This Is an extremely delicate test. Kastle12 detected blood in a dilution
of 1 part in 80,000,000. Glaister 1S found that the reaction was instantaneous
in dilutions of 1 part of blood in 800,000 of distilled water in medico-legal
cases. It .has, however, got its limitations. Traces of copper salts give an
equally good positive reaction. The negative result is, therefore, valuable
and is conclusive as to the absence of blood.
4. Leucomalachite Green Test.—This test which was recommended
by "Adler in 1904 is quite useful, but it is not so sensitive as the benzidine
xtest. It depends upon the fact that leucomalachite green is oxidized to
malachite^ green with a bluish-green or peacock blue colour by hydrogen
peroxide solution. The reaction occurs also with a solution of the blood
pigment previously boiled. On the other hand, the reaction is negative when
iron is removed from haemoglobin forming hsematoporphyrin.14
The reagent is prepared by dissolving 1 gramme of leucomalachite green
in 48 cc. of glacial acetic acid diluted with double distilled water and is then
made up to 250 cc. A drop of this reagent is placed on the stain and after
a few seconds a drop of hydrogen peroxide solution (3 to 3.6 per cent) is
added, when the characteristic colour of malachite green appears if blood is
present. It is not affected by those substances which interfere with the
These four chemical tests are based on the fact that peroxidase present
in haemoglobin acts as a carrier of oxygen from the hydrogen peroxide to
the active ingredients of the reagents (guaiacum resin, benzidine, phenol-
phthalein and leucomalachite green) and produces the characteristic coloured
compounds by oxidation. Oxidase and peroxidase are also present in all
animal cells, but they are destroyed by boiling, while the peroxidase of
haemoglobin is not affected by such treatment.
It is suggested that before applying these chemical tests the luminescence
test be used as a preliminary test for detecting small obscure blood stains
mixed with rust, mud, earth, ashes, oil, paint, fruit juices, etc., or changed
by weather, temperature and age. A few drops of a solution containing
either 3-amino-phthalic acid-hydrazide-hydrochloride 1 g., sodium peroxide
5 g. and distilled water 1,000 ml. or 3-amino-phthalic acid-hydrazide-
hydrochloride 1 g. sodium carbonate 50 g., hydrogen peroxide (10 vol.) 50 ml.
and distilled water 1,000 ml. are .sprayed on a blood stain, when a distinct
bluish-white luminescence is clearly visible in the dark. It is claimed that
this test is specific for haematin of blood,13 but Naidu and Pitchandi 1G are of
opinion that it is not specific for blood, as the reagent used for this test gives a
positive reaction with copper sulphate, verdigris, cobalt chloride, cobalt sul-
phate, bleaching powder, etc.
Microscopical Examination.—This is useful not only for the detection of
the red blood corpuscles but also for the recognition of pus cells, epithelial
cells, bacteria, faecal matter, etc., which are, sometimes, found mixed with
blood in^the suspected blood and other stains. 5fee~ presence of squamous
_egithelial cells from the vagina or columnar cells of the uterus in blood stains
may indicate the menstrual source~bf the blood. Similarly, the ^epithelial
cells of the respiratory tract with a large number of pus cells or food parti-
cles with sarcinae and other bacteria will help the examiner in expressing
an opinion as to the gastric source of the blood. The size, appearance and
other histological features of the red blood corpuscles may also reveal the
origin of the blood. Skill in micrometry is, therefore, essential for such work.
12. Bulletin, 51, Hygiene Laboratory, U.S., 1909.
13. Brit. Med. Jour., April 10, 1926, p. 650.
14. Allen, Commercial Organic Analysis, 1933, p. 33.
15. Me. Grath, Brit Med. Jour., Aug. 8, 1942, p. 156.
16. Jour, and Proc. Inst. Chem., Vol. XV, Sep. 1943, p. 94.