COPPER 



73 



CORNEA 



Copper. 1. Microchemical tests. Fix in 

 formalin or alcohol, use same hema- 

 toxylin or methylene blue stain as for 

 Lead. With former copper hemofuscin 

 is blue and hemosiderin (iron pigment) 

 is black, while with latter copper pig- 

 ment is pale blue and the iron pigment 

 uncolored (Mallory, F. B. and Parker, 

 F., Jr., Am. J. Path., 1939, 15, 517-522). 

 See also Okamoto, K., Utamura, M. and 

 Mikami, G., Acta Sch. Med., Univ. 

 Imp. in Kioto., 1939, 22, 335-360 (il- 

 lustrated in colors); Mendel, L. B. and 

 Bradley, H. C, Am. J. Physiol., 1905, 

 14, 313-327 (bromine test for) ; Claude, 

 A., Cold Spring Harbor Symposia on 

 Quantitative Biology, 1941, 9, 263-270 

 (copper of respiratory pigment) ; Hoag- 

 land, C. L. et al., J. Exper. Med., 1942, 

 76, 163-173 (copper and other substances 

 in virus elementary bodies). When 

 search is necessary for traces of copper 

 without need for microscopic localiza- 

 tion an emission spectrograph may give 

 the information quicklj^ see Histo- 

 spectrography. If quantitative deter- 

 minations of copper in small amounts 

 of tissue are required use the polaro- 

 graphic technique elaborated by Car- 

 ruthers, C, Indust. and Engin. Chem., 

 1945, 17, 398-399. Details for deter- 

 mination of copper in epidermis are 

 given by Carruthers, C. and SuntzeiT, 

 v., J. Biol. Chem., 1945, 159, 647-651_. 



2. As vital stain. Intravenous in- 

 jections of colloidal solutions of copper 

 in rabbits are described by Duhamel, 

 B. G., C. rend. Soc. de Biol., 1919, 82, 

 724-726. 



Copper Chrome Hematoxylin (Bensley's) 

 for mitochondria. Fix very small pieces 

 in x\ltmann*s Fluid or in Acetic-Osmic- 

 Bichromate fixative of Bensley 12-24 

 hrs. Wash, dehydrate, clear, imbed in 

 paraffin and cut sections at 4 or 5 mi- 

 crons. Deparaffinize. Sat. aq. copper 

 acetate, 5 min. Several changes aq. 

 dest., 1 min. 0.5% aq. hematoxylin, 1 

 min. After rinsing in aq. dest. pass to 

 5% aq. neutral potassium chromate, 1 

 min. which should turn sections dark 

 blue-black. If they are only light blue, 

 rinse in aq. dest. again place in copper 

 acetate and repeat if necessary several 

 times until no increase in color is ob- 

 tained. Wash in aq. dest. and treat for 

 few sec. with copper acetate. Wash in 

 aq. dest. and differentiate under the 

 microscope in Weigert's borax-ferri- 

 cyanide mixture (borax, 1 gm. ; potas- 

 sium ferricyanide, 1.25 gm.; aq. dest. 

 100 cc.) diluted with twice the volume 

 aq. dest. Wash in tap water, 6-8 hrs. 

 Dehydrate, clear and mount in balsam. 

 The mitochondria appear a beautiful 

 deep blue against a yellowish back- 

 ground. It is important to have good, 



ripe hematoxylin. It is usually made 

 by dilution from a 10% sol. in abs. al- 

 cohol. This method of staining should 

 be tried after fixation in Regaud's fluid. 



Coproporphyrin of megaloblasts in embryos, 

 see Porphyrins. 



Coreine 2R, see Celestin Blue B. 



Corinth Brown G, see Erie Garnet B. 



Coriphosphine O (CI. 787). An acridine dye 

 used as a fluorchrome (Metcalf, R. L. 

 and Patton, R. L., Stain Techn., 1944, 

 19, 11-27). 



Corn Blue B, see Victoria Blue R. 



Corn Blue BN, see Victoria Blue B. 



Cornea. This is a difficult tissue to prepare 

 in stained sections because of its curva- 

 ture and avascularity. A valuable sil- 

 ver method is minutely described by 

 Pullinger, B. D., J. Path, and Bact., 

 1943, 55, 97-99. 



Fix anterior and posterior surfaces 

 in 10% aq. neutral formalin before ex- 

 cision of eye, if possible, by flooding 

 anterior surface with fixative and by 

 injecting fixative into anterior chamber 

 through a hypodermic needle at the 

 same time withdrawing fluid from the 

 chamber likewise by hypodermic. Re- 

 move eye, inject fixative into vitreous 

 at same time removing fluid from it. 

 Leave in fixative 24 hrs. Excise cornea 

 along corneo-scleral margin, detach 

 iris, ciliary bodj'' and lens. Fix latter 

 separately and cornea for further 3 

 days, 4 altogether. Indicate location 

 future sections by nick in opposite edge. 

 Transfer cornea to aq. dest. avoiding 

 metal instruments then and thereafter. 

 Wash and leave over night in aq. dest. 

 + 3 drops ammonia (S. G. 0.8S) per, say, 

 50 cc. After washing in 2 or 3 changes 

 aq. dest., and pouring off last aq. dest., 

 filter onto cornea through cotton v/ool 

 moistened with aq. dest. 20 cc. del Rio- 

 Horiega's solution. To make this add 

 300 cc. 5% aq. sodium bicarbonate to 

 100 cc. 10% aq. silver nitrate in brown 

 glass stoppered bottle. Add few drops 

 ammonia waiting each time for smell 

 of ammonia to disappear until almost 

 but not all ppt. is dissolved. They 

 add 250 cc. aq. dest. 



Place container with cornea plus fil- 

 tered solution in incubator at 37°C. 

 4 hrs. Pour off solution and wash 

 cornea in several ciianges aq. dest. 

 Then reduce by pouring onto cornea 

 10% aq. neutral formalin, lo min. Cut 

 away "dome" of cornea with knife and 

 support its concave surface with the 

 lens, freeze and section at about 15 m- 

 parallel to surface. Take sections into 

 aq. dest.; mount at once in glycerin 

 jelly or pass through alcohols to abso- 

 lute, clear in creosote and mount in 

 balsam. Collagen pale yellow, nuclei 



