NITROCELLULOSE I N AMYL ACETATE AS AN EMBEDDING MEDIUM H. STANLEY B E N N E T T * Department of Anatomy, Harvard Medical School, Boston, Massachusetts I n 1934 Barron published a paper advocating the use of amyl acetate as a solvent for celloidin embedding material. Although he does not say so directly, the proportions of celloidin and amyl acetate he recommends indicate that he probably used Schering’s celloidin or one with similar properties. Hercules Powder Company low viscosity nitrocellulose s dissolved in alcohol-ether has been recommended as an embeddinq mass by Ruby ( ’33)’ Davenport and Swank ( ’34), Jlettler, Mettler and Strong (’36)’ Koneff and Lyons (’37), and Bensley and Bensley ( ’38). Both of these embedding media have been used successfully in this laboratory, but a combination of the two methods, utilizing Hercules Powder Company nitrocellulose dissolved in amyl acetate, has been found to possess many features which render it superior to either of the above two methods. The principal point on which the superiority of this new method hinges is that it is possible to make a very concentrated solution of low viscosity nitrocellulose in amyl acetate and still have a fluid, rapidly penetrating medium. A 16% solution ’ This work mas nided in p a r t by a grant from t h e Josiah Macy, J r . Foundation. the National Research Council. Hercules Powder Company dehydrated nitrocellulose, RS 1/2 see. viscosity, with 30% alcohol, can be purchased at low cost in 1-pound or 5-pound tins directlv from the IIexules Powder Company, Parlin, N. J. It can also b e procured through company sales offices in the Delaware Trust Building, Wilmington, Del. ; the McCormick Building, Chicago, Ill. ; the Standard Oil Building, San Francisco, Calif.: or through the following foreign agencies: H. J . McAdie, 1100 Craig Street, East, Montreal, Quebee, Canada; N. V. Hercules Powder Co., Korte Vijverberg 5 , The Hague, Holland; A. C. Hatrick, Pty., Ltd., 68-70 Mentmore Are., Roseberry, Sydney, N. S. W., -4nstralia. ’Fellow of 233 T H E AX ATOMIC AT^ RECORD. V O I . . 76, NO. 2 AND SVPPLLXENT NO. 2 234 H. STANLEY BENAETT of Scliering’s celloidin in amyl acetate is so viscous that it will barely flow. The most concentrated practical solution of lorn viscosity nitrocellulose in alcohol-ether is the ‘50% ’ solution used by Koneff and Lyons in their hot embedding method. This ‘50%’ solution is made up by dissolving 50 gm. of nitrocellulose in 100 cc. of solvent, and is hence equivalent to n 334% solution if reckoned according to the method of expressing percentages used in this paper. If one dissolves low viscosity nitrocellulose in amyl acetate, it is quite feasible to prepare and use a 50% solution (50 gm. moist nitrocellulose in 50 cc. amyl acetate), though for most purposes a 40% solution is adequate. I f necessary, freely flowing solutions of nitrocellulose in concentrations up to 60% can be prepared by using other solvents, as noted subsequently. According to the Hercules Powder Company (’38), estersoluble nitrocellulose of even lower viscosity (RS 1,/4 see. vise., and R S 18 to 25 cps) is available and may be valuable f o r certain anatomical purposes, though the material used in the present study (RS l / 2 see. vise.) has proved to be very satisfactory and perfectly adequate as a n embedding medium. This nitrocellulose comes as a loose, fluffy, cotton-like material moistened with denatured alcohol, and can be readi1;v handled and weighed directly on the scales. It requires no special preparation or drying, and can be dissolved in the solvent immediately after weighing. It dissolves very rapidly, the lower concentrations requiring only a few minutes or a n hour o r two f o r solution to reach completion, and the higher ones less than a day if they a r e agitated occasionally. The Hercules Powder Company ( ’38) (personal communication) wishes to make it very clear that this nitrocellulose, although perfectly safe if handled carefully, is highly inflammable and becomes explosive if the alcohol moistening it is allowed to dry. They therefore strongly advise that the nitrocellulose be removed from its container and dissolved in the solvent immediately after i t is received from the manufacturer. After solution is complete, it should be allowed to stand for a few hours or a few days, and the A RAPID NITROCELLULOSE EMBEDDING M E T H O D 235 supernatant fluid decanted off and stored in smaller bottles until used. I f sheets of tin-foil a re inserted under the cork, the material can be handled repeatedly without deterioration. It should also be pointed out that nitrocellulose should not be handled near a n open flame, no lighted cigarette should be allowed in the room where it is being used, and care must be taken to avoid electric sparks near a n open container of nitrocellulose, particularly if it is dry. The material is apt to deteriorate over a period of time if exposed to prolonged bright sunlight or to prolonged heat, and hence should be protected from warm radiators and pipes, and shaded from direct sunlight. METHOD Solutions a r e made up a s follows : 10% 20% 30% 40% solution-10 solution-20 solution-30 solutioii-40 gin. of gin. of gm. of gm. of nitrocellulose t o iiitrocellulose t o nitrocellulose to nitrocellulose t o 90 cc. amyl 80 cc. amyl 70 cc. ainyl 60 ce. aniyl acetate acetate acetate acetate F o r certain purposes a 50% solution iiiay be made up with 50 p i . of nitrocellulose to 50 cc. amyl acetate. F o r convenience in storage one can make u p stock solutions of 40% or 50% nitrocellulose, and later dilute to the lower concentrations as desired. It should be borne i n mind that tlic weight of the nitrocellulose weighed out directly from the can is not entirely due to pure nitrocellulose, since 30g1 of the weight of the fresh product is due to alcohol. The true percentage of nitrocellulose in the finished solutions will be somewhat less than those given above. The actual embedding process is very simple. After dehydrating through alcohols and running from absolute alcohol to pure amyl acetate through two or three gradations, the tissue is put into each of the four solutions in sequence for 24 hours each, and hardened in a block in chloroform. After hardening i n two changes of chloroforin for 12 to 24 hours each, the blocks can be stored in 80% alcohol or in a mixture of equal parts of 95% alcohol and glycerol. These blocks are 236 H. STANLEY BENXETT cut under 8056 alcohol. Sections can be stained separately or on the slide, cleared in oil of origanum, ancl mounted in gum clamar. If sections a r e to be stained on the slide, they can be readily mounted on albuminized slides b>- the usual oil of cloves method. Thick sections sometimes come off when stained after mounting with this method. This can he prevented by the following procedure. Blot tlie slides after mounting the sections, flooding with oil of cloves and hardening in 80% alcohol. Then flood them with a solution of 1% o r 2% Hercules Powder Coinpany nitrocellulose in acetone 01’ ethyl acetate, drain off the excess, allow the slide to dry momentarily, and then plunge again into 80% alcohol. After 4 hour o r so in 80% alcohol the slides can be put through iiiost staining solutions witliout clifficultp. Large pieces of tissue may h a r e to be embedded longer tlian 24 hours in each solution. I n this 1aborator;v pieces over 2 em. in diameter are usually left 2 days in each of tlir lop, 207% and 30% solutions, and 1 week in tlie 40% solution. I n many instances this time can be cut down without danger. Small pieces less than 1 em. in diameter can, if haste is tlesirable, be 1~111 tIiiwLigli the 2054 and 40% solutions alone for 24 hours apiece, omitting the other two concentrations altogether. This is not always adrisahle if very thin secatioiis a1-e desired. MODIFICATIOKS Ethyl acetate, acetone, methyl methacrylate, methyl salicylate, and methyl benzoate can all be used as satisfactory suhstitutes f o r amyl acetate with this variety of nitrocellulose up to 40% conccntration. Ethyl acetate and acetone give a solution with considerably less viscosity tlian amp1 acetatcnitrocellulose, and allow one t o niake fluid solutions with coiicentrations as high as 60% (60 gm. of nitrocellulose with denatured alcohol in 40 cc. of acetone or ethyl acetate). Such high concentrations a r e useful if very thin sections of friable, tough blocks of tissue a r e desired. Acetone has a disadvantage as a solvent in that in the hardening process it will float out of A RAPID XITEOCELLULOSE E M B E D D I S G N E T H O D 237 tlie paper cups when chloroform is poured over it unless it is allowed to stand in chloroform vapor or in the a i r until a n appreciable crust has formed over the surface of the solution in the cup. Both acetone and ethyl acetate will slowly evaporate from tightly stoppered bottles, and their more concentrated solutions cannot be kept f o r more than a few m-eeks xithout becoming excessively viscous. Hercules Powder Company nitrocellulose has also been used successfully in this laboratory instead of Schering’s celloidin in a modification of Pkterfi’s methyl benzoate-celloidiii-~araffin double embedding method. Tissues dehydrated through alcohols a r e talien directly from the secoiitl c1iaiig.e of absolute alcoliol and placed in a 27% solution of Hercules Powder Company nitrocellulose in methyl benzoate for 24 hours, and then into a 10% solution of nitrocellulose in methyl benzoate f o r another 24 hours. The tissue is then placed in two changes of benzene, $ hour each, into benzene saturated with paraffin, 4 t o 1 hour, and into melted paraffin and embedded in the usual way. This method gives a very good reinforcement to fragile tissues, and in my experience the sections do not curl or crinkle after cutting as they do so often wlien any but very low concentrations of Schering ’s celloidin a r e used in double embedding. ADVAXTAGE O F THIS METHOD Hercules Powder Company nitrocellulose in amyl acetate or i n ethyl acetate gives a n embedding niediurii just about as fast as Barron’s method o r the more rapid nitrocellulose in alcohol-ether procedures. If necessary the embedding time can be lessened considerably below the usual requirements. The method is very inexpensive. It will allow the use of far more concentrated celloidin solutions than any other method that has come to my attention. It yields a tough, hard block which does not shrink appreciably on hardening. Blocks made from 40% nitrocellulose i n amyl acetate or any other of the solvents mentioned can be cut readily in thick sections a t 100 to 500 p, or just as readily at 12, 10 or 6 p. Serial sections 238 at 1 2 H. STANLEY BENNETT 10 u have been obtained in this laboratory without difficulty from very large blocks of decalcified tissue, and froiii big blocks full of dense connective tissue which would be exceedingly difficult to cut with the usual celloidin methods. Vsiiig 40% or 50% solutions, 6 H sections can be readily produced, even from large blocks of tough tissue. Blocks of tissue 1 cm. in diameter h a r e been repeatedly sectioned serially a t G 1-1 without losing a section. Thinner sections down to 4 p can be cut, depending 011 the nature of the tissue. It is f a r simpler to prepare than the Lindsay Light Company celloidin recommended by Bensley and Beiisley ( '38), and makes possible the use of a higher concentration of nitrocellulose. Although Lindsay Light Company celloidin has not lxen used in this laboratory, Hercules Powder Company nitrocellulose in amyl acetate would seem to give as good cutting properties as those ascribed to the Lindsay Light Company celloidin. Tlic block obtained 11s. the present method is tougher and cuts better than that produced by 1270 or 14% Schering's celloidiii in amyl acetate, as recommended by Barron. It also has marked advantages over Hercules Powder Company celloidiii in alcohol-ether. Amy1 acetate as a solvent causes tlie nitrocellulose to dissolve niore rapidly, yields more concentrated solutions, allows more rapid embedding, and gives a tougher block. The blocks of anipl acetate-Hercules Powder Company nitrocellulose after liardeniiig in chloroform a r e transparent and almost water-eleai*. 01' SITM1\1AI1Y Hercules Powder Coiiipaiiy iiitroccllulose R S 112 Sec. viscwsitv dissolved in amyl acetate is recornmended a s an emhecldiiig medium, aiid directions f o r its use arc given. This method has distinct aclvantages over most other methods willi respect to expense, case of preparation, rapidity of infiltration a i i d embedding, transparency and toughness of the resulting block, and ease of cuttiiiq- in thick or thin sections. A RAPID NITROCELLULOSE EMBEDDIKG METHOD 239 LITERATURE CITED BARRON, D. H. 1934 Amy1 acetate: a useful solvent for einhcdding masses. Anat. Rec., vol. 59, pp. 1-3. BESSLEY,R. R., A N D S. H. RENSLEY 1938 Handbook of Histological and Cytological Technique. Univ. Chicago Press. D \T ENPORT, H. A., AND R. I,. SWANK1934 Embedding with low viscosity nitrocellulose. Stain Teclinology, vol. 9, pp. 137-139. HERCIJLES POWDER COMPANY1938 Nitrocellulose. Hercules Powder Company, Wilmington, Del. KOXEFF,A. A., AND W. R. LYONS 1937 Rapid embedding wit11 hot lowviscositp nitrocellulose. Stain Technology, vol. 12, pp. 57-39. IIETTLER, F. A., c. c. METTbER A N D F. c. STRONG 1936 The C e ~ ~ O S i ~ ~ - l l i t ~ O cellulose technique. Stain Technology, vol. 11, pp. 1G.7-166. RUBY,S. 1933 A low viscosity nitro-cellulose f o r embedding tissues. Anat. Rec., vol. 55, snppl. p. 74.