256 METHODS AND FORMULAS AF 13.1-21.1 



AF 13 FOR OTHER USES 



AF 13.1 Formulas 



13.1 Lhotka and Ferreira 1950 20540b, 25 :27 



formula: water 100, chloral hydrate 20 

 RECOMMENDED FOR: removal of "bound" formaldehyde from tissues. 



13.1 Overton 1890 23632, 7:10 



formula: ale. 70% 100-250, hydrogen peroxide 10 

 RECOMMENDED FOR: bleaching overfixed or blackened osmic acid preparations. 



13.1 Schridde 1906 h-sl. 1938 Mallory Mallory 1938, 40 



formula: water 70, 95% ale. 30, ammonium hydroxide 0.5 



RECOMMENDED FOR: removal of precipitates from formaldehyde-fixed tissues before 

 embedding. 



13.1 Verocay 1908 23681, 19:769 



formula: water 80, 96% ale. 20, potassium hydroxide 0.01 



RECOMMENDED FOR: prevention of precipitate in formaldehyde-fixed tissues before 

 embedding. 



AF 20 Decalcifying Fluids and Methods for Softening Chitin 



These two groups of methods have been thrown together for the reason that they solve 

 essentially the same problem, that is, to soften material normally so hard that it cannot be 

 sectioned. The removal of calcium has for many years been achieved principally with acids 

 in which the calcium carbonate and phosphate are soluble. These acids naturally lead to a 

 gross hydrolysis of the tissues, unless this hydrolysis is restrained by the addition of some 

 other agent. The agents used to restrain swelling are very varied and will be noticed as a 

 part of all the formulas given under the heading AF 21.1. Fairly recently attempts have been 

 made to remove the calcium in solutions that are either neutral or very slightly alkaline. 

 In 1938 Wilkes (14900, 142:958) proposed the removal of the calcium through a base 

 exchange similar to that used in the softening of water, and employed for this purpose a 

 30% solution of -sodium hexametaphosphate. Dotti, Taparo, and Clarke 1951 (Tech. Bull., 

 21 :475) used a base exchange resin (Win — 3000) in combination with 10 to 20% formic acid; 

 Birge and Imhoff 1952 {Tech. Bull, 22:16) use a slightly alkaline chelating agent. 



The softening of chitin (AF 23.1 below) is a problem which has not yet satisfactorily been 

 solved. Diaphanol was once recommended for the purpose, but is no longer on the market, 

 and the preparation of its principal constituent (chlorodioxyacetic acid) is so dangerous 

 that it cannot be recommended in the ordinary laboratory. The technique of Jurray 1937 

 has given better results in the author's hands than anj^ other, but it will only work on 

 certain insects, and then its reaction cannot always be forecast accurately. 



21 EMPLOYING ACID MEDIA FOR DECALCIFICATION 



21.1 Formulas 



21.1 Andeer 1884 23730,33:193 



formula: water 100, phloroglucinol 0.01, hydrochloric acid 3 



21.1 Anonymous 1946 4349,26:13 



formula: water 85, 40% formaldehyde 10, nitric acid 5 



21.1 Bayerl 1885 1780, 23:35 



formula: water 100, chromic acid 1.5, hydrochloric acid 0.5 



21.1 Belloni 1939 test. 1943 Cowdry Cowdry 1943, 35 



formula: water 100, formaldehyde 6, formic acid 50 



21.1 von Beust test. cire. 1938 Wellings Wellings circ. 1938, 230 



formula: water 100, 40% formaldehyde 10, formic acid 10 

 RECOMMENDED FOR: embryonic teeth. 



