MS 31.0 



METAL STAINS 



545 



the eyeball removed from the orbit. The 

 eye is then carefully washed in triple- 

 distilled water, and an area about one- 

 millimeter square is cut about three milli- 

 meters to one side of the point of entry of 

 the optic nerve. This usually demonstrates 

 best the nervous elements of the retina. 

 Many of these blocks may, of course, be 

 taken from the same eye. These blocks are 

 placed in the alcoholic pyridine fixing solu- 

 tion in a chemically clean vessel. About 25 

 miUiliters of the fixative should be used for 

 a piece of retina of the size indicated, and 

 overnight fixation will be ample. The small 

 pieces are removed directly from the fixa- 

 tive to absolute alcohol, in which they 

 should remain at least 12 hours, being 

 gently agitated at intervals to prevent the 

 accumulation of diluted alcohol on the 

 bottom of the chemically clean, stoppered 

 vessel containing them. The absolute alco- 

 hol should then be replaced for a period of 

 at least 24 hours by a mixture of equal 

 parts of absolute alcohol and ether. It can- 

 not be emphasized too frequently that the 

 ether used for dehydration of specimens 

 intended for celloidin embedding should 

 be of the grade sold as "dried over so- 

 dium" and not the water-saturated mate- 

 rial commonly sold for anesthesia. The 

 material is next transferred to the thinnest 

 of the three solutions of celloidin for a 

 period of not less than 12 hours. It is then 

 transferred to the intermediate solution of 

 ceUoidin for a further period of 24 hours, 

 and finally placed in the thickest solution 

 for a further 24 hours. With fragments as 

 small as this, the simplest hardening tech- 

 nique is to remove the piece from the 

 syrupy solution in a clean, dry pipet, and 

 to express the drop containing the piece 

 from the end of the pipet directly into 

 about ten milliliters of anhydrous chloro- 

 form. Again it must be emphasized that 

 this chloroform must be specially dried, 

 preferably over calcium chloride, if the 

 material is to cut well. This small drop of 

 celloidin should remain in the chloroform 

 only long enough to harden, and should 

 under no circumstances be permitted to 

 remain until the chloroform has pene- 

 trated to the contained fragment of retina. 

 A period of about 30 seconds will probably 

 be sufficient before the drop of celloidin is 



removed to clean, 70% alcohol, where it 

 may remain until it is convenient to con- 

 tinue the process. 



After not less than one hour in the 70 % 

 alcohol, the drop of hardened celloidin is 

 removed and trimmed until only about 

 one millimeter of celloidin remains on top 

 of the fragment of retina. The trimmed 

 block is now placed in the alcoholic ex- 

 tract of cork where it may remain almost 

 indefinitely. Balbuena, in his original 

 method, suggests between 2 and 20 days. 

 After the fragment has been (to quote 

 Balbuena) "sensitized" in the alcoholic 

 extract of cork, it is again removed to 

 clean, 70% alcohol and washed until the 

 greater part of the brown color has left 

 the superficial layers of celloidin. The 

 fragment is then oriented in thick syrupy 

 celloidin, on the surface of a block of wood 

 adapted to being held in the object holder 

 of a sHding microtome, and the block, 

 with the contained fragment, is then 

 placed under a bell jar with chloroform, 

 or allowed to evaporate in dry air until 

 such time as it is hard enough to cut. 

 Sections are cut with a knife which has 

 been moistened in 70% alcohol and then 

 accumulated in 70% alcohol until a suffi- 

 cient number have been secured. 



The sections are transferred directly 

 from 70% alcohol to a chemically clean 

 watch glass containing the silver-staining 

 solution. This is best held on a small tripod 

 and is warmed, after the sections have 

 been placed in it, until it begins to steam. 

 The flame is then removed, the reagent 

 allowed to cool down until steam is no 

 longer evident, and then reheated to 

 steaming. This cycle of heating and per- 

 mitting to cool is repeated until the sec- 

 tions have become yellowish. This may 

 take anywhere from three to ten minutes 

 according to the extent to which they 

 have been previously sensitized in the 

 alcoholic extract of cork. As soon as they 

 are yellow-brown, they are allowed to 

 cool in the silver solution. When this has 

 reached room temperature, two or three 

 drops of the alcoholic extract of amber are 

 added to render the solution cloudy. 

 Sufficient extract of amber should be 

 added to render the silver solution defi- 

 nitely milky but not creamy. 



