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Nitrocellulose in amyl acetate as an embedding medium.

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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.
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