Quality and Sensitifity of Presumtive Blood Test of Chicken (Gallus
gallus domesticus) Blood Using Leuco Malachite Green (LMG)
Nadia Yohana
1
, Bilqisthi Ari Putra
1
, Farah Aidah Nurreza
1
Djoko Legowo
2
1
Forensic Science, Postgraduate School, Universitas Airlangga, Jl. Airlangga No. 4-6, Surabaya, East Java, Indonesia
60115
2
Veterinary Pathology Department, Faculty of Veterinary Medicine,Universitas Airlangga, C Campus Mulyorejo,
Surabaya, East Java, Indonesia 60115
Keywords: Forensic, LMG test, blood, chicken
Abstract: Forensic Science is a branch of medical science that contains the application and utilization of science for law
enforcement and justice. Forensic laboratories have the task of supporting a component of case investigation,
identifying the case investigation component, the causes of death, known properties and signs for the benefit
of proof. The function of blood in the scene of the crime scene is to prove that it is blood of the victim or
blood that is not human blood. This research used Leuco Malachite Green (LMG) test. This study aims to
determine the quality of LMG reaction in chicken blood with different blood dilution. This research material
is LMG solution, chicken blood, aquadest solution. After blood is taken and dilution is performed 10-1 to 10-
5. The results of this study were obtained on the dilution of 10-1 obtained positive results (+), dilution 10-2
obtained positive results (+), dilution 10-3 obtained positive results (+), dilution 10-4 and dilution 10-5
obtained negative result (-). The conclusion of this study for the dilution of 10-1 obtained positive results (+),
dilution 10-2 obtained positive results (+), dilution 10-3 obtained positive results (+), dilution 10-4 obtained
negative results (-), and 10-5 dilution has negative results (-).
1 INTRODUCTION
Forensic Science is a branch of medical science
that contains the application and utilization of science
for law enforcement and justice (Idries et al, 2011).
Blood is probably the most commonly searched body
fluid in forensic casework (Takayama, 1912).
The Takayama or Hemochromogen test is used by
some forensic laboratories as a confirmatory test for
blood. The enhancement reagents often provide
valuable aid in crime scene analysis and laboratory
examination: in fact, they are able to detect minute
traces of blood even when attempts have been made
to wash away the incriminating evidence (Takayama,
1912). Leuco Malachite Green (LMG), leuko-crystal
violet (LCV), Lumiscene, Bluestar Forensics,
Fluorescein, and Luminol are widely used for
presumptive testing in casework at crime scene
investigations and in the laboratories. A presumptive
test will indicate if a biological substance such as
blood is present in a stain found. In this case, is the
stain found at a crime scene is blood or not (James et
al, 2005).
Chicken blood has larger erythrocytes than
mammals but it is smaller than mammalian
erythrocytes. The erythrocyte size of ungags
generally ranges from 10.7 x 6.1μm to 15.8 x 10.2μm.
In erythrocyte Chicken mature are elliptical shape
with a nucleus position in the middle (Bijanti, 2010).
Blood detection in animals in a study similar to
that of chicken blood has been performed on fish
blood using fluorescein test, Bluestar ©,
phenolphthalein, Hemastix®. The conclusion that can
be taken that fluorescein found the best results (ie,
low false positive rate, detecting the highest
proportion of the real positive). So it can be informed,
fluorescein testing can be further investigated to
improve its application in fisheries research. The use
of fluorescein, the injury can be detected up to 5 hours
after the injury occurred and the application of once
fluorescein, there is fluorescein detected much less
after one hour (Colotelo, 2009).
Yohana, N., Putra, B., Nureza, F. and Legowo, D.
Quality and Sensitifity of Presumtive Blood Test of Chicken (Gallus gallus domesticus) Blood Using Leuco Malachite Green (LMG).
DOI: 10.5220/0007543003650367
In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 365-367
ISBN: 978-989-758-348-3
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
365
2 MATERIALS AND METHODS
Materials:
Leuco Malacithe Green (LMG), H
2
0
2
, Pipette,
Chicken’s blood, Ice Container, Aquadest, Ethylene
DiamineTetraacetic Acid (EDTA), Syringe, Tube.
Sample:
Chicken’s blood (Gallus gallus domesticus) taken in
a brachial vein with a 3 cc syringe. Blood is inserted
in an anti-coagulant tube of Ethylene
DiamineTetraacetic Acid (EDTA). Then blood was
taken to Pathology Laboratory, Pathology
Department, Faculty of Veterinary Medicine,
Airlangga University for blood analysis.
Blood Dilution:
The blood was diluted with aquadest with dilution 10
-
1
, 10
-2
, 10
-3
, 10
-4
dan 10
-5
.
Figure 1: To Study Blood Detection Used Leuco Malachite
Green (LMG)
Reagen Test and Interpretation
Adapted from Andersson (2017), The LMG solution
was prepared by adding 150 ml of aquadest and 100
ml of concentrated acetic acid to 1 g of Leuco
Malachite Green into a brown chemical bottle. The
solution was stirred until all the Leuco Malachite
Green was dissolved and the solution was filtrated.
The prepared LMG solution was afterwards
transferred from the brown chemical bottle with LMG
solution into a smaller separate tube. A 30 % H
2
0
2
(MERCK®) was diluted with deionized water to a 10
% H
2
0
2
solution and the H
2
0
2
was then transferred to
a smaller separate tube. A volume of 25 μl of LMG
solution and 25 μl of H
2
0
2
solution was used
throughout this study. When not in use, all solutions
where kept in a refrigerator.
Methods:
Leuco Malachithe Green (LMG) and H
2
O
2
were
prepared according to Manufacturer guideline and
Forensic Laboratory of Indonesian’s Police
Department as well. All reagents were used according
to the manufacturer’s guidelines. Positive controls
were taken by applying the reagent to a bloodstained
piece of filter paper. Negative controls were
performed by applying the reagents to a fresh piece of
filter paper with no trace of blood.
Sensitivity Testing Autoclaved bottles and distilled
H
2
O were used. Water was measured using a
graduated cylinder and blood was added using a
Gilson pipette. Differing low concentrations of blood
were achieved by making a stock solution of blood
and distilled water. Solutions of 1:10
-1
; 1:10
-2
; 1:10
-3
;
1:10
-4
; and 1:10
-5
were prepared. A set of 35
3cmx3cm pieces of filter paper were placed in each of
the diluted blood solutions for each of the
presumptive reagents tested. The pieces of filter paper
were then removed and allowed to dry for 72h. Each
of the pieces of filter paper was then tested with its
corresponding reagent to see whether the blood
present was detectable. The reagents were added
directly to the 1 cm
2
pieces of filter paper. The time
taken for the reagent to register a positive result was
determined and recorded. Tests were considered
negative if reagents failed to react within 4min of
exposure to the blood-stained filter paper.
3 RESULT AND DISCUSSION
The results of this study were obtained on the dilution
of 10
-1
obtained positive results (+), dilution 10
-2
obtained positive results (+), dilution 10
-3
obtained
positive results (+), dilution 10
-4
and dilution 10
-5
obtained negative result (-).
Figure 2: positive results (+) of presumptive test using
LMG.
ICPS 2018 - 2nd International Conference Postgraduate School
366
Sensitivity
Blood
Dilution
10
-1
10
-2
10
-3
10
-4
10
-5
Chicken
+
+
+
-
-
The table above shows that LMG in Chiken’s blood
can still react to dilution 10
-3
.
Descriptive
N
Mean
Min.
Max.
Chicken
35
1.857
.0
4.0
Quality
Kruskal-Wallis Test
Dilution
N
Mean Rank
Chicken
10
-1
7
29.50
10
-2
7
27.50
10
-3
7
18.00
10
-4
7
7.50
10
-5
7
7.50
Total
35
All the data were analyzed by Kruskal-Wallis test
with significance p<0.05 for all groups. This study
resulted that the more dilution of chicken blood, the
more negative the LMG test results
The result of this study for the Blood Chicken
dilution of 10
-1
obtained positive results (+), dilution
10
-2
obtained positive results (+), dilution 10
-3
obtained positive results (+), dilution 10
-4
obtained
negative results (-), and 10
-5
dilution has negative
results (-).
Haemoglobin concentration can affect the quality
of the LMG test reaction. At higher dilutions it causes
a decrease in haemoglobin concentration. Based on
Swenson and William (1993), the amount of
haemoglobin chicken ranges from 6.50-9.00 g/dL. It
is estimated that haemoglobin can no longer be
detected by LMG at dilutions of more than 10
-3
, but
can be re-examined in the narrower range between 10
-
3
to 10
-5
.
4 CONCLUSIONS
The conclusion of this study for sensitivity test and
quality test the Blood Chicken dilution of 10
-
1
obtained positive results (+), dilution 10
-
2 obtained
positive results (+), dilution 10
-
3 obtained positive
results (+), dilution 10
-
4 obtained negative results (-),
and 10
-
5 dilution has negative results (-).
ACKNOWLEDGEMENTS
The author would like to thank Bilqisthi Ari Putra,
DVM, Djoko Legowo, DVM and Farah Aidah
Nurreza, DVM as partner team for this research.
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Quality and Sensitifity of Presumtive Blood Test of Chicken (Gallus gallus domesticus) Blood Using Leuco Malachite Green (LMG)
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