[ From The Johns Hopkins Hospital Bulletin, No. 49, April, 1895.] 
A RAPID METHOD OF MAKING PERMANENT SPECIMENS 
FROM FROZEN SECTIONS BY THE USE OF FORMALIN. 
Thos. S. Cullen, M. B. 
Any one who has hardened tissues in formalin will be im- 
pressed with the rapidity of its action, with the firm consist- 
ence of the tissue, and with the absence of the contraction of 
the specimen so often seen when alcohol is used as the 
hardening medium. Microscopical examination of a specimen 
hardened in formalin, as we all know, shows almost perfect 
preservation of the cellular structure. Recently it occurred 
to me that formalin might be used in the preparation of frozen 
sections. 
One of the greatest difficulties experienced in rendering- 
frozen sections permanent lies in the fact that when passed 
through alcohol the section frequently not only contracts but 
contracts irregularly, distorting the specimen; further, such 
specimens will often stain imperfectly. The use of formalin 
will obviate these difficulties, allowing one to make an excel- 
lent permanent specimen from the frozen section. My 
method is as follows: The tissue to be examined is frozen 
with carbonic acid or ether and then cut; the sections are 
then placed in 5 per cent, watery solution of formalin for 3 
to 5 minutes, or longer if desired; in 50 per cent, alcohol 3 
minutes, and in absolute alcohol 1 minute. The tissue is now 
thoroughly hardened and can be treated as an ordinary cel- 
loidin section, being stained and mounted in the usual way. 
On examining this mounted section one might readily take it 
for a well preserved alcoholic specimen. Supposing we stain 
with haamatoxylin and eosin, the entire process is as follows: 
a. Place the frozen section in 5 per cent. aq. sol. formalin 
for 3 to 5 minutes. 
b. Leave in 50 per cent, alcohol 3 minutes. 
c. In absolute alcohol 1 minute. 2 
d. Wash out in water. 
e. Stain in haematoxylin for 3 minutes. 
/. Decolorize in acid alcohol. 
g. Rinse in water. 
h. Stain with eosin. 
i. Transfer to 95 per cent, alcohol. 
j. Pass through absolute alcohol, then through either creo- 
sote or oil of cloves, and mount in Canada balsam. 
The blood is lost in frozen sections. To overcome this Prof. 
Welch suggested that the specimen be first fixed in formalin 
and then frozen. I tried this and found that we were able 
to preserve the blood, but that it did not stain very distinctly. 
For convenience this second procedure will be called method 
11. The essential factor is the same in each case. The latter 
process, however, requires at least two hours. A small piece 
of the tissue is thrown into 10 per cent, solution formalin for 
two or three hours. It is then put on the freezing microtome 
and thin sections can be readily made. The sections''are 
stained in the usual way. The detailed procedure of method 
II is as follows: 
a. A piece of tissue 1x.5x.3 cm. is placed in 10 per cent. aq. 
sol. formalin for 3 hours. 
h. Frozen sections are made. 
c. Left in 50 per cent alcohol 3 minutes. 
d. In absolute alcohol 1 minute. 
e. The sections are now run through water and stained in 
hsematoxylin for 3 minutes. 
f. Decolorized in acid alcohol. 
g. Rinsed in water. 
h. Stained in eosin. 
i. Transferred to 95 per cent, alcohol 
j. Passed through absolute alcohol, then either through 
creosote or oil of cloves, and mounted in Canada balsam. 
For ordinary use method lis all that is required. Given a 
piece of tumor from the operating room, it is possible to give 
as definite a report in 15 minutes as one would be able to give 
after examining the alcoholic or Muller’s fluid specimens at 
the expiration of two weeks. Method II is of especial value 
in the examination of uterine scrapings. Instead of putting 
them in the 95 per cent, alcohol in the operating room, they 3 
may be immediately dropped into 10 per cent. aq. sol. formalin. 
By the time the pathologist receives them, which is at least 
two hours afterwards, they are firm enough to be frozen with- 
out difficulty, and permanent sections can be immediately 
made. The second method is to be recommended for all deli- 
cate tissues. In employing these methods one must remember, 
as for example in epithelioma, that some of the cell-nests will 
drop out, there not being anything to hold them in situ, as 
there is when celloidin is used. We have, however, hardened 
and stained epithelioma of the cervix by this method without 
the slightest difficulty.