PRACTICAL OF IMMUNOLOGY (Lab-3)

SUBMITTED BY

AZIMA FATIMA, 008

Msc Biotechnology, 2nd Sem

Submitted to

Dr. Rizwanul Haque

Associate Professor

Session-2019-21

Central University Of South Bihar

Gaya

[1]
CONTENT- * EXPERIMENTS

1. To separate serum from blood…………………………………….. 3-4

2.To separate plasma from blood sample………………………………5-7

3. Estimation of the percentage of live and WBCs in the blood sample after
RBCs lysis using trypan blue…………………………………………8-10

4.Lysis of RBC in the blood sample…………………………………….11-13

5.Identification and estimation of the percentage of live and dead cells in the
blood sample using trypan blue…………………………………………14-16

6.Differential staining of the cells in the blood sample using Wright- Giemsa
staining method………………………………………………………….15-19

[2]
Experiment no.-1

AIM:- To separate serum from blood.

REQUIREMENTS:- centrifuge test tube, test tube stand, eppendorf,

alcohol, cotton and blood sample.

THEORY:- the separated serum from blood contains particles or solute

either in the form of protein or cell which are present in different
states. When the centrifuge force is applied the particles in the sample
starts settling down and finally settle at the bottom in the form of
pellet. This occurs when force of gravity overcomes the force of
diffusion.
PROCEDURE:-
i. Took blood sample in centrifuge tube and kept it at room temperature.
ii. Centrifuged the tube at 1000 rpm for 5 minutes at25oC.
iii. Took out the tube along with supernatant.
iv. Separated the supernatant (yellow coloured serum from blood).
v. Collected serum in a separate tube.

Observation:-
After the centrifugation, yellow color fluid was obtained, i.e., serum
which was collected in separate tube.

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PRECAUTIONS:-
i. Balance the centrifuge carefully and set the rotation accordingly.

[4]
 Experiment no.-2

AIM:- To separate plasma from blood sample.

REQUIREMENTS:- Centrifuge test tubes, test tube stand, alcohol,

cotton, syringe, heparin as anticoagulant, blood.

THEORY:-Plasma is the colorless watery liquid of the blood and lymph

that contains no cells but in the blood cells i.e. erythrocytes, leucocytes
and thrombocytes are suspended. It makes up approx. 55% of total
blood volume. It is made primary of water with small amount of
minerals, salts, ions, nutrients and protein in solution the large variety
of protein including albumin, immunoglobins and clotting proteins such
as fibrinogen are present in it. It is the main contribution to osmotic
pressure in it. the blood coagulated, collected and is centrifuge by
adding few drops of anticoagulant i.e. heparin at room temp and
plasma is separated to anticoagulant to blood doesn‘t clot the cells
including RBC‘s, WBC‘s, platelets and other clotting factors. It is the clot
that makes the difference between serum andplasma.
PROCEDURE:-

1. The blood sample was taken by 5ml. Now put in centrifugetube,

added few drops of heparin.

2. The centrifuge tube that were put in the centrifuge and tubes
were centrifuged at 2000 rpm for 10min.
3. The tubes were then taken out and clearly visible out light
yellow fluid were taken in another tube with help of pipettes.
4. The supernatant separated from blood settle at bottom.
5. kept the supernatant in another tube at 4oC for further use purpose.

Observation:-
The separated supernatant at the above in the tube was observed which
was plasma.

PRECAUTIONS:-
Centrifuge should be properly balanced and rotations should be set
accordingly.
 Experiment no.-3

AIM:- Estimation of the percentage of live and dead WBCs in the blood
sample after RBCs lysis using trypan blue.

Requirements:- hemocytometer, pipette, microscope, coverslip, trypan

blue, ammonium chloride (0.85%), nacl(0.9%), blood sample, centrifuge
machine, tip box, ethanol, tissue paper, falcon tube, eppendroff tube.

Theory:- The trypan blue dye exclusion test is used to determine the
number of viable cells present in a cell suspension. It is based on the
principle that live cells possess intact cell membranes that exclude
certain dyes, such as trypan blue, eosin, or propidium, whereas dead
cells do not. A hemocytometer consists of a thick glass microscope slide
wit a grid of perpendicular lines etched in the middle. The grid has
specified dimensions so that the area covered by the lines is known,
which makes it possible to count the number of cells in a specific
volume of solution.WBC count is performed with a Neubauer
hemocytometer. Using the x10 microscope magnification, count WBC
using the four outer large squares on the outer sections of the counting
chamber.

procedure:-

i. Took the 0.5 ml blood in the centrifuge tube from thetube.

ii. Added 4.5 ml ammonium chloride(0.85%) in that centrifuge tube
made it the volume 5ml in 15ml falcontube.
iii. Mixed the contents by inversion for 10 minute at room
temperature until the liquid was not clearlymixed.
iv. It must be diluted 1:10 at roomtemperature.
 v. Left the falcon tube for 10 minute before put in centrifuge
machine.
vi. After that centrifuged at 1250 rpm for 5min.
vii. Discarded the supernatant and added the ammoniumchloride.
viii. Maintained the total volume5ml.
ix. Repeated the above method 4-5times.
x. Then discarded the supernatant and observed whitepellet.
xi. Then added 1 ml of 0.9% Nacl in pellet and mixedit.
xii. Then took out 10սl sample and added 10սl trypan blue in
eppendrofftube.
xiii. Mixed itwell.
xiv. With the help of micropipette transferred a small amount (10ul)
of the sample in the eppendroff tube with a small amount (10ul)
of the trypan blue to a chamber on thehemocytometer.
xv. This was done by carefully touching the edge of the cover slip
with the pipette tip and allowing the chamber to fill by capillary
action.
xvi. Covered the hemocytometer with coverslip.
xvii. Observed the live and dead cell under the microscope at
10x,40x,60x.
xviii. Count cells within one large grid (“A” with very small squares). It is
easiest to move to one of the corner grids (A,B,C,D) and increase
the magnification of the microscope so the grid takes up the
entirespace.
xix. Count the viable cells (opaque) in the upper left 1-mm square grid
(“A” with 16 very small squares). Repeated with each corner grid
A,B,C,D so that we had four numbers. Average thenumbers.
xx. Did the same but only count dead (blue)cells.
Observation:-

Those cells which appeared purple/blue in color under the microscope

were dead cells and those appeared transparent and opaque in shape
were live/viable cells.

Calculations:-

Calculate the viable cells per ml using the formula-

The average of cells in one grid x 10000x dilution factor = number of

cells/ml.

Calculate the percentage of viable cells by dividing the viable cells in

one grid by the viable+dead cells in one grid and multiply by100.

In the first box, the total dead WBC had 255, in second box-269, in third
box-279, and in the last forth box-379

The total dead cell number= 1182,

Average dead cell number= 295.5,

The total dead cell number= 295.5 x 1000 x 104

= 295.5 x107cells/ml.

Precaution:-

i. Clean the coverslip and hemocytometer withethanol.

ii. Do not overfill or under fill thechambers.
iii. Do not directly pipette into thechamber.
iv. Carefully handle thehemocytometer.
v. 80% viability or less suggest cells are nothealthy.
[10]
 Experiment no.-4

AIM: Lysis of RBC in the blood sample

Requirement:- blood sample, centrifuge machine, tip box, falcon tube,

pipette, ammonium chloride (0.85%), nacl (0.9%).

Theory:- This method is applied to freshly collected whole blood

samples in order to isolate white blood cells for a number of
applications: total WBC DNA extraction, WBC culturing without red
cells` interference, and comet assays using comet chip. The basic
mechanism of hemolysis by isotonic ammonium chloride is as follows.
NH3diffuses freely through the cell membrane and increases the
concentration of intracellular OH-. OH-reacts with intracellular CO2to
form HCO3-. In red blood cells, the intracellular HCO 3-is exchanged with
the extracellular Cl-through the Cl-/HCO3-trans-membrane anion
exchanger of RBCs (a.k.a. Band 3 anion channel). The result is an influx
NH4Cl inside RBCs, which causes cellular swelling and eventually rupture
of the cell membrane. In this protocol, freshly collected whole blood an
by effectively hemolyzed after 3-5 minutes of incubation in a
homemade ammonium chloride hemolysis buffer at37oc.

Procedure:-

i. Took the 0.5 ml blood (using heparin or EDTA as the anti-

coagulant).
ii. Added the 4.5 ml ammonium chloride (0.85%) to maintain the
total volume 5 ml in 15 ml falcontube.
iii. It must be diluted 1:10 at roomtemperature.

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 iv. Mixed the contents by inversion for 10 minute at room
temperature until the liquid was not clearlymixed.
v. Left the falcon tube for 10 minute before put in centrifuge
machine.
vi. After that centrifuged immediately at 2000 rpm for 5minute.
vii. Decanted thesupernatant.
viii. Repeated the above procedure 4-5 times until the white pelletnot
shown at thebottom.
ix. Then added 0.9% nacl for further purpose orexperiment.
x. Stored at 15o-25oc for 1 month, 2o-8oc or{-15o-(-25oc)}.

Observation:-

The white pellet which was settled in the bottom of tube, were lysis of
RBC.

Precautions:-

i. Do not vortex.
ii. Repeated the procedure whenever the white pellet does notsee.
 Experiment:-5

AIM:- Identification and estimation of the percentage of live and dead

cells in the blood sample using trypan blue.

Requirements:- blood sample, trypan blue, pipette, tip box, tissue

paper, ethanol, hemocytometer, microscope, coverslip, eppendroff
tube.

Theory:- Very large numbers of RBCs are present in the Blood

specimen. Practically, counting this amount of Red Cells directly under
the microscope is highly impossible. So, the RBCs are counted by using
a special type of chamber, designed for the counting of blood cells in
the specimen, known as Hemocytometer or Neubauer`s chamber. For
this, the blood specimen is diluted (usually 1:100, 1:1000) with the help
of RBC diluting fluid (Hayem`s fluid, nacl) which preserve and fix the the
Red blood cells. The nacl, Hayem`s fluid is isotonic to the RBCs and
does not cause any damage to it. The Normal saline solutions can also
be used for this but it causes the slight creation of RBCs and allows
rouleaux formation which may cause the errors in results. After diluting
the specimen, the content is charged on Hemocytometer/ Neubauer`s
chamber and the cells are counted in the areas specific for RBC count.
Nowadays, two types of RBC diluting fluid are commonly used in
laboratories:-

 Hayem`s RBC diluting

 Fluidformalin citrate diluting fluid.

Procedure:-
 i. Made an appropriate dilutions of the red blood cells 1:1000
dilution of whole blood is adequate (using heparin or EDTA as an
anticoagulant).
ii. Took out 10սl of blood from it and added 10սl of trypan blue in
eppendroff tube.
iii. Cleaned the coverslip and hemocytometer with ethanol. Let it
dried.
iv. Placed the coverslip on the hemocytometer.
v. The solution allowed about 20սl to be taken under the slide on
each slide using capillary action.
vi. Immediately placed the hemocytometer on the stage of the
microscope and located the grids at low power.
vii. This was done by carefully touching the edge of the coverslip with
the pipette tip and allowing the chamber to fill by capillary action.
viii. Count the number of red blood cells, using the large center
square (5 square). This large square is divided into 25 small
squares. Count the red blood cells in 5 of these smaller (e.g. the
four corners and the centersquare).

Calculation:-

Cells/ml= number in 5 squares x 5x104/ml x 1/dilution.

Observation:-

Those cells which appeared purple/blue in color under the microscope

were dead cells and those which appeared opaque in shape and
transparent were live or viable cells.

Precautions:-

i. Do not overfill or under fill the chambers.

 ii. Do not directly pipette into the chamber.
iii. Carefully handle the hemocytometer.
iv. Before use the hemocytometer it must be clean alcohol.
v. 80% viability or less suggest cells are not healthy.
 Experiment.-6

AIM:- Differential staining of the cells in the blood sample using Wright-
Giemsa staining method.

Requirements:- slide, blood, coplin jar, Geimsa-stain, PBS, buffered

water, microscope.

Theory:- Geimsa stain is a differential stain and contains a mixture of

Azure, methylene blue, and Eosin dye. It is specific for the phosphate
groups of DNA and attaches itself to where there are high amounts of
adenine-thymine bonding. Azure and eosin are acidic dye which
variably stains the basic components of the cells like the cytoplasm,
granules etc. Methylene blue acts as the basic dye, which stains the
acidic components, especially the nucleus of the cell. Methanol act as a
fixative as well as the cellular stain. The fixative does not allow any
further change in the cells and makes them adhere to the glassslide.

Procedure:-

i. Took one drop of blood and left fordry.

ii. Then fixed the blood by making the thin film smear on theslide.
iii. Fixed air-dried film in absolute methanol by dipping the film in a
coplin jar containing absolute methanol, left for it 5minute.
iv. Removed and let air dry the slide to fix thecell.
v. Then stained with diluted Geimsa stain for 5 minute in coplin jar
containing Geimsastain.
vi. Removed and air dry theslide.
vii. Then pour the slide in coplin jar of PBS for 5minute.
viii. Removed and air dry the slide toneutralize.
ix. Washed by briefly dipping the slide in and out of a coplin jar of
buffered water.
x. Let air dried in a vertical position.
xi. Observed the cells under the microscope first at 10x, and then
40x.

Observations:-

Cell components Color observed after staining

Red blood cells Mauve-pink
Neutrophils Reddish purple nuclei with pink cytoplasm
Eosinophils Purple nuclei, faintly pink cytoplasm and red to
orange granules
Basophils Purple nuclei, blue coarse granules
Lymphocytes Dark blue nucleus with light blue cytoplasm
Monocytes Pink cytoplasm with a purple color nucleus
platelets Violet to purple color granules
Nuclei of host cells Dark purple

Precautions:-

i. Excessive washing will decolorize thefilm.

ii. Smear must bethin.
iii. Excessive stain may color the cell more. So do not leave the smear
in Geimsa stain for longperiod.