from the American Journal of the Medical Sciences for January, 1874.] 
ON THE GUAIACUM PROCESS 
FOR THE 
DETECTION OF BLOOD AS A VALUABLE AID IN DISTINGUISHING 
NUCLEATED FROM NON-NUCLEATED RED BLOOD DISKS. 
By R. M. BERTOLET, M.D., 
MICROSCOPIST TO THE PHILADELPHIA HOSPITAL. 
The large size, elliptical shape, and the central oval nucleus, charac- 
terizing the red blood disks of birds, fishes, and reptiles, are generally 
considered as ample land-marks to enable even the merest novice in micro- 
scopy to distinguish the blood of any animal belonging to any one of 
these three great classes from the flat, biconcave, non-nucleated disks 
characterizing human and mammalian blood. Such is the case, at least, 
as far as fresh specimens are concerned, but it is far from an easy matter 
to discriminate between these two kinds of blood when they have once 
become clotted and thoroughly dried. What great changes this process of 
drying implies ! The corpuscles are no longer flat and smooth, but 
shrunken and crenated ; their outlines are no longer regular and sharply 
defined, but irregular, jagged, or stellate; they are so intimately aggluti- 
nated with one another, and hidden in the meshes of the coagulated 
fibrin, that it becomes difficult, even in thin fragments, to recognize them. 
When blood is very thinly spread out and rapidly dried upon a glass 
slide, then the disks remain firmly stretched and the diameters are not 
materially altered;1 but when left to dry in any other manner, the great 
shrinkage and other alterations in the corpuscles just enumerated take 
place. Under these circumstances it becomes an extremely difficult task 
1 Vide Strieker, Gewebe Lelire, Leipsig, 1871, p. 280. 2 
to decide which of the two kinds of blood we have to deal with. The 
size and shape of the dried corpuscles are not to be depended upon ; to 
resuscitate them exactly to their original condition by the imbibition of 
fluids is an impossibility, especially so when irrigation with pure water 
is resorted to, as recommended in Wharton and Stille's Medical Juris- 
prudence, § 758, 3d ed., 1873. Distilled water not only completely 
abstracts the colouring matter from the red corpuscles, so that the author 
of the article on “ Microscopic Evidence” found it necessary to render 
them “ more obvious” by adding iodine or red aniline solutions, but it 
also finally causes both the colourless and coloured disks to disintegrate 
and eventually to disappear.1 These are sufficient reasons to condemn 
the use of water in the examination of dried blood. There are other 
less objectionable fluids that may be employed—the sulphate of soda in 
solution is excellent; I have found none, however, so admirably adapted 
as slightly acidulated pure glycerine. It does not evaporate, so that 
specimens mounted in it can be kept an indefinite time for study. Its 
slight acidity will render the nuclei more distinctly visible when present. 
How rarely, though it must occur in medico-legal cases, that the micro- 
scopic expert has given to him blood that has not already been more or 
less completely dried. In very many of these cases it is of the utmost 
importance to determine which of the two kinds of blood it is, and the 
determination of the presence or absence of the nuclei in the red corpus- 
cles is of more importance than all the other features possessed by the 
different bloods, frequently virtually deciding the guilt or innocence of the 
accused. 
The addition of serum-like and other liquids to small fragments of the 
questionable blood, mounted beneath the microscope, is not to be neglected, 
yet very unsatisfactory, for as yet there is no liquid known which will 
precisely restore the size and shape of the once dried corpuscles. Those 
from the mammalian animals can be made to swell out, but become circular 
in outline instead of biconcave as seen in the fresh state; while the 
elliptical disks become more nearly round and their outlines pale and 
indistinct. Hence the great importance attached to the nucleus, for it 
frequently is the only distinctive mark between the two kinds of blood. In 
cases where the blood stains are alleged to be those of some animal having 
the nucleated disks, it behooves the examiner to use every exertion to 
develop, if possible, the nucleus. 
Although few experienced microscopists would hesitate in pronouncing 
upon the existence or non-existence of a nucleus in any given corpuscle, 
it is an extremely difficult matter to establish the fact in the minds of the 
jury, and still more difficult to demonstrate it clearly to those who are 
unaccustomed to making microscopic observations. 
1 Dr. Kline, in Burdon-Sandersou’s Handbook for tlie Physiological Laboratory 
1873, p. 29. 3 
Any method, therefore, which will enable the examiner to demonstrate 
without the shadow of a doubt, not only in his own mind but also to a 
jury, the presence or absence of the nucleus cannot fail to be accepted as 
a most valuable corroborative proof. Such a method for differentiating 
between the two kinds of blood, I believe is to be found in the well-known 
gnaiacum test for detecting blood stains. 
If a microscopic preparation, mounted in feebly acidulated glycerine or 
other suitable fluid, is carefully irrigated with a properly prepared alcoholic 
solution of guaiacum resin ; then when a very small quantity of the ethereal 
solution of the peroxide of hydrogen (ozonic ether) is introduced beneath 
the glass cover, a bluing, varying from a light sapphire to a deep indigo 
shade, will be observed in the several corpuscles of mammalian blood. 
Though the degree of coloration of the different corpuscles presents con- 
siderable variety, yet all parts of the individual corpuscle itself are coloured 
uniformly. If the blood has been derived from an animal having nucleated 
corpuscles, an entirely different picture is presented; when treated by these 
same reagents, the nucleus is seen as a sharply defined, dark blue body, 
ivhile the protoplasm surrounding it assumes a more delicate violet hue. 
Sometimes the other parts of the blood-globules are not the least coloured ; 
in all instances, however, the nucleus is deeply stained and easily recog- 
nizable when present, even by the uneducated eye. 
The charming picture, that can thus be produced by differentiating the 
nucleus from the other parts of the corpuscles, may be of invaluable service 
where it is desirable to convince the jury by optical proof of the existence 
of the nucleated blood-globules. Furthermore, in those cases where the 
blood is alleged to pertain to an animal having the nucleated disks 
and found not to be such by the ordinary methods of investigation, I be- 
lieve it to be the duty of the expert to employ this method of searching 
for the nucleus, as stated in my testimony at the trial of Talifero, at Mar- 
tinsburg.1 
The attention of the profession has not yet, as far as I can ascertain, 
been called to this, the ultimate application of the guaiacum process for 
the detection of blood. The original papers of Dr. Day, oLQeelong. Aus- 
tralia, who first established the reliability of the guaiacum process as a blood 
test, have been inaccessible to me, yet Dr. Alfred S. Taylor, to whom they 
were referred, and who has himself investigated with this process, states:— 
“ It simply enables the operator to say whether that which he is examining 
is the colouring matter of a red-blooded animal. It throws no light on the 
class of animal to which the blood belongs; it may be hot blooded or cold- 
blooded, a mammal or a reptile.”2 “So again the process does not 
enable the examiner to detect the blood-globules, or cells, or even to speak of 
their presence.” 
This is certainly an underestimation of its value, for with proper pre- 
1 Vide Martinsburg “Independent,” Nov. 22, 1873. 
2 Guy’s Uosp. Reports, vol. xiii. p. 440, 3d series, 1867. 4 
cautions the blue-coloured disks or blood-globules can be obtained with this 
process; the examiner is thereby enabled to solve the question whether 
the blood is human or from a bird, fish, or reptile ; but it affords him no 
light upon the question which so frequently arises, namely, whether the 
blood is human or from a mammalian animal. 
In the fresh condition it is a comparatively easy matter to see and 
manipulate, so to speak, the nucleus. By the addition of a two per cent, 
solution of boracic acid, the elliptical blood disk swells and acquires a cir- 
cular contour ;* the oval nucleus becomes more and more spheroidal and 
yellow, while, at the same time, it increases in size. Here and there cor- 
puscles are met with in which the yellow central body is not round, but 
beset with processes which stretch like rays towards the periphery ; this is 
especially the case when a tannic acid solution of the same strength is 
employed ;a the nuclei are apt to assume, also, a more eccentric position 
and after a sufficient length of time, the observer is sure to see many 
corpuscles in which they lie, some partly, some entirely outside of the 
outline of the pale disk. None of these phenomena are observed when 
dried blood is acted upon by these agents. Acetic acid* gives a beautiful 
red (hoematine) tint to the fresh nucleus. A neutral solution of carmine,4 
as well as aniline colours and other substances stain, more or less intensely, 
the disks and their nuclei, but none of them are quite as satisfactory as 
that produced by the guaiacum process. It may here be observed that 
ether, since it is employed in obtaining this blue colouration, has the 
effect upon amphibian5 and like blood of somewhat diminishing the size 
of the corpuscle, but, on the other hand, the thickness of the margins has 
increased and the nuclei are very clearly and sharply defined. 
Another reason, besides its superiority, for employing the guaiacum 
resin and the ozonic ether in order to colour the nucleus, lies in the fact 
that the examiner is not dealing with substances involving the use of 
additional strange technical terms, but with such that he has already 
thoroughly familiarized his audience with. For it is not only necessary in 
medico-legal cases that he himself be satisfied that he is dealing with blood, 
but he ongkrtT rr-iKlr1fr convince all that the matter under consideration 
is nothing else than blood. This can readily be accomplished, even with 
a small quantity of blood, by the exhibition of the oxidation of the 
guaiacum upon a white porcelain surface, or paper; by the production 
of haematin crystals, which also can again most readily be proved to be 
such by the same bluing process ; finally, it should be employed under the 
microscope to show the presence or absence of fthe nucleus. 
1 Dr. Kline, 1. c. p. 30. 
2 Boettcher, Virchow’s Archiv, Bd. 36, p. 342. 
3 Heule, Allgemeine Anatomie, p. 431. 
4 A. Rollett, Strieker’s Gewebe Lehre, p. 293. 
5 A. Rollett, Strieker’s Gewebe Lehre, p. 291.