Y1(ft. ft 
A RfiSUMfi OF 
THE USES OF FORMALIN. 
BY 
GEORGE C. FREEBORN, M. D., 
Instructor in Normal Histology in the Medical Department of 
Columbia University in the City of New York ; Patholo- 
gist to the Woman's Hospital in the State of New 
York ; Pathologist to the New York 
Obstetrical Society. 
REPRINTED FROM THB 
Mete Xorfc iHe'Oual journal 
for June 13, 1896. Reprinted from the New York Medical Journal, 
for June 13, 1896. 
A BfiSUMR OF THE USES OF FORMALIN* 
By GEORGE C. FREEBORN, M. D., 
INSTRUCTOR IN NORMAL HISTOLOGY IN THE 
MEDICAL DEPARTMENT OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK ; 
PATHOLOGIST TO THE WOMAN'S HOSPITAL IN THE STATE OF NEW YORK J 
PATHOLOGIST TO THE NEW YORK OBSTETRICAL SOCIETY. 
This reagent is also known in commerce under the 
names of formol and formalose. It is a forty-per-cent, so- 
lution of the gaseous body, formic aldehyde (IICOH), in 
water. It is prepared by oxidizing methylic alcohol and 
bringing the resulting gas into solution in water. It is 
non-inflammable. It mixes in all proportions with alcohol 
and water. Its power of penetration is good. Its keeping 
properties are good. A series of experiments were insti- 
tuted to determine this point, with the following results: 
A forty-per cent, solution was kept in open and closed ves- 
sels, daily tests being made. The results of these experi- 
ments showed that the solutions did not decompose. There 
was a loss of 1'6 per cent, of formalin and an increase of 
OT per cent, of formic acid. Polymerization took place 
with the formation of a butterlike mass containing sixty 
per cent, of formic aldehyde; this dried up into a hard 
mass which contained eighty-five per cent, of formic alde- 
* Read before the New York Pathological Society, March 23, 1896. 
Copyright, 1896, by D. Appleton and Company. 2 
A RESUME OF THE USES OF FORMALIN. 
hyde. Fish and others advise that it be kept in darkened 
bottles, as the light may decompose it. In an experience 
of two years I have not noted any appreciable change in 
the solutions. 
Attention was first called to the antiseptic properties of 
formalin by F. Blum, in 1893. In 1894 Pottevin found 
that when formalin was added to cultures of bacteria, their 
growth was arrested. Cohn also found that solutions and 
the vapor of formalin killed bacteria both in the vegetative 
and in the spore stage, but that it had but little action on 
molds unless used in strong solutions. This want of action 
on molds has also been noted by many other observers. 
Miquel, in experimenting with gaseous formic aldehyde, 
found that it acted as a disinfectant for small and loose ob- 
jects confined in small spaces, but was not reliable for dis- 
infecting large rooms. 
Cambier and Brochet also experimented with the gase- 
ous form, with results similar to those of Miquel. Their 
laboratory experiments were satisfactory, but their attempt 
to disinfect a large room did not give perfect results. They, 
however, demonstrated the fact that layers of dust a cen- 
timetre thick were rendered sterile. They also devised a 
portable apparatus for producing the gas. 
Alleger made quite an extensive series of experiments 
in order to determine the germicidal action of formalin on 
bacteria. He made use of cultures of the bacillus of diph- 
theria iu Petri dishes. The surfaces of these dishes were 
sprayed with solutions of formalin varying in strength from 
1 to 10,000 to 1 to 100. He found that a solution of 1 to 
2,000 prevented the growth of the bacillus but not that of 
molds. Another series of experiments were made with 
stick cultures in test tubes. Five drops of solutions of 
formalin, varying in strength from 1 to 20,000 to 1 to 100, 
were placed on the surface of the culture media in each A RESUME OF THE USES OF FORMALIN. 
3 
tube. At the end of forty-eight hours none of the tubes 
which had been treated with a 1-to-100 or stronger solu- 
tion showed any growth. A third series of experiments 
were made with smear cultures, which were allowed to 
grow for from twenty-four to forty-eight hours and then 
they were treated with the above mentioned solutions of 
formalin for a few minutes. Cultures were then made from 
these, with the result that no growth took place from those 
treated with the stronger solutions. 
Formalin has also been used in surgery, obstetrics, and 
gymecology as an antiseptic, but has had to be abandoned 
on account of its irritating properties. 
As a preserving agent formalin was first used by the 
botanists. Cohn experimented with it extensively, and 
found that the green and red colors of plants were not ex- 
tracted. At the end of five months his specimens still 
retained their natural colors and were not shriveled. The 
botanists Sadebeck and Holfert recommend it highly. 
It was first introduced into the zoological technique by 
F. Blum, who obtained excellent results with it as a pre- 
servative agent, and it has now come into general use. 
The excellent results obtained by the botanists and zool- 
ogists with formalin as a preservative soon resulted in its 
introduction into the anatomical and histological technique, 
and at the present time it is quite generally used. 
As a preservative agent for gross specimens, it is used 
in the strength of two per cent.,* though weaker solu- 
* Bolles Lee {Anat. Anz., xi, 1895, p. 253) calls attention to what 
he considers an inaccurate use of the terms formol, formalin, and for- 
maldehyde ; also to the manner of stating the percentages used. He 
maintains that the proper way of stating the strength of the solutions 
is to say "formol or formalin diluted with so many volumes of 
water." 
Parker and Floyd (Anat. Anz., xi, 1896, p. 567) reply to the criti- 
cism made by Bolles Lee in the above-cited article. They contend 4 
A RESUME OF THE USES OF FORMALIN. 
tions, from three quarters to one per cent., have been used. 
These weaker solutions are objectionable on account of the 
likelihood of the growth of molds, and because they cause 
more or less swelling of the tissues. As the result of the 
experience of numerous observers, it appears that five-per- 
cent. solutions give better results. 
The quantity of the solution should be large-a hun- 
dred times the volume of the specimen-and the fluid 
should be renewed at the end of twenty-four hours. In 
some cases it is well to renew the fluid a second or even a 
third time. 
Formalin used in this manner preserves the natural 
form, the transparency, and, to a certain extent, the natu- 
ral color of the specimens. In some specimens the blood- 
color appears to be bleached out, but if the preparation is 
placed in strong alcohol this is nearly if not entirely re- 
stored. 
For preserving the blood-color of specimens, Johres 
makes use of the following procedure and fluid : 
Sodium chloride 1 part; 
Magnesium sulphate 2 parts ; 
Sodium sulphate 2 " 
Water 100 " 
To this mixture are added from five to ten parts of a 
forty-per-cent, solution of formalin. After the specimen 
has become sufficiently hardened, pour off the formalin 
solution, wash the specimen in ninety-five-per-cent, alcohol, 
then keep it in ninety-five-per-cent, alcohol until the blood- 
" that for the sake of consistency the same method of expression ought 
to be used for alcohol-i. e., ninety-five volumes of alcohol and five 
volumes of water. These expressions seem to us unnecessarily cum- 
bersome, and as they are in no way more precise or less ambiguous to 
one familiar with the meaning of per cent, than the expressions we 
used, we prefer them." A RESUME OF THE USES OF FORMALIN. 
5 
color becomes restored, and finally preserve it in a mixture 
of equal parts of glycerin and water. 
Fish makes objection to the use of formalin as a perma- 
nent preservative on account of the large amount of water 
present, which might cause freezing, and advises the addi- 
tion of an equal volume of alcohol. Hodenpyl,* in using 
formalin for making sections on the freezing microtome (see 
below), found that the least trace of formalin left in the 
specimen prevents its freezing. It would therefore seem 
that Fish's objection is not valid. 
Koehler and Lumibre found that if from fifty to a hun- 
dred and fifty cubic centimetres of a solution of one volume 
of formalin diluted with four volumes of water were injected 
into the gastro-intestinal canal of small animals by the 
mouth and anus, also into the carotid artery, and the ani- 
mal was kept hung up in the air, in a dry place, for some 
weeks, it was perfectly preserved without distortion. They 
performed an autopsy on an animal-a guinea pig-treated 
in this manner, four months after, and found the tissues 
and organs perfectly preserved. Dr. Henry Power f has 
treated the bodies of children in a manner similar to this 
with good results. 
Professor George S. Huntington informs me that he has 
used formalin for the preservation of organs. He injects a 
solution of from two to twenty-five per cent, into the blood- 
vessels, and the result is a perfect preservation of the form 
and color of the organ. He has found that it is of no use 
for preserving dissecting material. 
For the preservation of brains formalin has given excel- 
lent results. The fresh brain is placed in a ten-per-cent, 
solution, and at the end of ten days it will have sufficiently 
hardened to permit of the making of thick sections for 
demonstration of the gross anatomy, the distinction be- 
* Personal communication. f Ibid. 6 
A RESUME OF THE USES OF FORMALIN. 
tween the white and gray matter being more sharply defined 
than when alcohol is used. 
Parker and Floyd confirm the observations of Lanzi- 
!otti-Bu>msanti, Hoyer, Hoffer, and others, in regard to the 
swelling of the brain when formalin alone is used. In a 
sheep's brain they found this swelling to be forty per cent, 
•of its original volume. In order to correct this defect they 
■experimented with various reagents in combination with 
formalin. They finally found that a mixture of six volumes 
of ninety-five per-cent, alcohol and four volumes of a two- 
per-cent. solution of formalin gave nearly perfect results. 
Sheep's brains hardened in this mixture retained their origi- 
nal color and form, and were very little increased in volume. 
" A brain that before treatment (June 20th) measured one 
hundred and one cubic centimetres, when finally prepared 
(July 15th) measured one hundred and three cubic centi- 
metres." 
Fish states that an excellent hardening of the brain may 
be obtained with the following mixture : 
Water 2,000 c. c.; 
Formalin 50 " 
Sodium chloride lOOgrms.; 
Zinc chloride 15 " 
The specific gravity should be about l"05. The brain 
is left in this mixture for a week or ten days. The blood- 
vessels and cavities should be injected with the fluid if pos- 
sible. After the end of the ten days the brain is trans- 
ferred to formalin, 50 cubic centimetres, and water, 2,000 
cubic centimetres, where it may be kept indefinitely; or, 
after being a week in this fluid, it may be first transferred 
to fifty-per-cent., then to ninety-per cent., and finally to 
ninety-five-per-cent, alcohol. He has also treated portions 
of the adult central nervous system by this method, and A RESUME OF THE USES OF FORMALIN. 
7 
afterward with mercuric chloride, picro-aceto-sublimate, 
and chromacetic-acid mixtures, with good results. 
For hardening eyes Leber used formalin mixed with 
water in the proportion of one to ten. The natural color 
and transparency of the organ were retained. The cornea 
and lens became but slightly cloudy. In his opinion, the 
fine structure was as well preserved as with Muller's fluid. 
If the eyes were placed in alcohol the cornea and lens be- 
came opaque. I have employed formalin in a five-per-cent, 
solution for this purpose with the same results. 
As a hardening agent for microscopic work, formalin 
has been used very extensively, the strength of the solutions 
employed varying from one per cent, to the full strength- 
forty per cent. As the results of many observations, it 
may now be said, with possibly one or two exceptions, that 
formalin alone is an unfit reagent for hardening tissues for 
microscopic work. It was condemned by Hermann in 1893 ; 
Lachi states that it has an injurious effect on connective 
tissues, smooth and striated muscle, and embryos. Many 
other observers condemn its use without being so specific 
as Lachi. 
The exceptions, where it gives satisfactory results, are 
mucous membranes and the central nervous system. I 
have used it in five-per-cent, solution for hardening cystic 
adenoma of the ovary, with good results; also for the mu- 
cous membrane of the uterus. 
Lachi, who has condemned its use for all other tis- 
sues, speaks well of its action on the central nervous 
system. 
Van Gieson has employed it in four-, six-, and ten-per- 
cent. solutions for hardening the central nervous system. 
The ganglion and nerve fibres were well fixed. Sections 
stain well with Weigert's haematoxylin method. He has 
also used it for hardening the central nervous system for 8 
A RESUME OF THE USES OF FORMALIN. 
after-staining with Rehm's modification of Nissl's method. 
The results were good, but not quite so sharp as with alco- 
holic hardening. 
The best results for microscopic work are obtained when 
formalin is combined with other fixing reagents. When it 
is used in combination with the chrome salts more rapid 
penetration is obtained, whereby the time required for hard- 
ening is shortened. I have used a solution of formalin in 
Muller's fluid made as follows: 
Potassium dichromate 2 grammes; 
Sodium sulphate 2'5 " 
Two-per-cent, solution of formalin.. 100 c. c. 
With this fluid I have obtained excellent preservation of 
the ovary, the uterus, etc. At the end of forty-eight hours 
the specimen is cut into slices an eighth of an inch thick; 
these are washed in water for two hours; they are then 
placed in alcohol for twelve hours, and then carried through 
the usual processes of imbedding in celloidin. Specimens 
hardened in this manner show no shrinkage, and the tissue 
elements are well preserved. 
Landowsky recommends the following fixing fluids for 
mitotic figures in cells : 
1. Water 20 c. c.; 
Alcohol (ninety-five per cent.).... 10 " 
Formalin 3 " 
Hydric acetate 0'5 " 
2. Water 30 c. c.; 
Alcohol (ninety-five per cent.).... 15 " 
Formalin 5 " 
Hydric acetate 1 " 
Probably the most successful use of formalin in histo- 
logical technique is its substitution for osmic acid in the 
osmium-dichromate fluid used in Golgi's silver method for 
the central nervous system. A RESUME OF THE USES OF FORMALIN. 
9 
This substitution was probably first made by Dr. O. S. 
Strong, though it has been recommended by Lachi and 
others. Strong employs the following mixture : 
Potassium dichromate (3'5- to five- 
per-cent. solution) lOOvols.; 
Formalin 2'5 to 5 vols. 
After the specimen has been in the solution for several 
days it is transferred to a one-per-cent, silver-nitrate solu- 
tion ; or at the end of two days it is transferred from the 
formalin-dichromate mixture to the following: 
Potassium dichromate (five-per-cent. sol.)... 2 vols.; 
Formalin 1 vol. 
After remaining in this fluid for from twelve to twenty-four 
hours it is placed in the silver solution. The advantages 
of this method are, that the stage of hardening is pro- 
longed, the stage favorable to impregnation lasts longer, 
and the results are more certain. For embryonic tissue 
he does not consider it as good as the osmic-dichromate 
mixture. 
Fish has used the above-described method, but thinks 
he has obtained better results with the following: 
Muller's fluid 100 c. c.; 
Formalin (ten per cent.) 2 " 
Osmic acid (one per cent.) 2 " 
Strong has also used formalin as an injection medium for 
hardening brains in situ. He uses formalin diluted with 
an equal volume of water. This he injects into the cere- 
bral vessels until it runs out of the cut jugulars. After a 
few minutes he makes a second injection, then a third, and 
even a fourth, at intervals of fifteen minutes. The brain 
is then removed from the cavity of the skull. The swell- 
ing which usually occurs when formalin is used does not 
take place. Sections from brains hardened in this manner 
may be stained by either the Weigert or the Golgi method. 10 
A RESUME OF THE USES OF F011MALIN. 
When the Golgi method of staining only is to be used 
an equal volume of a ten-per-cent, solution of potassium 
dichiomate is added to the formalin in place of the 
water. 
Dr. T. S. Cullen has devised two methods for using 
formalin in connection with frozen sections. They are as 
follows: 
Method I. 
1. Keep sections made with the freezing microtome in 
a five-per-cent, aqueous solution of formalin for three to 
five minutes. 
2. Keep them in fifty-per-cent, alcohol for one minute. 
3. Keep them in absolute alcohol for one minute. 
4. Wash them in water. 
5. Stain them in haematoxylin for two minutes. 
6. Decolorize them in acid alcohol (1*5 per cent. HC1). 
1. Wash them in water. 
8. Stain them with eosin for twenty seconds. 
9. Place them in ninety-five-per-cent, alcohol. 
10. Pass them through absolute alcohol, clear them in 
creosote or oil of cloves, and mount them in Canada 
balsam. 
The blood being lost in the frozen sections, the defect 
was overcome by fixing the tissue in formalin, and then 
making frozen sections as in 
Method II. 
1. A piece of tissue 1X2X5 centimetres is kept in 
a twenty-per-cent, aqueous solution of formalin for two 
hours. 
2. Frozen sections are made. 
3. Keep them in fifty-per-cent, alcohol for three minutes. 
4. Keep them in absolute alcohol one minute. A RESUME OF THE USES OF FORMALIN. 
11 
5. Wash them in water and stain them in haematoxylin 
for two minutes. 
6. Decolorize them in acid alcohol (P5 per cent. HC1). 
7. Wash them in water. 
8. Stain them in eosin for twenty seconds. 
9. Place them in ninety-five per-cent, alcohol. 
10. Pass them through absolute alcohol, clear them in 
creosote or oil of cloves, and mount them in Canada balsam. 
Method I is used for diagnosticating bits from tumors, 
and it is possible to make a report in fifteen minutes. 
Method II is used mostly for the examination of uterine 
curettings. The author's practice is to have bottles con- 
taining a ten per-cent, solution of formalin in the operating 
room. The curettings are immediately placed in one of 
these, and by the time they reach the pathologist they are 
hard enough to make frozen sections of. 
Bender has also used formalin for making frozen sec- 
tions, not for preliminary hardening, as in Cullen's method, 
but for completing the hardening of specimens that have 
already been in alcohol. lie places pieces of tissues, two 
millimetres thick, that have been in alcohol, in a one-per- 
cent. solution of formalin, and keeps them there until the 
alcohol is completely removed. This requires from half an 
hour to an hour. He then washes them well in water and 
makes frozen sections. The tissue, he states, is rendered 
soap-like in consistence by the action of the formalin. 
Ohlmacher states that formalin, when used in from two- 
to four per-cent, solutions, acts as a powerful mordant for 
aniline dyes. Cover-glass preparations are treated for one 
minute with the solution, washed well in water, and then 
stained in the cold. Or it may be used instead of aniline 
oil or carbolic acid as a menstruum for dissolving the dyes. 
One gramme of fuchsine or other aniline dye is dissolved in 
ten cubic centimetres of alcohol, and this is added to one 12 
A RESUME OF THE USES OF FORMALIN. 
hundred cubic centimetres of a four-per-cent, solution of 
formalin. Formalin methylene blue, made by dissolving 
one gramme of methylene blue in one humdred cubic cen- 
timetres of a four-per-cent, solution of formalin, makes an 
effective stain. A saturated solution of safranin in a four- 
per-cent. solution of formalin gives a beautiful double stain 
when used after the formalin methylene blue. Nuclei stain 
blue, plasma stains reddish. 
S. II. Gage has used the following solution as a dis- 
sociating agent with good results : 
Normal salt solution 1,000 c. c.; 
Formalin (forty per cent.) 2 " 
Formalin has been used by Hauser for preserving plate 
and tube cultures of bacteria. His method is as follows: 
Plate cultures, in Peri's dishes, have a piece of filter paper 
placed under the cover, which has been moistened with ten 
to fifteen drops of formalin. The plates are then placed in 
a closed vessel in the bottom of which is laid paper or 
cotton saturated with foimalin. After twenty-four hours 
the cultures are fixed. Test-tube cultures are closed with 
a plug of cotton that has been wet with formalin and then 
placed in a closed chamber as above. After twenty-four 
hours they are removed and sealed with sealing wax, when 
a permanent preparation is obtained. Colonies from plate 
cultures may be permanently mounted by the following 
procedure: The selected colony is cut out of the plate and 
placed on a slide and covered, and then a little of the melted 
medium is run under the cover. The slide is then exposed 
to the action of the vapor of formalin for twelve hours. 
Formalin renders ordinary culture media, gelatin, and that 
fluidified by bacteria non-liquefiable by heat. The above- 
mentioned method of preserving bacteria has been employed 
successfully by Alleger, Cheesman, and many others. I 
am informed by Dr. Cheesman that cultures treated in A RESUME OF THE USES OF FORMALIN. 
13 
this manner by him a year ago are still well preserved, but 
some of the chromogenic forms have lost their color to 
some extent. 
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15 
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A RESUME OF THE USES OF FORMALIN. 
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schungsberichte aus d. Biol. Stat. Pion, iii, 1895, p. 209. The New York Medical Journal. 
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