OSSIFICATION 



181 



OXIDASE 



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 Griff en, A. B., Stain Techn., 



14, 7-11. This staining of ossification 

 centers with alizarin red can be com- 

 bined with the coloration of the cartil- 

 aginous skeleton with toluidin blue to 

 make quite brilliant specimens (Wil- 

 liams, T. \V., Stain Techn., 1941, 16, 23- 

 25). _ 



Ossification, intense glycogenesis during 

 (Gendre, H., Bull. d'Hist. Appl., 1938, 



15, 165-178). 



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

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



Ova, concentration of parasitic ova in Feces. 



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 

 IMicrodissection 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- 

 per, H., Arch. Path., 1942, 36, 647-662 

 have indeed investigated variations in 

 ovarian fluorescence during cyclical 

 changes. 



Owen's Blue (British Drug Houses Ltd.), a 

 dis-azo djx similar in composition to 

 IVIanchester blue. Used best in alco- 

 holic solution (H. G. Cannan, J. Roy. 

 Micr. Soc, 1941,61,88-94). 



Oxalate Solutions, see Anticoagulant Solu- 

 tions. 



Oxazins. Dyes resembling the thiazins but 

 in which sulphur atom is replaced by 

 oxygen. Examples : brilliant cresyl 

 blue, celestin blue B, cresyl violet, gal- 

 lamin blue, gallocyanin, Nile blue sul- 

 phate, resorcin blue. 



Oxidase. Unfortunately, as Lison (p. 263) 

 points out, histologists and biochemists 

 are not always agreed as to terms. The 

 latter include under the designation 

 "oxidases" all enzymes capable of cata- 

 Ij^sing a reaction of oxidation, for in- 

 stance the phenolases, purinoxidases, 

 succinoxidase, tyrosinase, etc. ; whereas 

 what the former describe as "oxidases" 



are in reality phenolases and thus only a 

 part of the whole group of oxidases. 

 The action of oxidase (or phenolase) in 

 the presence of O2 is the same as a per- 

 oxidase in the presence of H2O2. But 

 the particular oxidases are more delicate 

 and easil}' modified in their action by 

 variations in temperature, pH and other 

 factors. The following methods are 

 from Lison, much abbreviated. 



1. M. nadi oxidase reaction (Graff) 

 = oxidase reaction, modification A (W- 

 II. Schultze) and stabile oxidase reac- 

 tion (V. Gierke). Make 2 solutions: A. 

 Boil 1 gm. anaphthol in 100 cc. aq. dest. 

 Add drop by drop 25% aq. potassium 

 hydroxide until melted a naphthol is 

 dissolved. Cool. Can be kept in dark 

 at least 1 month. B. Obtain good 

 sample dimethyl - p - phenylenediamine 

 furnished in sealed tubes. It blackens 

 quickly when secured in bulk. Graff 

 advised, as more stable, dimethyl-p- 

 phenylenediamine hydrochloride. Make 

 1% solution of either in aq. dest. Boil 

 and cool. Keeps 2-3 weeks in dark. 

 Immediately before using take equal 

 parts A and B, filter and employ filtrate. 

 Place frozen sections of formalin fixed 

 tissues or smears (after fixing for 2 hrs. 

 in formalin vapor or in formol, 10 cc. 

 -+- 96% alcohol, 40 cc.) in above mixture 

 of A and B in a thin layer at the bottom 

 of a Petri dish. Agitate a little to per- 

 mit oxygenation of the fluid. Blue 

 granules quickly appear (1-5 min.). 

 Rinse in water and examine. To make 

 more permanent treat with Lugol's 

 iodine diluted one third, 2-3 min., 

 which makes the blue granules brown. 

 Restore blue by washing in aq. dest. 

 4- few drops sat. aq. lithium carbonate. 

 Counterstain with hemalum or safranin, 

 mount in glycerin. Schmorl advised 

 instead of Lugol's a cone. aq. sol. am- 

 monium molybdate. 



2. G. nadi oxidase reaction (Graff) 

 = labile oxidase reaction (V. Gierke). 



This more difficult method is for fresh 

 tissues. The nadi reagent is prepared 

 without addition of alkali. The re- 

 quired pH depends on the cells investi- 

 gated. For animal tissues Lison recom- 

 mends about 8.2, 8.1 and 7.8 and for 

 plants 3.4-5.9. Directions are given 

 by Graff (S., Die Mikromorphologischen 

 Methoden der Fermentforschung, Ab- 

 derhalden's Handb., 1936, 4 (1), 93-142). 



3. Naphthol reaction of Loele. This 

 is not, in the opinion of Lison, strictly 

 speaking a microchemical reaction, but 

 it is as simple. Place small amount 

 a naphthol in a test tube. Add drop by 

 drop 10% aq. potassium hydroxide until 

 naphthol is completely dissolved. Add 

 200 cc. aq. dest. Solution may be used 

 after 24 hrs. It will last about 3 weeks. 



