Immunology and cell culture techniques

Sardar Hussain ,Asst.Prof.Biotechnology
GSC CTA [Company address]
LAB MANUAL ON
IMMUNOLOGY AND CELL
CULTURE

SARDAR HUSSAIN ,ASST.PROF.BIOTECHNOLOGY 1
1 Lab manual on Immunology and cell culture
Contents
Sl no Title Page No.
1 Study of Blood grouping 2-3
2 Study of Rh factor 4
3 Study of percentage of haemoglobin by Sahli’s Method 5-6
4 Differential leucocyte count 7-9
5 Enumeration of RBC by Neubars chamber 10-11
6 Enumeration of WBC by Neubars chamber 12-13
7 Erythrocyte sedimentation rate 14-16
8 Method of Determination of Packed Cell Volume (PCV) 17-18
9 Study of ELISA by kit method 19-22
10 Study of Ouchterlony double diffusion 23-26
11 Extraction of Plasma and Serum from blood 27-28
12 Basic cell culture techniques 29-31
13 Preparation of balanced salt solutions 32-33

Experiment no. 1: Blood grouping
Introduction: It was in 1901, that Austrian-American immunologist and pathologist Karl
Landsteiner discovered human blood groups. Karl Landsteiner's work helps to determine blood groups
and thus opened a way for blood transfusions which can be carried out safely. He was awarded
the Nobel Prize in Physiology or Medicine in 1930 for this discovery. Based on the presence of A and B
antigens in the red blood cells (RBCs), the blood has been categorized into four types: A, B, AB, O.
according to whether RBCs have antigen A, antigen B, or both A and B antigens or neither the antigens.
Type O blood could be used in any transfusions because it lacks both the antigens and the person
of this blood group type is called a universal donor. A person with blood type AB called a universal
recipient can receive any type of blood because it lack both antibodies A and B. Group O is commonest
group and group AB is the rarest in human population around the world.
ABO grouping is performed with antisera containing high titer of anti-A and anti-B antibodies by
slide or tube method, in which drop of each kind of antiserum is added to 2-3 drops of saline diluted blood
sample and observed for slide agglutination reaction.
Principle: ABO blood group system is based on principle of agglutination, type of reaction that occur
between particulate antigen and specific antibodies (agglutinins) that lead to clumping or agglutination
of cells. Here the cells involved in agglutination are RBC so the process is known as hemagglutination and
is used for matching blood cell types.
Requirements:
 Blood sample
 Antiserum A and antiserum-B
 Spirit or 70% alcohol
 Microscopic slides
 Blood lancets or needle (sterile)
 Toothpicks
 Cotton, marker.
Procedure:
 Take a microscopic slide and divide in half by drawing a line in the middle of the slide. Mark the
letter X on one side and Y on another side.
 Clean middle finger tip with cotton moistened with 70% alcohol and allow it to dry.
 Prick the finger with a sterile lancet.
 Place a drops of blood on the slide. ( X and Y)
 Add a drop of antiserum A to X and antiserum B to Y to the blood suspension.
 Using separate tooth picks, mix the antisera and blood, rock the slide back and forth for two
minutes between your fingers.
 Examine the two zones X and Y for clumping of RBCs and results are recorded.
Result: The blood group of the given blood sample is …………

Interpretation: a clear clumping of RBCs indicates positive agglutination reaction. Agglutination is
the result of clumping of RBCs and serum having antibodies to the antigen of the cell. A antibody
reveals A blood group, B antibody reveals B blood group, if clumping takes place in both A and B it is
AB blood group and if no clumping in both the A and B it is O blood group.
Fig: Shows various possible A, B and O blood group & Rh factors.

Experiment no: 2: Determination of Rh factor
Introduction: The Rh blood group system (including the Rh factor) is one of thirty-five current human
blood group systems. It is the second most important blood group system, after ABO. At present, the Rh
blood group system consists of 50 defined blood-group antigens, among which the five antigens D, C, c,
E, and e are the most important. The commonly used terms Rh factor, Rh positive and Rh negative refer
to the D antigen only. Besides its role in blood transfusion, the Rh blood group system—specifically, the
D antigen—is used to determine the risk of hemolytic disease of the newborn (or erythroblastosis
fetalis) as prevention is the best approach to the management of this condition.
Rh derives from rhesus and the terms rhesus blood group system, Rhesus factor, rhesus
positive and rhesus negative are also used.
An individual either has, or does not have, the "Rh factor" on the surface of their red blood cells.
This term strictly refers only to the most immunogenic D antigen of the Rh blood group system, or the
Rh− blood group system. The status is usually indicated by Rh positive (Rh+ does have the D antigen)
or Rh negative (Rh− does not have the D antigen) suffix to the ABO blood type. However, other antigens
of this blood group system are also clinically relevant. These antigens are listed separately. In contrast to
the ABO blood group, immunization against Rh can generally only occur through blood transfusion or
placental exposure during pregnancy in women.
Principle: Blood diluted with saline is not used in this test because the antibody used in the typing sera is
of the incomplete albumin variety and the dilution will stop the agglutination of RBCs.
The Rh antigen present on the RBCs reacts with anti-D or Rh antiserum resulting in the
agglutination of RBCs. The agglutination reaction between antibody and antigen results in finer clumps,
hence closer examination is essential.
Requirements:
 Finger prick blood sample
 Anti-D or Rh antiserum
 Glass slide
 Blood lancet
 Spirit , Spirit lamp
 Cotton and applicator stick
Procedure:
 Add a drop of anti-D serum on a clean glass slide.
 Add 3drops of blood on slide where antisera D was placed.
 Mix the blood with serum thoroughly with an applicator stick.
 Rock the slide back and forth for two mins.
 Examine the slide for macroscopic agglutination of RBCs.
Result: …………………………….. Agglutination observed.
Interpretation: A positive agglutination reaction between RBCs and Rh antiserum indicates that the
person is Rh +ve and no agglutination reaction between RBCs and Rh antiserum indicates that the person
is Rh –ve. The person having Rh antigen in the blood is designates as Rh+ve , the person having no Rh
antigen in the blood is designates as Rh-ve.

Experiment no. 03: Estimation of percentage of Haemoglobin.
Aim: Estimation of Haemoglobin with the help of Sahli's Haemoglobinometer or
Hb count by Sahli's haemoglobinometer.
Principle: Anti-coagulated blood is added to the 0.1 N HCl and kept for 5-7 minutes to form acid haematin.
The color of this acid haematin should be matched with the solution, present in the calibration tube.
Distilled water is added to the acid haematin until the color matches and the final reading is directly noted
from the graduation in the calibration tube. [Please note that 100 percent on the scale corresponds to
14.5gm % to 15gm %].
Requirements: Blood sample, Sahli’s haemoglobinometer, Hydrochloric acid, distilled water.
Procedure:
 Place N/10 HCL in diluting tube up to the mark 20.
 Take blood in the haemoglobin pipette up to 20-cubic-mm-mark and blow it into diluting tube and
rinse well.
 After 10 minutes add distilled water in drops and mix the tube until it has exactly the same color
as the comparison standards. Note the reading, which indicates the percentage of haemoglobin.
Result: The Hb % in the given sample is..... g/100 ml of blood/.....g/dl of blood/.....G%.
Precautions:
I.Pipetting of blood should be done cautiously.
ii. Mix the blood properly with HCl by using stirrer
iii. Match the color cautiously.

Experiment no. 04: Differential Leukocyte count
Introduction: Differential blood count gives relative percentage of each type of white blood cell and also
helps reveal abnormal white blood cell populations (eg. immature granulocytes, or circulating lymphoma
cells in the peripheral blood).
Reference ranges for differential white blood cell count in normal adults is as follows:
 Neutrophils - 2.0–7.0×10 9/l (40–80%)
 Lymphocytes - 1.0–3.0×10 9/l (20–40%)
 Monocytes - 0.2–1.0×10 9/l (2–10%)
 Eosinophils - 0.02–0.5×10 9/l (1–6%)
 Basophils - 0.02–0.1×10 9/l (< 1–2%)
The reference ranges may vary depending on population studies, the individual laboratory, instruments,
and methods.
Requirements:
There are 3 stains that used in differential leukocyte count:
Wright stain / Leishman stain / Giemsa’s stain, glass slides, 70% alcohol, blood sample, sterile needle,
cotton, and microscope.
The composition of Giemsa’s stain:
 Giemsa powder 0.3 gm
 Glycerin 25 ml
 Methyl alcohol 25 ml
This makes stock solution and before use it has to be diluted by adding 1 ml (stain) to 9 ml of buffered
water.
Procedure: Preparation of the
slide:
1) A fresh (non-heparinized)
sample of blood is added to one
side of the slide
2) The edge of another slide is pushed against
the drop of blood and smeared onto the rest of
the slide (see 3 and 4 below).
3) 4)

The smeared slide is allowed to dry.
5) Fixative and stain was added, observed under microscope.
Observation and results
Tabulation of the count
Results:

Interpretation
Differential blood count is also used along with leukocyte count (WBC) to generate an absolute value for
each type of white blood cells (eg, absolute neutrophil count, absolute lymphocyte count, or absolute
eosinophil count), which usually gives more meaningful information than the percentage of each, since
relative percentage can be misleading. Expressing absolute values are also useful for monitoring (eg,
monitoring neutropenia during chemotherapy or bone marrow transplantation). Using absolute values,
conditions such as neutropenia, neutrophilia, lymphopenia, lymphocytosis, monocytopenia,
monocytosis, eosinophilia, and basophilia can be identified, which can help differential diagnosis of
patient’s underlying disorders
Differential blood count is not a part of complete blood count (CBC) but is interpreted together with CBC
to help support or exclude a suspected diagnosis. For example, the presence of anemia along with
thrombocytopenia with a low or high white blood cell count may suggest bone marrow involvement by
leukemia.
Immature granulocyte (IG)
Immature granulocytes (IGs) encompass immature cells of granulocytic lineages, including
metamyelocytes, myelocytes, and promyelocytes, which are easily recognized morphologically and are
reported by automated analyzer as IG altogether. IG normally absent from peripheral blood.
Increased IG occurs accompanied by an increase in neutrophils in the following conditions:
 Bacterial infections
 Acute inflammatory diseases
 Cancer (particularly with marrow metastasis)
 Tissue necrosis
 Acute transplant rejection
 Surgical and orthopedic trauma
 Myeloproliferative diseases
 Steroid use
 Pregnancy (mainly during the third trimester)
Increased IG may occur without an accompanied increase in neutrophils in some conditions, particularly
elderly patients, neonates, and patients with myelosuppression. In these situations, isolated increase in
IGs (>2%) can be useful for recognizing an acute infection, even when not suspected.

Experiment No.5 RBC counting using improved Neubauer's chamber
Aim: To count the RBCs In the given blood sample
Theory: Erythrocytes, also known as red blood corpuscles, contain the haemoglobin (Hb) which carries
the oxygen to cells and tissues, is responsible for the red color of blood. The life span of normal RBC is
120 days. The RBC is produced in red bone marrow in adult. Before birth, the production of RBC
(erythropoiesis) first occurs in the yolk sac of the embryo and later in the liver, spleen, thymus, and lymph
nodes of a fetus up to seven months. Thereafter, production of RBC occurs in red bone marrow. The
agents, required for the synthesis of blood cells are called haematinic agents (iron, folic acid, vitamin B12,
erythropoietin, colony stimulating factors).
Fig 1. Steps of Erythropoiesis
Normal ranges:
Male: about 5.4 million per µl or cubic mm of blood
Female: about 4.8 million per µl or cubic mm of blood
Clinical significances: The decreased count of RBC indicates several conditions like iron deficiency
anaemia, megaloblastic anaemia, pernicious anaemia, thalassemia, sickle cell anaemia, aplastic anaemia,
chronic renal failure etc. Increased RBC production (Polycythemia vera) occurs in burn, lack of oxygen
during rock climbing, respiratory disorders etc., when the viscosity of the blood increases.
Materials: 0.9% w/v NaCl (normal saline), pipette, EDTA blood, Microscope, test tube, Neubauer's
chamber.
Method:
1. Make a 1:200 Dilution using EDTA Whole blood and saline
2. Put one drop of the solution in the Neubauer's chamber, using a pipette
3. Count the center square (R marked) of the chamber.
Calculation:
1. 80 small squares are counted.
2. Volume of fluid in each square = 1/4000 mm3
3. Average number of cell in each square = N/80
Where N= no. of RBCs in 80 small squares
Therefore 1/4000 mm3 contains, N/80 RBCs
So, 1 mm3 contains, N/80 X 4000
4. The dilution is 1 in 200
Therefore RBC count = N/80 X 4000 X 200 per mm3
= N X 50 X 200
= ………………………………million/ mm3

Result:
Diagram
Neubauer's chamber

Experiment No.6 Total Leukocyte Count with improved Neubauer's chamber
Aim: To count the WBC in the given blood sample
Principle: The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid (water: glacial acetic
acid: gentian violet = 97:2:1) and the cells are counted under low power of the microscope (10X) by using
a counting chamber. The glacial acetic acid lyses the red cells while the gentian violet slightly stains the
nuclei of the leukocytes to locate the WBC under microscope.
Theory: White blood cells, present in plasma take part in body defense against invading micro-organisms.
They are produced from the pluripotent stem cell in the bone marrow in adults. In case of foetus
haemopoiesis occurs in liver and spleen.
Normal ranges:
Table 1. Normal ranges of total leukocyte count:
Table 2. Differential leukocyte count:
Requirements:
 Microscope, Improved Neubauer's Chamber, WBC pipette, WBC diluting fluid
a) Glacial acetic acid: 2.0 ml,
b) 1 % (w/v) gentian violet: 1.0 ml,
c) Distilled water: 97 ml. This solution is stable at room temperature (25°C ± 5°C).
 A pinch of thymol may be added as preservative
 Double oxalated or EDTA blood.
Procedure: Make a 1:20 dilution of blood. Cork the tube tightly and mix the suspension by rotating in a
cell-suspension mixer for at least 1 minute. Fill the Neubauer’s counting chamber by means of a Pasteur
pipette or glass capillary (the depth of the fluid should be 0.1 mm). Focus on one of the 'W' marked areas
(each having 16 small squares) by turning objective to low power (10 X). Count cells in all four W marked
corners.
Calculations:
1. 4 large squares are counted.
2. Volume of fluid in each square = 1/10 mm3
3. Average number of cell in each square = N
Where N= no. of WBCs in 4 large squares
Therefore 4/10 mm3 contains, N number of WBCs

So, 1 mm3 contains, N X 10/4
4. The dilution is 20 times
Therefore WBC count = N X 10/4 X 20 per mm3
= ……………………………… / µl
Discussion and interpretation:
Clinical Significances of total leukocyte count: Increase in total leukocyte count of more than 10,000/cu
mm (µl) is known as leukocytosis and decrease of less than 4 000 cu mm (µl) as leukopenia.
Causes of leukocytosis:
i. it is common for a transient period in infections (bacterial, protozoal (malaria), or parasitic),
ii. Leukocytosis is also observed in severe hemorrhage and in leukemia
iii. During high temperature
iv. Severe pain
v. Accidental brain damage.
Causes of Leucopenia:
i. certain viral (hepatitis, influenza, measles, etc.), and bacterial (typhoid, paratyphoid, tuberculosis, etc)
infections
ii. Primary bone marrow depression (aplastic anaemia)
iii. Secondary bone marrow depression (due to drugs, radiation, etc.)
iv. Anaemia (iron deficiency megaloblastic etc.).
WBC count chamber Clinical significances of differential leukocyte count
Result:

Experiment no. 07: Erythrocyte sedimentation rate (ESR)
The ESR is a simple non-specific screening test that indirectly measures the presence of inflammation in
the body. It reflects the tendency of red blood cells to settle more rapidly in the face of some disease
states, usually because of increases in plasma fibrinogen, immunoglobulins, and other acute-phase
reaction proteins. Changes in red cell shape or numbers may also affect the ESR.
Method
When anticoagulated whole blood is allowed to stand in a narrow vertical tube for a
period of time, the RBCs – under the influence of gravity - settle out from the plasma.
The rate at which they settle is measured as the number of millimeters of clear plasma
present at the top of the column after one hour (mm/hr).
The Wintrobe sedimentation rack
Westergren method:
The Westergren method requires collecting 2 ml of venous blood into a tube containing 0 .5 ml of
sodium citrate. It should be stored no longer than 2 hours at room temperature or 6 hours at 4 °C. The
blood is drawn into a Westergren-Katz tube to the 200 mm mark. The tube is placed in a rack in a
strictly vertical position for 1 hour at room temperature, at which time the distance from the lowest
point of the surface meniscus to the upper limit of the red cell sediment is measured. The distance of
fall of erythrocytes, expressed as millimeters in 1 hour, is the ESR.

Wintrobe method:
The Wintrobe method is performed similarly except that the Wintrobe tube is smaller in diameter than
the Westergren tube and only 100 mm long. EDTA anticoagulated blood without extra diluent is drawn
into the tube, and the rate of fall of red blood cells is measured in millimeters after 1 hour. The shorter
column makes this method less sensitive than the Westergren method because the maximal possible
abnormal value is lower. However, this method is more practical for demonstration purposes.
This picture shows a rack holding Wintrobe Red blood cells have settled, leaving plasma at
the
Tubes, in which anticoagulated whole blood top of the tube. Reading: 18 mm/hour
Has just been added. (Time: one hour)
(Time: 0)
Average values in healthy men are: <15mm/hr; in healthy females, they are somewhat higher: <20mm.
The values are slightly higher in old age, in both genders.
Average values in healthy men are: <15mm/hr; in healthy females, they are somewhat
higher: <20mm. The values are slightly higher in old age, in both genders.

Theoretical considerations
The RBCs sediment because their density is
greater than that of plasma; this is
particularly so, when there is an alteration
in the distribution of charges on the surface
of the RBC (which normally keeps them
separate form each other) resulting in their
coming together to form large aggregates
known as rouleaux.
Rouleaux formation is determined largely
by increased levels of plasma fibrinogen and
globulins, and so the ESR reflects mainly
changes in the plasma proteins that
accompany acute and chronic infections,
some tumors and degenerative diseases. In
such situations, the ESR values are much
greater than 20mm/hr. Note that the ESR
denotes merely the presence of tissue
damage or disease, but not its severity; it
may be used to follow the progress of the
diseased state, or monitor the effectiveness
of treatment.
Some interferences which increase ESR:
 Increased level of fibrinogen, gamma globulins.
 Technical factors: tilted ESR tube, high room temperature.
Some interferences which decrease ESR:
 Abnormally shaped RBC (sickle cells, spherocytosis).
 Technical factors: short ESR tubes, low room temperature, delay in test
performance (>2 hours), clotted blood sample, excess anticoagulant, bubbles in
tube.
Chronic inflammatory disease (collagen and vascular diseases) increases ESR.
Polycythemia decreases ESR.
Report:

Experiment no. 08: Method of Determination of Packed Cell Volume (PCV)
Aim: to determine the PCV of the given blood sample
Introduction
When anti-coagulated blood is centrifuged at a standard speed, erythrocytes, which are heavier than
white cells and plasma, will settle down at bottom. This red cells volume is known as Haematocrit or
Packed Cell Volume (PCV).
Haematocrit or PCV is the volume of red cells expressed as a percentage of whole blood.
Methods:
There are two methods, used for the determination of haematocrit:
1. Macrohaematocrit
2. Microhaematocrit
Macrohaematocrit:
A large volume of blood is required in this method. Approximately 2 to 4 ml is required.
Principle:
Anticoagulated blood is taken in a Wintrobe tube. Filled upto the uppermost mark and then rotate for
desired length of time.
The packed cell volume (PCV) of red cells is directly read from the graduated tube as %
Requirement:
1. Blood specimen:
EDTA or double oxalated anti-coagulated blood is used in this method. Determine P.C.V. within six hr. of
blood collection.
2. Wintrobe Tube:
It is 110 mm in length and 2.5 mm in diameter. The lower 100 mm are graduated or marked, from 100 at
top and 0 (zero) at bottom for PCV.
3. Long necked pasture pipette or a special type of syringes is used for filling Wintrobe tube.
4. Centrifuge machine with known speed.
Procedure:
Mix 0.4 ml of EDTA with 2 ml blood. Fill the Wintrobe tube upto upper most mark with the help of
pasture pipette or syringe. Fill another Wintrobe tube to balance first one. If the blood sample is not
available, fill the tube with water.
Place the Wintrobe tube in opposite side in centrifuge. Turn the centrifuge to slow speed, then slowly
increase the speed to 3,000 rpm. Centrifuge for 30 min. at 3,000 rpm. After 30 min. switch off the
centrifuge and allow it to stop by itself. Take out the Wintrobe tube and read PCV directly with the help
of graduation mark given on the tube.
Normal Value:
i. In male – 42 to 50%
ii. In female – 36 to 38%
Microhaematocrit:
This method requires small amount of blood, 2 to 3 drops only. The blood can be obtained by finger
puncture.
Principle:
Anti-coagulated blood is centrifuged in a sealed capillary tube, and then PCV is determined by a special
haematocrit reader.
Requirement

1. Blood Specimen:
Blood from finger puncture may be used or EDTA or double oxalate venous blood can also be used.
2. Capillary Tube:
Use plain capillary tube for anti-coagulated venous blood and use heparinised capillary tube (Coated
with heparin internally) for blood obtained from finger puncture. The capillary tube is approximately 75
mm in length.
3. Microhaematocrit Centrifuge:
This is a special type of centrifuge. It has speed about 15,000 r.p.m. The top of centrifuge is flat with
grooves. The centrifuge also has timer, which is usually set for 5 min.
4. Haematocrit Reader:
There are several types of readers used for reading hct. The simplest method is use of card reader,
which can be made by hand.
5. Clay:
This is used to seal the end of capillary tube.
Procedure:
Draw the blood sample into appropriate capillary tube with capillary action. Use plain tube for anti-
coagulated blood and heparinized tube for plain blood. In case of finger puncture, the blood should
flow freely with little pressure. Now wipe off the first drop and then collect the blood specimen.
Fill the tube about 3/4th length with blood. Seal another end of the tube with clay or wax or ultimately
by heating. The sealing should be about 2 mm deep. Place two hct tubes in the groove of centrifuge
exactly opposite to each other.
It is not necessary that the capillary tube have exact amount of blood level. In case, if there is no filled
capillary tube to balance we can use an empty capillary tube. Centrifuge at 13,000 ± 2000 rpm.
Remove capillary tube from centrifuge. It will show three layers. Top layer is of plasma or serum; the
middle layer is thin creamy white in colour and is known as Buffy coat. It is a layer of WBC; the last layer
is the column of RBC. Use the hct reader for finding the value of hct.
Card Reader:
The reader is used as, hold the tube against Scale so that the bottom of red cell is matched with 0
(zero) line of the card. Move the tube across the card until the uppermost line of plasma is matched to
100% line of card.
Check to make sure that bottom of red cell column is still in the line of zero and the tube should be
straight and vertical. The line that passes through the top of the column of RBC gives the hct. Value.
Importance:
Decrease in PCV is suitable measure for anaemia. The fall in hct may be seen in decreased oxygen
supply to cells, heart disease or malignant condition, hct also rise in case of dehydration

Experiment no. 9: study of ELISA by kit method

Experiment no. 10: study of Ouchterlony double diffusion by kit method

Experiment no. 11: Extraction of serum and plasma from blood
Aim: Effective Separation of blood products
Purpose: To standardize separating procedures so that research samples will be uniform in quality The
decision to collect anticoagulated (plasma, buffy coat, RBC) or coagulated (serum, clot) blood samples
must be made prior to collection so that proper blood draw tubes will be used.
Serum (needs clot time) A serum separator tube (SST, tiger top tube). Let the blood sit for 30 minutes to
one hour at room temperature to clot before spinning and separating.
A delay in centrifugation may have a detrimental effect on the sample quality and may result inaccurate
results. Avoid hemolysis.
Separating plasma (time sensitive) Tube with an anti-coagulant eg: Edta (lavender top)sodium heparin
(green top), sodium citrate (blue top) are used for separating Plasma You need to spin and separate
within one hour of receiving the specimen (time sensitive)
Note: Universal Precautions must be used when working with blood. Use of personnel protective
equipment is mandatory. Use of eye protection is mandatory unless blood tubes are being opened and
serum/plasma/whole blood are being aliquoted inside a BL2 safety cabinet.
*Keep blood on wet ice and process within one hour of blood draw
Separation of plasma
1) Blood will be collected into purple top EDTA tubes and centrifuged (2000 rpm) at 4 degrees centigrade
for 20 minutes.
2) After centrifugation using clean pipette technique place 1.0ml of plasma into 1.5ml eppendorf tube
labeled with tracking number and “plasma”
3) Freeze immediately at –80 degree freezer
Separation of Serum
1. A 10 ml tube of whole blood will be collected following standard procedures using a serum separator
tube (SST, tiger top tube) from each patient.
2. Allow samples to clot for one hour at room temperature
3. Centrifuge for 10 minutes at approximately 1000g
4. Using clean pipette technique Aliquot 210ul of serum into labeled cryovials.
5. Immediately freeze vials of serum at –80-degree freezer
Aliquoting whole blood Whole blood will be aliquoted into sterile tubes upon receipt by carefully
inverting the blood tube so that it is gently mixed before pipetting appropriate amounts (protocol
specific) of whole blood into appropriate storage tubes using clean pipette tipps between each patient.
• Gently invert the tube of blood to mix contents

• Carefully open blood tube (universal precautions; gloves, eye protection)
• With clean pipette tip aliquot appropriate amount of whole blood into clean/labeled storage tubes.
• Freeze in –80 freezer

Experiment no. 12: Basic cell culture techniques.
There are number of methods that can be used for organ culture. These include;
 Plasma clot method - Here, the method involves the use of a clot that is composed of plasma
and chick embryo extract (or any other extract) in a watch glass. This method is particularly used
for the purposes of studying morphogenesis in embryonic organ rudiments and more recently
for studying the actions of various hormones, vitamins and carcinogens of adult mammalian
tissues.

 Grid method - Grid method, as the name suggests involves the use of perforated stainless steel
sheet, on which the tissue of interest is placed before being placed in a culture chamber
containing fluid medium.

Experiment no. 13: Preparation of Balanced Salt Solutions.
A balanced salt solution (BSS) is a solution made to a physiological pH and isotonic salt concentration.
Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. Balanced salt
solutions are used for washing tissues and cells and are usually combined with other agents to treat the
tissues and cells. They provide the cells with water and inorganic ions, while maintaining a physiological
pH and osmotic pressure.
Sometimes glucose is added as an energy source and phenol red is used as a pH indicator.
In medicine, balanced salt solutions can be used as an irrigation solution such as
during intraocular surgery and to replace intraocular fluids.
Hanks' salts: Is a collective group of salts rich in bicarbonate ions, formulated in 1940 by the
microbiologist John H. Hanks. Typically, they are used as a buffer system in cell culture media and aid in
maintaining the optimum physiological pH (roughly 7.0–7.4) for cellular growth. Due to their
poorly reactive nature and small concentration in solution, Hanks' salts are mainly used in media that are
exposed to atmospheric conditions as opposed to CO2 incubation.
Role of phenol red in cell culture.
Phenol Red, typically used at 11 mg/L, is used widely in culture media to identify changes from neutral to
acidic pH values, the latter signifying contamination or cell over-growth. It imparts an increasingly yellow
color as pH drops below 6.4, and a deepening red color at a pH of 8.2 and above.
Table 1. Required components
Component Mass Molarity
NaCl (mw: 58.4 g/mol) 8 g 0.14 M
KCl (mw: 74.551 g/mol) 400 mg 0.005 M
CaCl2 (mw: 110.98 g/mol) 140 mg 0.001 M
MgSO4-7H2O (mw: 246.475 g/mol) 100 mg 0.0004 M
MgCl2-6H2O (mw: 203.303 g/mol) 100 mg 0.0005 M
Na2HPO4-2H2O (mw: 177.99 g/mol) 60 mg 0.0003 M
KH2PO4 (mw: 136.086 g/mol) 60 mg 0.0004 M
Glucose (mw: 180.156 g/mol) 1 g 0.006 M
NaHCO3 (mw: 84.007 g/mol) 350 mg 0.004 M
1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 400 mg of KCl to the solution.
4. Add 140 mg of CaCl2 to the solution.
5. Add 100 mg of MgSO4-7H2O to the solution.
6. Add 100 mg of MgCl2-6H2O to the solution.
7. Add 60 mg of Na2HPO4-2H2O to the solution.
8. Add 60 mg of KH2PO4 to the solution.
9. Add 1 g of Glucose to the solution.
10. Add 350 mg of NaHCO3 to the solution.
11. Add distilled water until volume is 1 L.

Result:

Immunology and cell culture techniques

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