

 clearing in toluol and final storage in 

 anise oil saturated with naphthalene is 

 presented by Cumley, R. W., Crow, J. 

 F. and Griffen, A. B., Stain Techn., 



14, 7-11. This staining of ossification 

 centers with alizarin red can be com- 

 bined with the coloration of the carti- 

 laginous skeleton with toluidin blue to 

 make quite brilliant specimens (Wil- 

 liams, T. W., Stain Techn., 1941, 16, 23- 

 25). 



Ossification, intense glycogenesis during 

 (Gendre, H., Bull. d'Hist. AppL, 193S, 



15, 165-178). 



Otoliths, technique for (Johnston, M., J. 

 Roy. Micr. Soc, 1938, 58, 112-119). 



Ova, concentration of parasitic ova in Feces. 



Ovalocytosis, see Pencil Red Cells. 



Ovary. For routine purposes fixation in 

 Zenker's Fluid and coloration by Mal- 

 lory's Connective Tissue stain or by 

 Masson's Trichrome technique is in- 

 dicated. Follicular atresia can be beau- 

 tifully demonstrated by Vital Staining 

 with trypan blue or by other similar 

 dyes, see Evans, H. M. and Swezy, D. 

 R., Memoirs Univ. California, 1931, 

 9, 119-224. For_ the utilization of 

 Microdissection in determination of 

 the physical properties of the follicular 

 wall see Thanhoffer, L., Zeit. f. Anat. 

 u. Entw., 1933, 100, 559-562. The in- 

 teresting fluorescence studies on the 

 ovary by Policard, A., C. rend. Acad, 

 d. Sci., 1924, 179, 1287 are likely to be 

 extended now that the possibilities of 

 Fluorescence Microscopy are better 

 appreciated. Ragins, A. R. and Pop- 



