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Experiment No.

7
Object:- Smear Technique to observe sex chromosome (Barr Body) in
the buccal epithelial of human.
Principle:-
Barr body-Human being have 46 Chromosomes in their somatic cells In
human males there are 44 autosome and two sex chromosomes termed "X"
and "Y". In human females besides 44 autosomes there are two sex
chromosome termed "XX". This means that human females have the
potential to produce twice as much X-chromosome product as human males.
But this does not happen, because out of the two X chromosome in somatic
cells of mammalian females, One X chromosome is inactivated and
generally remain attached to the nuclear membrane in interphase cell called
called Barr body.
The inactive X-chromosome or Barr body represent a mechanism by which
the dosage compensation for X-linked gene products is achieved. In other
words female also produce the same X-chromosome gene products as males
despite having 2 X-chromosomes.
Material required
Tooth pick.
Glass slide.
Cover glass.
Coplin jar.
Giemsa stain
90% alcohol.
6N HCL
Distilled water
Xylene.
DPx
Compound microscope.
Procedure :
1. Scrap the inner side of your cheek with the help of broad side of a
tooth pick.
2. Spread the scraping on clean and dried slide. Allow it to air dry.
3. Fix the cells by dipping the slide in a Coplin jar containing 90%
alcohol for a minute. Allow it to air dry.
4. Tansfer the slide in a Coplin jar containing 6N HCL for 10 minute at
room temperature. This step of hydrolysis removes the debris from the
smear.
5. Stain the slide with distilled water.
6. Stain the slide with Phosphate buffered 4% Giemsa stain for 10-15
minute.
7. Transfer the slid into 1-2 minute for differentiation. Allow it to dry.
8. Clear the slide in Xylene.
9. Mount the slide with DPX and cover with cover glass.

Result Showing Barr body indicated by arrow in buccal epithelial cells of


human female whereas male has no Barr body.
Experiment No. 8
Object:- To study different stage of Mitosis in rat done marrow. To
observe Metaphase chromosomes of rat bone marrow cells.
Principle:-
All cells come from pre-existing cells. A Cell reproduces by performing an
orderly sequence of event in which it duplicates its contents and then divides
no to two. This cycle of duplication and division is known as Cells Cycle.
Cell division in needed for growth Reproduction, Regeneration and
Replacement of cells that die. The most basic function of cell cycle each to
duplicate accurately the vast amount of DNA in the chromosome and them
segregate the copies precisely into two genetically identical daughter cells.
These processes (synthetic) phase and it requires 10-12 hours and occupies
about half of the cell cycle time in typical mammalian cell. After S phase
chromosomal segregation and cell division occur in M (Mitosis) phase,
which requires much less time about an hour. M phase comprises two major
events, Nuclear Division or Mitosis during which the copies chromosome
are distributed into a part of daughter nuclei, and cytoplasmic division or
cytokinesis, when cell divides into two. S and M phase are separated by Gap
phases called G1 and G2. M phase is dividing into four stages, Prophase,
Anaphase and Telophase.
The marrow from long bones like femur provides the handiest material for
analysis of cell cycle (Mitosis), Metaphase chromosome and Karyotype
preparation, mutagenesis and cell physiology of small animals Bone marrow
cells can be incubated in vitro for at least one cycle without any exogenous
mitotic stimulation and can be use as very accurate representative of in vitro
condition. Such preparation can be made even in the field with the help on
hand centrifuge and burner as a substitute for incubator.
Apparatus Required :
Centrifuge, incubator, syringe, needle, Pasteur pipette, test tube,
coupling jar agitator, dissecting tray, dissecting set.
Chemical Solution
Hypotonic solution 0.56% KCl (Pre-warmed to 370 C before use)
Caroy's Fixative (Glacial acetic acid : Methanol 1:3)
Giemsa Station.
Stock Solution
Giemsa powder - 380 mg
Methanol - 25 ml
Glycerol - 25 ml
Leave overnight at 370 C Filters and store
Working solution Stock solution - 2.5 ml
Mechanol - 1.5 ml

Procedure :-
1. Intramuscular injection of colchicine (0.2 mg/kg body weight) is
given to the rat 2-3 hours prior to sacrifice for metaphase
preparation; Colchicine is not needed for experiment related to cell
cycle studies.
2. Animal is sacrificed cervical dislocation and femur bone is dissected
out.
3. Both the ends of the bone are cut.
4. 0.5 ml of pre warmed (370 C) hypotonic solution is taken in 5ml
syring with #18 needles.
5. The needle is then inserted through one of the cut ends of the femur
bone and the marrow is flushed forcefully into the centrifugation
tube.
6. The marrow is then agitated with the help of rubber agitator to
obtain a single cell suspension. More prewarmed (370 C) hypotonic
solution is added to make the volume upto 10 ml. The solution is
then incubated at (370 C) in an incubator for 30 minutes.
7. Process 4, 5 and 6 are repeated for 2nd famur bone.
8. Centrifuge the cell suspension at 1500 rpm in a centrifuge for 10
minutes to obtain the cell pellets in both the tubes. Discard the
supernatant.
9. Fix the cell by adding the fixative drop by drop and constantly
agitating the tube to obtain a suspension. Make the volume to 10 ml
with the fixative in both the tubes. Allow fixation for 10 munutes.
10.Again centrifuge to obtain pellet.
11.Re-suspend the pellet in fixative making the volume to 10 ml.
12.Again centrifuge to obtain pellet.
13.Re-suspend the pellet in small volume of fixative 2 ml in each
centrifugation tube.
14.Take a clean glass slide and place 2-3 drop of the solution (13) on
the slide with the help of Pasteur pipette. Gently flame the slide.
15.Allow the slide to dry.
16.Stain the slide with Giemsa for 10 minute. Give 2 rinses in distilled
water then allow the slide to dry in air.
17.Clear in xylol and mount in DPX.
18.Observe the slide a microscope to identify different stages of
mitosis.
19.Observe the metaphase plates under oil immersion lens.
Observation
Different stages of mitosis was observed.
Interphase : Nucleus appears highly granular
Metaphase : The chromosomes are aligned at the equator. The total number
could be counted as 42 chromosomes. Sister chromatids can be seen.
Anaphase : The sister chromatids synchronously separate to form two
daughter chromosome and each is pulled opposite poles.
Telophase: The two sets of daughter chromosomes arrive at the poles. New
nuclear envelope reassembles around each set. Two daughter nuclei each
with same number of chromosome is seen. Chromosome undergoes coiling.
The cytokinesis begins in which cytoplasm is divided into two and two
daughter cells are formed.
Observation
9 3 8 10 2 2 5 5
7 6 6 8 6 6 3 8
5 7 3 8 8 7 3 6
5 7 3 8 8 7 3 6
7 8 9 7 8 6 9 4
174 68

7 6 2 4
2 4 4 7
3 8 7 6
5 5 6 5
84

8 5 5 5 6 1 15 6
6 8 5 2 9 8 5 9
1 8 4 5 4 7 5 7
2 6 8 6 8 6 5 6
84 107

Calculation:- If x is the total number of RBCs in 5 chamber so.


X = 147 + 88 + 84 + 84 + 107 = 537
Then the total no. of RBCs in counting camber = 537x50=26850
Since dilution factor is = 200
Terefore total no. of RBC = 5370000
= 5.37x106 mm3
Experiment No. 5
Object:- To determine rate of oxygen consumption in cockroach by
using respirometer.
Principle:-
Aerobic organism requires a continuous supply of oxygen for the oxidation
of food stuff to produce energy. In this process, CO 2 and water are released
as by products. In this process, the utilization of O 2 and release of CO2 are
the two major components. In the present experiment we will quantity the
rate of O2 consumed by a cockroach with the help of a simple respirometer.
Aerial respiration is studied by manometric techniques.
Warburg's manometer is the favorite instrument for such studies.
However this instrument is quite expensive. Therefore we many construct a
simple device of our own to measurement of O2 consumption in cockroach.
Material required
Four ounance bottle
One holed rubber stopper, 2ml graduated pipette
Filter paper bits
Small pieces of wire gauze
15% KOH solution and cockroaches.
Procedure :
1. The bottle is fitted with a hold-rubber stopper.
2. A 2 ml graduated pipette is inserted into the bottle in such a way
that only 1/5th to 1/4th of the pipettee is inside the bottle.
3. Some filter paper bits soaked in 15% KOH solution were placed at
the bottom of the bottle. The wire gauze can be used to wrap the
filter paper bits.
4. A cockroach was weighed on an electronic balance.
5. The weighed cockroaches were introduced into the self-made
respirometer and the instrument was made air tight.
6. Another respirometer was made without cockroach as controlled.
7. Both controlled and experimental respirometerwhere increased in a
tray containing water.
Observation :
The slowly only of water into pipette was observed. The releases
KOH. The water serves as the manometric fluid. The volume of water
interred into the pipette in 1 hour is recorded. This volume represents the
volume of Os consumed by cockroach in 1 hour.
Calculate
Weight of cup = 1.120 g
Weight of cup + cockroach = 1.767g
Weight of Cockroach = .647 g
Volume of O2 consumed in 10 minutes = 0.8 ml
Volume of O2 consumed in one hour = 0.8 4.8
Volume of O2 consumed in1hour ( ml ) 4.8
Rate of Re spiration
Weight of cockroach ( g .) .647

= 7.42 ml/g/her
Result :- Rate of O2 consumption in cockroach was 7.42 ml/g. Body
weight.
Experiment No. 2
Object:- To carry out micrometry.
Principle:-
Micrometry is a technique to measure the size of objects under a
microscope. We may measure the length and breadth of Paramecium.
Material required
Compound microscope.
An ocular micrometer (OM)
A stage micrometer (SM)
Few prepared slides
Procedure :
1. The nose piece of the microscope turned to bring the 10x objective to
position and ocular micrometer fitted into ocular lens.
2. 1mm. line of stage micrometer was bringing to focus. We observe
under the microscope. we should be able to see two set of lines. One
of stage micrometer and other of ocular lens.
3. Looking through the microscope adjust ocular lens and the stage
micrometer (SM) appropriately so that the zero of two scales coincide.
4. Starting from zero position moves the eyes toward the right, look far
and read lines of coincidence. That division is the stage micrometer
which consider with the only micrometer was recorded.
5. Now we have to remove the stage micrometer very carefully.

Observation
Calculate is follows and determine the value of one Ocular micrometer
(OM) division.
Stage micrometer reading
Value of one Oclar divison 10
Ocular micrometer reading

Where SM and OM reading refer to number relating to the lines of


coincidence between the state and the ocular micrometer and 10 refers to the
value of 1SM division if you have recorded five reading you many have
calculated 5 different OM values. Obtain mean

10
Value of one Ocular division 10
10

= 10
Length of Value of One Actual value of
S.No. Magnification
Parameclum OM Division Measurement
1. 10 10 16 10
2. 10 10 16 10
3. 10 10 16 10 16 10 = 160
4. 10 10 16 10
5. 10 10 16 10

Length of Value of One Actual value of


S.No. Magnification
Parameclum OM Division Measurement
1. 10 10 9 10
2. 10 10 9 10
3. 10 10 9 10 9 10 = 90
4. 10 10 9 10
5. 10 10 9 10
Result : The length of Paramecium was 160 and breadth 90.

Experiment No. 9
Object:- To estimate hemoglobin content (g%) of own blood.
Material required
0.1 HCL
Distilled water
Haemocytomer (Haemometer)
Hypodermic needle
Spirit
Micropipette
Principle:-
Haemoglobinforms acid haematin in the presence of 0.1N HCL. This
compound than match with suspension of acid haematin in standard tube.
Haemometer: The Haemometer consist of scaled lateral comparison tube
containing suspension of acid haematin. These are held in a black frame
against a white brown glass. Besides, a graduated test of the same diameter
is also provided which can fit in the haemometer in between the two side
tube. The graduation on the experiment tube refers to percentage of
haemogobin in blood i.e. g/100 ml of blood.

Procedure :
1. Took .1 N HCL in haemometer graduated tube up to mark 2
2. Sterlize the finger tip with rectified spirit and prick it with the help of
sterilized hypodermic needle.
3. Sucked the blood up to mark 20 in micropipette.
4. Transfer the blood of microscope into graduated experiment tube of
the haemopmeter.
5. Stired the solution truffle with help of the glass rod and allow standing
of 10 minute.
6. Added distilled water drop by drop into the solution stir the solution
with glass rod with standard sealed tubers.
7. d
8. The mark up to the which the blood is diluted gives the percentage of
haemoglobin blood or g. Weight of haemoglobin/100 ml of blood.
Result : Haemoglobin contact of my own blood was ............ 8 ............%
Precaution
1. Clean the tube carefully before use.
2. There should be no air bubbes in micropipette.
3. Sterlize the finger and needle with 90% or absolute alcohol before
taking blood.
Experiment No. 10
Object:- To seprate Amino acids by ascending paper chromatography.
Material required
Chemicals- Different amino acids, Solvent (n-butanol, acetic acid,
water 4:1:3). Ninhydrin (0.2% in n-butanol)
Miscellaneous Glass jar, Pencil, Scale, Capillary tube,
Chromatography paper, Whattman paper no. 1)
Principal
Paper chromatography is technique in which the compounds or
molecules of a mixture are allow migrating at different rates through a
porous matrix which involves equilibration and partitioning of a compound
between stationary and mobile phase. Thus paper chromatography is type of
partition chromatography.
In paper chromatography hydrated cellulose molecules of paper is
serve as stationary phase and solvent act as mobile phase.
Procedure :
1. Fill the chromatography jar with solvent before the process to
saturated to jar
2. Take Whattman paper No. 1 strip (22cm7cm) and draw a line with
pencil 1cm. above the bottomo edge of paper.
3. Make 3 points on the line. Marks point as 1, 2 and 3.
4. Load the sample once by capillary tube on points by one and late it to
air dry.
5. After dryness apply the sample again in the same way two times
more.
6. After complete dryness of sample make two holes on the top of strip
and insert of thread into it.
7. Hang the strip in the jar in such a manner that the lower bottom of the
strip to suck the solvent but marked the line should not directly come
in to contact with solvent.
8. Close the jar with and keep it for 2-3 hour for chromatographic
separation.
9. After separation remove the strip form the jar and keep in over for
drying at 30-800C.
10.After complete drying spray ninhydrin over the strip uniformly and let
it dry in overn.
Observation :
No. of Spots
Spot Colour Rf.
observed
Experiment No. 3
Object:- Preparation of single cell suspension of spleen and
determination of cell viability
Material required
Rat
Haemocytometer
Microscope
Complete culture medium (RPMI 1640)
0.4% Trypan blue
Principal :-
Trypan blue exclusion test for viability
The dye exclusive test is used to determine the number of viable cells
present in a cell suspension. It is based on the principal that live cells posses
intact cell membrane that excludes certain membrane impermeant dyes such
as Trypan blue. In case of dead cell membrane will not be intact, so Trypan
blue dye can permeate through membrance and stain the cytoplasmic part.
Thus, live cells will have clear cyto plasm and colores, while dead cells will
have blue cytoplasm, in this viability test, a cell suspension in simply mixed
with dye and then visually exmined under microscope to determine whether
cells take up or exclude dye.
Neubauer counting chamber (Haemocytometer)
Observation
14/4 19/2 14/4 18/5 13/2 23/3 25/6 25/4
17/4 13/1 18/3 17/4 23/3 29/1 24/3 12/3
15/3 12/3 19/4 21/8 15/2 28/3 14/5 26/4
19/2 14/1 10/4 26/4 17/2 16/3 18/2 16/3
720/56 470/49
19/4 15/5 13/5 8/2 9/3 14/5 13/3 20/3
16/1 18/5 17/4 21/3 12/4 17/5 9/3 18/8
15/3 8/3 14/4 17/2 14/3 18/6 7/4 12/7
20/4 11/1 10/4 17/3 13/3 11/3 17/5 10/4
540/53 725/25
Experiment No. 6
Object:- Estimation of RBC count of mammalian blood.
Material required
Haemocytometer
Hypodermic needle
Cotton
Rectified spirit
Hayman's diluting fluid (NaCl lg, HgCl2 0.5g, distilled water 200ml)
Procedure :
1. Sterilized the finger tip with rectified spirit.
2. Pricked the finger tip with sterilized hypodermic needle to let blood
oozen out.
3. Pipette up the oozed blood directly into clean and dry RBC pipettee
up to mark 5, if the blood has been sucked above this mark removed
by wiping the end of popette by cotton or blotting paper.
4. Without delay sucked Ham's diluting fluid up to mark 1.01, mixed
thruway by gentle shaking and rotating the pipette for 3-5 minutes. If
any clot appears reject the whole sample and repeat from the start.
5. Reject first few drops from pipette and then put a drop diluted blood
over counting chamber on the central plate of slide of
Haemocytometer.
6. Put the cover glass in such a way that it must be placed on the side
pillars of central platform.
7. Count the erythrocyte in RBC counting chamber under the high power
objective of a compound microscope.
8. The RBC lying on the middle of the line of square to your side to the
right is also to be counted in the total while those laying on the upper
and left side of square are not to be counted. RBC counting chamber
had 25 small squares each of which again divided into 16 small areas.
out of 25 squares counting may be limited to only with central and
four corners squares. Each having 16 smaller squares totally 80 in all.
Experiment No. 4
Object:- Isolation of lymphocyte from blood and spleen using density
gardient centrifugation
Material required
Rat
Syringe
High Speed cooling centrifuge
Centrifuge tube.
HiSep-1.077 (Lymphocyte sepration medium)
Haemocytomer
Microscope
Heprin
complete culture medium (RPMI 1640)
Principle :
Typically a sucrose density gradient is created by gently overlying lower
concentration of sucrose of higher concentration in centrifuge tube. For
example, sucrose greatly may consist of layers extending from 70% sucrose
to 20% sucrose in 10% increments (through this is highly variable
depending on sample to be purified). In the life science a special technique
called density gardient separation in used for isolation and purifying cells,
viruses and such cellular paracles.
The Sample containing the cells of interest is placed on top of the gradient
and centrifuged respective g. The particle travel through the gradient until
they reach the point in the gradient at which their density matches that of the
surrounding sucrose. This fraction can then be removed and analyzed.
Procedure
1. A rat anaesthetized with ether and vivisected to open the heart. Blood
is collected in heparinised tube and kept at 4 0 C. Spleen is excised
aseptically and kept in cool (40 C) culture medium.
2. Under aseptic condition, excised spleen is macerated through a nylon
strainer of pore size<100 m into complete culture medium to get
single cell suspension under a sterile laminar flow hood.
3. Isolation of splenic lymphocytes.
4. Splenic signle cell supension prepared as above is suspended in
complete culture medium Solenic lymphocytes are isolated density
gradient centrifuge using HiSep (Hi Media: Density 1.077g/ml)
5.
6.
7. Lymphocytes layer at the interface between medium and HiSep was
carefully aspired washed three times with BS and pellateted. Cell is
responded in culture medium (RPMI 1640) counted with the help of
Haemocytometer.
8. Viability is assessed by trypan blue exclusive test. Viable cells (>95%)
were adjusted to 2x104 cells/ml with complete culture medium and
used for further study of immunological response.

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