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Haemin Crystal Practical Preparation
Haemin Crystal Practical Preparation
EXERCISE 1
PREPARATION OF HAEMIN
CRYSTALS
Structure
1.1 Introduction 1.5 Observations
1.4 Procedure
1.1 INTRODUCTION
Haemin or haemin (Ferric chloride heme) is an iron-containing porphyrin with
chlorine that can be formed from a haem group, such as haem group in
haemoglobin of human blood. The purpose of this exercise is to enable you to
understand how can we prepare crystals from the haem part of the
haemoglobin.
Objectives
After completing this exercise, you will be able to:
Preparation of Nippe’s Reagent: Dissolve 100 mg KCl, 100 mg KBr and 100
mg KI in 100 mL of glacial acetic acid. 5
BZYCL-136 Physiology and Biochemistry: Laboratory
1.3 PRINCLIPLE
Haemoglobin is present in RBCs which imparts red colour to the blood and
helps in the transport of gases. Haemoglobin is a conjugated chromoprotein
which is composed of two parts- non-protein haem and protein globin. Haem is
made up of four pyrrole rings and a central iron ion in ferrous state.
The haemin crystals are prepared by heating of the blood with Nippe’s
reagent. This ruptures the RBCs and the haemoglobin is released. Also, in this
process the ferrous form of iron is converted to ferric form. The globin protein
gets denatured by heating with acetic acid; while, heme is converted to
oxidized heme called haematin. The haematin combines with halogens such
as chloride ions to form insoluble haemin which appear as rhombic crystal of
chocolate brown colour. The haemin crystals or hydrochloride of heme (Fig.
1.1) are characteristic of blood and are thus, used for identification of blood
stains.
1.4 PROCEDURE
1. Sterilize the tip of finger with cotton swab soaked in 90% alcohol.
3. Place a drop of blood on a clean slide and spread it to form a thin film.
4. Add 2-3 drops of the Nippe’s reagent on the blood and place a cover
slip.
5. Gently heat the slide over low flame until the reagent starts boiling.
6. Remove the slide from the flame and add few drops of Nippe’s reagent
from the side of the coverslip. Heat it again for few more seconds.
7. Cool the slide and observe under the microscope, first under low
6 magnification (10x) and then under high magnification (40x).
Exercise 1 Preparation of Hemin Crystals
1.5 OBSERVATIONS
Rhomboid, chocolate brown crystals as shown in Figure 1. 2. are observed.
Porphyria is a group
Crystals are homogeneous solids, bounded by plane faces and having a of diseases in which
geometric shape. substances called
porphyrins build up,
A lyophilized form of haemin is used as a pharmacological agent in certain negatively affecting
cases for the treatment of porphyria attacks. the nervous system.
1.6 DISCUSSION
Haemin crystals are used in medico-legal practices to distinguish fresh or
dried blood stains from other red-coloured stains. The shape of haemin
crystals varies in different species and thus, blood stains of human can be
confirmed. Another advantage of the haemin test is that it can be performed
with the dried blood stains.
1.7 PRECAUTIONS
• Clean the finger with alcohol before pricking.
Acknowledgement of Figures
Fig.1. 2: (a) Self-Clicked
(b) https://www.flickr.com/photos/internetarchivebookimages
/14780801025