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354
A MICRO-CHEMICAL TEST .FOR CHROMATIN.
By H. lVI. WOODCOCK, D.Se.LoND.
Fellow of University Oollege.
(Medical Resea1'ch Oouncil grantee; Jenner Memorial Research student.)
(Li8ter In8titute of Preventive Medicine.)
THE best method for demonstrating chromatin hitherto available to
microscopic workers has been the classic one of the addition of dilute,
acidulated methyl-green to the fresh, un fixed cells. The same stain is also
very serviceable for material fixed in certain ways,as it is used in the
Ehrlich-Biondi-Heidenhain combination. But, elective (for practical
purposes) as is methyl-green, and though I have found it to be most useful
in the course of my own work, it has· one or two drawbacks, when the
question is not so much of staining, with a view to distinguishing nuclear
material the presence of which is known, as of determining whether a particular body or granule contains or consists of chromatin. In the first
place the method remains purely a staining process; it is not in the nature
of a precise microchemical test such as is available for the microscopic
. detection of iron, by means of the prussian-blue (or ammonium sulphide)
reaction. Secondly, when examining minute granules, suspected of being
chromatinic, one is sometimes in doubt as. to whether a particular granule
is faintly stained or whether an apparent greenish tint is due to all optical
effect; in such a case a method of staining which is also a microchemical
test is greatly needed.
I think, therefore, it is worth while to draw attention to a microchemical
reaction, recently described, which promises to be a 'most valuable aid in
the determination whether a body or granule does or does not contain·
chromatin. The method is known as Feulgen's nucleal-reaction, being
based upon certain reactions of thymonucleic acid observed by him some
years ago.' The method has since been further elaborated and experimentally tested by Feulgen and Rossenbeck (Zs. Physiol. Ghemie, 135,
1924, p. 202), and Feulgen-Brauns (P/lugel"s Al'chiv, 203,1924, p. 415), to
whose papers those interested may be referred for full details. The principle of the method depends on two chemical reactions. Firstly, by means
of partial acid hydrolysis, certaiu purin-bodies, guanine and adenine, contained in the purin-nucleotides of thymonucleic acid, are broken down, so
that reducing groups of carbohydrate-like constitution are set free. These
carbohydrate groups are, according to Feulgen, of aldehyde nature. This
is shown by a particular reaction given with fuchsin-sulphurous acid
(Schiff's reaction), which has been found (Wieland and Scheuing) to be one
1 In 1924, Feulgen published a paper (in German) on "New Methods for the Biological.
histological Study of Cell·nuclei," etc. (Ber. ges. PhY8iol., 22, p.489.)
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H. M. Woodcock
355
of the most infallible means of detecting aldehyde groups. By this reaction
a dye or colour-body is formed of a red to violet tint; redder when seen in
greater depth or thickness, more violet in tone when seen thinner tinder the
microscope. The reaction is of great specificity. Sugars, for instance, do
not give it, neither do pentoses, the . latter being a point of practical
importance in connexion with this new method (see below).
The details of the technique followed have varied slightly as improvements in it have been made. I give here the method adopted by FeulgenBrauns, who has made exhaustive experiments to ascertain the optimum
working conditions of both the reactions concerned. As regards the
hydrolysis, the optimum for all objects, animal and plant nuclei, whether
in sections or smears, is immersion in normal HCI, maintained at a
constant temperature of 60° C. for four minutes. The operation is best
performed in a beaker, heated over a micro-burner. 'l'he slides are then
quickly rinsed with distilled water and place a immediately in the fuchsinsulphurous acid solution. The mode of preparation of this reagent is as
follows: One .gramme of fuchsin (" Diamantfuchsin," Merck, or parafuchsin) is dissolved in 200 cubic centimetres of boiling, distilled water
(i.e., 0'5 per cent). The solution is cooled to 50 0 C., filtered, and to it are
added twenty cubic centimetres of normal HCI. After further cooling to
25 D C., one gramme of dry, chemically pure sodium bisulphite is added.
As the bisulphite is dissolved, the solution gradually loses its fuchsincolour and Ultimately becomes of a sligl. tly yellow tint. 'l'his reagent
should be kept in a well-stoppered bottle in the dark. It is important that
it should contain sulphurous acid in excess, otherwise the fuchsin tends
to be dissociated, and the liquid takes on a reddish tint. It may be noted
that the above composition of the reagent differs from Schmidt's formula
(" Pharmac. Chemie "-H, Org. Chemie, p. 344, 1922), in that less HCl is
used (0'36 per cent instead of one per cent), which is considered to be an
advantage. Feulgen-Brauns found that the optimum period for the action
of the fuchsin-sulphurous acid is, for all animal cell-nuclei, from one to
one and a half hours, and for those of plants about three hours. The
preparation is then well rinsed, either, if a thin section or film, with tapwater, or, preferably, especially if rather thick sections are concerned, .
with water containing S02 in solution. The composition of the S02 water
is given as follows: 200 cubic centimetres tap-water, ten cubic centimetres
sodium bisulphite, and ten cubic centimetres normal HCl The preparation, if a smear, can either be dried or treated as are sections in the usual
way, i.e., dehydrated with alcohol, cleared and mounted. The stain is
remarkably resistant and permanent.
According to the workers, this nucleal-reaction is "of a specificity
hitherto unknown" 'for chromatin; hence its great value. There is, however, one slight apparent difficulty, which will be mentioned below. There
is no danger of" overstaining." Indeed, as Feulgen-Brauns remarks, the
maximum stain-formation is the optimum, for when all the liberated
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356
A Micro-OheJnical Test jor Oh1'O'lnatin
aldehyde-groups are united with the fuchsin-sulphurous acid to form the
colour-body, lIO more can be formed. Hence, I would point out, as she
also llotes, that preparations can in some cases be left with advantage
much longer in the staining reagent, up to twenty-hours, for instance. I
have found that, in the case of muscle-cell nuclei, this longer period gives
better results. One extremely important point is that no damage to the
morphological character of the nuclei is caused by the hydrolysis, unless·
this is continued for a much longer time than is necessary. The resulting
microscopic appearance is that seen after ordinary staining with chromatinic
stains. As regards many types of tissue-cell nuclei, I calJ confirm this
statement. Another great advantage of this method is that there is no
possibility of mistaking iron-containing material or granules for chromatin:
. a mistake which may readily occur with one of the best of nuclear stains,
namely hrematoxylin.
As regards fixation, sublimate-acetic mixtures, and also Zenker's fiuid,
are noted as being very suita,ble. It is stated that material should not be
fixed in formalin, as this is itself of aldehyde character. But I have
obtained excellent results in the case of sections of nervous tissue, which
had been fixed in formalin. Since the hardening process, through the
changes of alcohol, removes all traces of tile formalin, I fail to see any
disadvantage in using this fixative. This brings me to the apparent
difficulty, commented on by Feulgen and Rossenbeck.They found that
sections of fresh material, on being cut with a freezing microtome,
showed a diffuse coloration in the cytoplasm after treatment with the
Ilucleal reagent. But if the sections were passed through the various
grades of alcohol and embedded in paraffin in the usual manner, no such
staining was evident after the treatment, the substance which produced it
having been dissolved during the prior procedure. The authors conclude
that there may be aldehyde bodies in the cytoplasm, which they regard as
representing products of the normal metabolism. To get rid of these
aldehyde bodies, they recommend leaving the sections for some time in
strong alcohol (96 per cent), before bringing down to water for the test.
Smears of blood, for instance, should he fixed in methyl-alcohol. In no
case should they be fixed by heat if this test is to be used (see also below).
Most unfortunately, this n llcleal reaction is not universally applicable.
Not all chromatin gives the reaction; but all chromatin which contains
thymonucleic acid. Strictly speaking, the method is, therefore, a test fo]'
tbymonucleic acid. Now, yeast-cells do not give the reaction, because their
chromatin does not contain thymollucleic acid, but a pentose-nucleic acid.
I have tried the reaction several times upon Saccharomyces cerevisice in the
hope that it might succeed, because I have been unable to obtain any good
evidenc,e of a discrete, properly constituted nucleus in yeast-cells. After
staining with methyl-green, there is a diffllse coloration, as in the case
of the ordinary bacteria which I have tried, alld I consider that the chromatin
is uniformly distribu·ted throughout the protoplasm of the yeast-cell.
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H. M. Woodcock
357
Purther, hitherto no thymonucleic acid had been found in the nuclei of
ordinary plant-cells. but now Peulgen and Rossenbeck have shown. by
means of this reaction, that some plant-cells, at allY rate, do contain
thymonucleic acid (e.g., the. cellcnuclei of a bean-.embryo). As regards
animal cells, muscle-cell nuclei have been found to contain thymonucleic
acid in addition to inosinic acid, hence the reaction is also well given by
their chromatin. Altogether very many types of alllmal-cell nucleus have
been investigated, with positive result, and it is probable that the method
may prove of general application in their case.
I should like to give, in conclasion, a few examples of the results of
this reaction, as they bear upon work which I have done alld my conclusions resulting therefrom. Peulgen and Rossenbeck state that they could
not obtain the reaction in the case of bacteria, or in that of trypanosomes,
but they obtained it in the case of ciliJ.tes. Certainly some bacteria do
give it, as I accidentally ascertained. The test was tried by me on the
spores of Glugea lophii. At the first time of trying, the only material
which happened at the moment to he available was an emulsioll in saline
of a ripe spore-containing, cystic mass, which had been left standing some
little time and had become contaminated. Smears were made, and as a
result of the nucleal reaction the bacteria were definitely pinkish in colour,
while there was no sign of colour in the spores. Miss Robertson kindly
?,llows me to say that, similarly, the bacteria in her cultures of Bodo also
give the reaction quite well. Again, as regards trypanosomes, Bresslau
and Scremin (Arch. Protistenk., 48, 1924, p. 509) have found that both the
nuclear elements give the reaction, which is, indeed, particularly marl<ed
in the case of the kinetonucleus. These observations, I may also add, have
been confirmed by Miss Robertson, in the case of the forms of the skatetrypanosome in the leech (Pontobdella). Moreover, in the case of the freeliving binucleate, Bodo (cf. saltans) she also gets decisive positive results.
These observations afford, I consider, strong evidence in favour of the view
originally taken by Schaudinn, with which the late Professor Minchin and I
always agreed, that the conspicuous, intensely staining body near the origin
of the fiagellum in the trypanosomes and allied forms is, in fact, of nuclear
character; and Bresslau and Scremin also regard this point as now settled.
Hence the terms of tropholll;lCleus and kinetonucleus, which I originally
proposed for the two nuclear bodies in the binucleates, are seen to be completely justified. Bresslau and Scremin consider that the failure of Peulgen
and Rossenbeck to obtain a successful, positive result was due to insnfficient
duration of the hydrolysis; they found that, while hydrolysis for four
minutes was unsuccessful, if the process were continued for six or seven
minutes a positive result was obtained: Miss Robertson has suggested to
me that the failure may also be in part due to fixation of the smears by
heat, and this may apply to their failure in the case of bacteria also.
Heating may alter the chemical composition of the nuclear material.
Purther, in a recent paper (A1'Ch. PToi'istenk., 52, 1925, p. 394), Thiel
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358
A MicTO-Chemical Test for ChTomatin
states that he has' tried the nucleal reaction upon Rickettsia rnelophagi,
with entirely negative results. Unfortunately, Thiel does not say whether
his smears, were fixed by heating or otherwise before applying this test,
although, in connection with another point, he fixed them in formalin.
And, in any case, until rl10re is known as to whether bacteria in general
give the reaction, i.e., contain thymonucleic acid in their chromatin, this
negative result cannot be regarded as a proof of the absence of chromatin.
Still, taken in conjunction with my failure to stain another" Rickettsia"
(R. pediculi) with methyl-green, I think Thiel's observation is of considerable importance. [Vide also letter to Editor (p. 397) in this connection].
Lastly, ill the course of my present work upon G. lophii, of the anglerfish, in relation to Encephalitozoon, and upon Sarcocystis, I have tried
the nucleal reaction in all these cases, and in none of them have I been
able to obtain a positive result. In the case of G. lophii, not only the
spores have been tried, but sections of young cysts, containing much
material that is not yet of the nature of spores. And that the reaction
was carried out successfully is shown by the positive result in the case of
the cell-nuclei of the surrounding nervous tissue. I will not say more upon
this question here, beyond remarking that, in some smears of a true
Microsporidian, Thelohania sp., from the' larva of a mosquito, kindly
given me by Dr. Wenyon, no difficulty was experienced ill obtaining the
nuclear material well stained, both in the early, spore-forming stages and
in the spores themselves.
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A Micro-Chemical Test for
Chromatin
H. M. Woodcock
J R Army Med Corps 1926 46: 354-358
doi: 10.1136/jramc-46-05-04
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