DEPARTMENT OF BIOTECHNOLOGY

Student Name :
Registration No :
Subject Code : 21BT38P
Subject Name : Cell Biology Laboratory
Year & Semester : II YEAR & III
Regulation : 2021
Faculty I/c : Ms. S. DHIVYA
 Course code 21BT38P Semester III
Category Program Core Course (PCC) L T P C
Course Title CELL BIOLOGY LABORATORY 0 0 4 2

COURSEOBJECTIVES

 To learn the various techniques for morphology, identification and propagation of

cells

PREREQUISITE

 Biochemistry Laboratory

COURSEOUTCOMES

CO. No. Course Outcome Blooms level

At the end of the course students will be able to
C208.1 Outline the awareness on the sterilization techniques and K2
microscopy handling
C208.2 Experiment with differential staining in order to understand K3
the internal components of a cell
C208.3 Demonstrate the ability to propagate cells and perform cell K2
division
C208.4 Distinguish the viable cells from dead cells usingcell viability K4
assays.

MAPPING WITH PROGRAM OUTCOMES AND PROGRAM SPECIFIC OUTCOMES

CO No. PO-1 PO-2 PO-3 PO-4 PO-5 PO-6 PO-7 PO-8 PO-9 PO-10 PO-11 PO-12 PSO-1 PSO-2 PSO-3 PSO-4

C208.1 2 1 - - - - - - 1 - - - 2 - - -
C208.2 3 2 1 - - - - - 1 - - - 2 - - -
C208.3 2 1 - - - - - - 1 - - - 2 - - -
C208.4 3 3 1 - - - - - 1 - - - 2 - - -
C208 3 2 - - - - - - 1 - - - 2 - - -

Note: 1: Slight 2: Moderate 3: Substantial

 LIST OF EXPERIMENT No. of Credits:2
1. Principles of sterilization and handling of microscopes
2. Studying the diversity of cells using permanent slides
3. Differentiate plant cells from animal cells using a staining procedures
4. Stain and differentiate gram positive and gram-negative bacteria
5. Stain and differentiate nucleated and non-nucleated blood cells
6. Staining method to identify blood parasites.
7. Cell lysis through Osmosis and Tonicity
8. Peripheral Blood Mononuclear Cell Separation from Blood
9. Growing root tips of Allium cepa and study different stages of Mitosis
10. Study of Meiotic Cell Division using permanent slides
11. Thin Layer Chromatography
12. Cell Viability: Tryphan Blue Assay

LEARNING RESOURCES

TEXT BOOKS

1. Rickwood, D. and J.R. Harris “Cell Biology: Essential Techniques”, Johnwiley,1996.

2. Davis, J.M. “Basic Cell Culture: A Practical Approach”, IRL,1994.

REFERENCES

1. Roitt, I., Brostoff, J. and Male, D. “Immunology”, 6th Edition, Mosby, 2001.
Exp. No 1a Date:
INTRODUCTION TO PRINCIPLES OF STERLISATION TECHNIQUES
Aim: To study different sterilization techniques and applications sterilization.
Sterilization refers to any process that effectively kills of eliminates transmissible agents such as
fungi, bacteria, viruses and spore forms. Sterilization however does not remove prions.
Applications
Food canning: It is a method of preserving foods in which food is processed and sealed in
airtight containers.
Medicine and surgery: In general, surgical instruments and medications that enter in and already
sterile part of the body (such as blood beneath the skin must of such instruments include scalpels,
artificial pace makers etc. sterilization can be achieved through application of
i) Heat ii) Chemical iii) radiation iv) Filtration
Heat sterilization can be achieved by application of dry heat, moist heat.
Dry heat sterilization
It is one of the earliest forms of sterilization practiced. Dry heat as the name indicates utilizes dry
heat (i.e.) either free from water vapor or as very little of it and where moisture plays minimal or
no role in the sterilization process. Dry heat sterilization process is accompanied by conditions
where heat is absorbed by either surface of material and then passed inward to the next layer.
Eventually the entire material reaches the proper temperature needed to achieve sterilization.
Proper time and temperature for dry heat sterilization is 100 oC for two hours and 170o C for 1
hour. Instruments should be dry before sterilization since water will interfere within the process.
Dry heat destroys microorganisms by coagulating proteins causes oxidative free radical damage,
causes drying of cells and can even burn them into ashes as in incineration.
Flaming, incineration and hot air oven utilizes dry heat to achieve sterilization.
Flaming: It is done to inoculation loops in microbiology lab showing the loops in Bunsen burner
or alcohol lamp until it glows ensure that any infections agents get inactivated. This is commonly
used for all metal or glass objects.
Incineration: It will burn any organisms to ash. It is used to sanitize medical and other bio-
Hazardous wastes before it is discarded with non-hazardous wastes.
Hot air oven : They are electrical devices used in sterilization. The oven uses dry heat to
sterilize articles. Generally, they can be operated from 50-300 oC. They have thermostat which
controls the temperature. These are digitally controlled to maintain the temperature. A double
walled insulation keeps the heat in and conserves the temperature. The inner layer being poor
conductor and the outer layer being metallic. There is an air-filled space in between to aid
incinerations. Air circulation helps in uniform distribution of heat. These are filled with the
adjustable wire mesh plated trays or aluminium trays. And may have on/off rocker switch
indicator and control for temperature and folding time.
Advantages
They do not require water and there is no much pressure building within the oven unlike an
autoclave making the safer to work with. They are much smaller than autoclaves but can still be
effective.
Disadvantages
As oven used dry heat instead of wet heat prions may not be killed by them every time.
Wet Heat Sterilization
As the name indicates wet heat utilizes air that is heavily laden with water vapor and where the
moisture plays an important role in sterilization methods used.
i) Below 100 o C eg: water bath (50 o C for 60 min).
ii) At 100oC eg: Boiling (10-30 min)
iii) At above 100oC eg: Autoclave
Wet heat coagulates proteins in any organisms and is aided by water vapor that has very high
penetrating property leading to death. It also causes oxidative free radical change. This can kill
prions even at high inert temperature.
Autoclave
It was developed by Chamber land (1884), here the air is initially forced to enter the chamber
and filled with saturated steam and the outlets are closed. Hot saturated steam continuously
enters into the chamber until it reaches 15lbs at 121 oC. At this temperature the saturated steam
destroys all the endospores in the small volume of liquid within 10-15 mins. This treatment is
continued for 15 minutes to provide a margin of safety.
Chemical sterilization:
Although heating provides the most reliable way to get rid of objects of all transmissible agents,
it is not always appropriate because it will damage heat sensitive material such as biological
materials fiber optics, electronics and many plastics.
Ethylene oxide, ozone, H2O2, chlorine, formaldehyde, gluteraldehyde, are some of the chemical
agents frequently used for sterilization.
Ethylene oxide : This gas is used to sterilize objects which are sensitive to temperature greater
than 60oC such as plastics, optics and electrics. Ethylene oxide treatment is usually carried out
between 30-60 oC. Ethylene oxide penetrates well moving through paper cloth and some plastic
films and is highly effective.
It can kill all known viruses, bacteria and fungi including bacterial spores and is suitable for
medical devices. It is toxic and flammable (requires long time 10-15 hrs to sterilize them in any
heat treatment).
Also require post sterilization, aeration to remove toxic wastes. It is most common sterilization
method used for over 70% of total sterilization and for 50% of all medical devices.
Ozone: It is used in industrial settings to sterilize water and air, as well as disinfectant for
surfaces. It eliminates microbes by its ability to oxidize most organic matter. It is toxic and
unstable so ozone must be produced on sight so it is not practical to use it in many settings.
Hydrogen peroxide (H2O2)
It is relatively non-toxic when diluted to low concentrations. H 2O2 is strong oxidant and these
oxidizing properties allow it to destroy a wide range of pathogens. In medical sterilization, H 2O2
is used as high concentrations ranging from 35-90%. Biggest advantage of using H 2O2 as
sterilant is its short cycled time.
Chlorine:
Chlorine bleach is another accepted liquid sterilizing agent. House hold bleach consists of 5.25%
sodium hypochlorite. Prions can also be inactivated by diluting the chlorine solution by 1 of 2.5
parts. Chlorine bleach will kill many organisms immediately. But for complete sterilization it
should be allowed to react for 20 mins. It cannot eliminate spores completely and also it is highly
corrosive. Bleach decomposes overtime when exposed to air, so fresh solution should be made
daily.
Gluteraldehyde and formaldehyde
These two solutions are accepted as liquid sterilant provided that immersion time is sufficiently
long. To kill all spores in a clear liquid can take up to 12 hrs with Glutaraldehyde and even take
longer with formaldehyde. Presence of solid particles may lengthen the required period. Since
these solutions are volatile, they are toxic when they are inhaled and have skin contact.
Irradiation Sterilization:
Methods of sterilization exists using sterilization such as X-rays, gamma rays, electron beam or
subatomic particles.
X rays: X-rays are the form of ionizing energy allowing irradiating large packages. These
penetrations are sufficient to treat multiple pallet loads of packages. As X-ray sterilization is an
electricity passed process it can be termed on or off when needed.
Gamma rays: Gamma radiation requires shielding for the safety operators. They also require a
storage of a radioisotope usually Cobalt- 60 which continuously emits gamma rays. Since
gamma rays are highly penetrating, they are commonly used for sterilization of disposal medical
equipment such as needles, syringes, etc,
Electron beam processing:
It is commonly used for sterilization of medical equipment. Electron beam gives an On/Off
technology and provide much higher dazing rays than gamma & X-rays. Due to higher dosage
rate less exposure time is needed and thereby any potential degradation to polymers is reduced.
Ultra Violet radiation:
Ultra violet radiation is useful only for sterilization of surfaces and some transparent objects. UV
radiation is routinely used to sterilize the interior of biological safety cabins.
Filter sterilization
It cannot kill microbes but removes them. Clear liquids that would be damaged by heat,
irradiation or chemical sterilization can be sterilized by mechanical filtration. This method is
commonly used for sensitive pharmaceuticals and protein solution in biological research. The
pore size of filter ranges between 0.1mm-0.2mm diameter and they are used to remove microbes
from 1ml to several liters of volume. Prions cannot be removed by filtration. The filtration
equipment and the filter themselves may be purchased as presterilised disposal units or must be
sterilized disposal units that can be sterilized by user generally by autoclaving at a temperature
that does not damage fragile filter membrane. To ensure the best results pharmaceuticals,
steroids filtration is performed in a room with highly filtered air or in a laminar flow cabinet, a
device which produce a laminostream of HEPA filtered air.

Result:
Thus the principle of various sterilization techniques was studied.
Exp. No.1 b Date:
PRINCIPLES OF MICROSCOPY, PHASE CONTRAST & FLUORESCENT
MICROSCOPY
Aim
To study the principle, working and application of compound microscope and light microscope.
Principle
A microscope is an optical instrument used to magnify minute images of objects and organisms.
Most effective microscope should provide magnification, resolution and clarity of the image
Magnification of object or a specimen by a compound microscope occurs in two phases. The
first lens in the system (one close to system) is object lens or eyepiece. The objective lens forms
the initial image of the specimen called real image when this image is projected up through the
microscope body to plane eyepiece. The ocular lens forms the second image called the virtual
image. The magnifying power of objective lens usually varies from 40X to 100X. The total
power of magnification of final image formed by the combined lens is the product of separate
power.
Power of objective x power of ocular = Total magnification
10X (low power) x 10X = 100X
40 X (high power) x10X = 400X
100X (oil immersion) x10X = 1000X
Description of parts:
The parts of compound microscope
i) Reflecting mirror-
It is present in the basement of the microscope
ii) It is used to focus external light source on the specimen
2) Condenser- The condenser usually enhances a quality of light focused on specimen by
allowing only the required intensity of light to fall.
3) Objective lens- Magnifying lens of compound microscope objectives of various magnification
powers can be chosen according to requirements.
4) Eye piece - It magnifies the image produced by the objective the virtual image produced
reaches the observer.
5) Body tube - It provides the support for the objective and eyepiece at the each end.
6) Coarse adjustments- It moves the body tube up and down for exact focusing.
7) Stage- The plate formed below the objective lens with hole on the centre to allow light from
condenser to fall on the specimen. The slide is placed on stage.
8) Stage clips and nose piece -There are arrangements to hold the slide on to the stage. A rotating
disc which contains 3 to 4 objective lens are used to select the appropriate lens for magnification.
9) Arm -Supports the body tubes and adjustments
10) Inclination joins- Permits the tilting of upper portion of microscope.

Terms used:
Some of the following terms are used to describe the efficiency of the microscope.
1) Resolving power- It is simply defined as the ability to distinguish two adjacent points as
distinct and separate. In general, it is an optical properly essential for viewing a specimen
clearly. Since microorganisms are closely located a microscope must have appropriate resolution
to distinguish them clearly.
Formula:
Resolution power = Wavelength of light in nm X NA of optical lens
2
2) Numerical Aperture- The angle subtended by an axis outer layer still covered by objective
measures as aperture angle. The sine of this aperture angle is multiplied by refractive index of
the medium filling space between front lens and cover slip is measured as numerical aperture.
The value of NA is usually determined while designing the microscope and ranges from 0-1025
(oil immersion)
3) Limit of resolution: It is the smallest distance by which the two objects can be separate objects
4) Magnification- The phenomenon by which the micro organism can be viewed in large
proportion it can be measure as
Magnification = size of image lens in microscope / retinal image with an aided eye
5) Fluorescent microscope- The fluorescent microscope function is based on the presence of
chemical substances in cells which absorbs light and fluorescence.
Functions:
The practical function : Microorganisms are stained with fluorescent acid illuminated with blue
light which is absorbed and green light is unsettled by the dye. The function of exit filter is to
remove all. But the blue light the barrier filter blocks out blue light and allows green light to pass
through and reach the eye. The barrier filters are selected based on the basis of dye used.
Application:
It is used to identify the specific proteins present in cell based on antibody and antigen technique.
PHASE CONTRAST MICROSCOPE
It is valuable for studying living unstained cells and widely used in applied theory and biological
studies. It has a conventional light microscope fitted with phase contrast condenser. This special
optical system makes it possible to distinguish unstained structure within the cell which differs
slightly in its refractive index or the thickness.
Functions
Phase contrast microscope is based on the principle that rays of light moves at different speeds
through the material of different refractive index so light ray passing through the specimen are
out of phase, to varying degrees with the rays that are around the specimen. When these out of
phase rays are brought back together they will generate a contrast by interference meaning that
peaks of the light rays that arrive at all near the same time arrange on another.
Applications
The major advantages of phase contrast microscopy is avoidance of the necessity of fixing and
staining so it can be used to study activities and properties of live cells including cellular
movements and internal structure and hence it could not be distorted. It is also used to examine
the strained specimen.
Result

Thus, the principle, function and applications of various microscope are studied.
Exp. No.2 Date:
IDENTIFICATION OF PLANT, ANIMAL AND BACTERIAL CELL AND THEIR
COMPONENTS BY MICROSCOPY
Aim
To observe the permanent slides of plant cell, animals cell and bacterial cells in laboratory using
compound light microscope.
Materials required
Plant cell (specimen slide), animal cell (specimen slide) bacterial cell (specimen slide)
compound microscope.
Procedure
Preparation of plant cell
Plant cells are observed by sectioning tender plants. The stem cells or leaf cells can be observed
for this thin section of leaf, stem is taken. It is kept in water for few minutes so that the cell may
absorb some water and become bulk which enables us to have a clear view when observed under
microscope. The section is kept on the slide and also mounted with water to prevent them from
drying. It is observed under microscope.
Description
The plant cell is made up of cellulose. The cell membrane is found inside the cell wall. Nucleus
is large and oval in shape. The nuclear membrane surrounds the nucleus. Cytoplasm is thick jelly
like structure. Cell organelles are found inside it. Endoplasmic reticulum is a network of
membrane surrounding the nucleus Ribosome are small molecular bodies attached to the
endoplasmic reticulum, which helps in protein synthesis. Mitochondria are bean shaped and it
helps in breakdown of sugar molecules into energy. Vacuoles are few and large and food
lysosome is absent chloroplast is green, oval pigment containing chlorophyll.
Preparation of Animal cell
The animal cell was observed under microscope. The saliva drops are kept on the glass slide and
then smeared on the surface of glass slide. Allow it to dry for a few minutes. If strains are added
and checked the cells can be observed clearly. So some amount of stain is added to the slide and
allowed it to dry. After few minutes it becomes dried and then slide is observed under
microscope.
Description
Cell wall is absent. Cell membrane is the outer layer. Nucleus is large, oval in shape. Nuclear
membrane surrounds the nucleus. The cytoplasm is thick, jelly like substance. Cell organelles
are present in it. Endoplasmic reticulum is network of bodies attached to the nucleus which help
in protein synthesis. Mitochondria help us in the breakdown of sugar molecules into energy.
Vacuoles are many and smaller in size. Chloroplast is absent.
Preparation of bacterial cell
The bacterial cell is taken from bacterial culture. This culture is taken using a loop and placed on
the surface of the glass slide. Then heat it and fix it on the slide. Then the slide is stained and
observed under microscope.
Description
Cell wall is made up of peptidoglycan. Cell membrane is found inside the cell wall. Nucleus is
not bounded by the nuclear membrane. And the nucleolus is absent. Cytoplasm is thick liquid
with naked chromosome and the other basic cell components. Endoplasmic reticulum is absent.
Ribosome is present and it is distributed in the cytoplasm.

Result

Thus the plant, animal and bacterial cells are seen under microscope and identified.
Exp. No.3 Date:
Differentiate plant cells from animal cells using a staining procedures
Aim
To differentiate plant cell from animal cells by staining them.
Principle
Plants and animals specimens are stained with dyes before observing them through a microscope
because cells appear transparent under the microscope and to make the internal organelles and
other structures clearly visible, different stains or dyes are used for different parts of the cells.
Iodine binds to starch, making a blue-black color. Since there are starch molecules in onion
epidermal cells, the addition of iodine solution to a specimen of these cells allows the iodine to
infiltrate the epidermal cells and bind to the starch molecules present.

Materials required
glass slides ,slide covers, toothpick, Paper towel pieces, Methylene Blue Stain ,Iodine
Stain ,Paper towels, Microscope .
Procedure:
Procedure:
Part 1 – Cheek cells (animal)
1. Gently scrape the inside of your cheek with a toothpick. You may not be able to see
anything on the toothpick but cells are there.
2. Place one drop of water on a slide and mix the scrapings with the drop of water.
Cover the mixture with a cover slip
3. Place a drop of methylene blue stain near the edge of the cover slip and place a small
piece of paper towel to the opposite edge of the cover slip – this will draw the stain
under the cover slip and stain the cells.
4. Record the observation.

Part II Onion Cells (plant)

1. Remove a thin piece of onion epidermal tissue from the inner surface of the onion.
 2. Place one drop of water on a slide and place the onion skin flat in the water.
3. Place a drop of iodine stain near the edge of the cover slip using a piece of paper
towel.
4. Record the Observation.

Result:
Thus, the basic structure of animal and plant cell was differentiated using staining technique.
Exp. No.4 Date:
STAIN AND DIFFERENTIATE GRAM POSITIVE AND GRAM-NEGATIVE
BACTERIA

Aim
To perform a procedure for differentiating between two principle groups of bacteria- gram
positive and gram negative.
Principle
The most important differential stains used in microbiology is the gram stain named after
Dr. Christian gram. The gram stain reaction is based on the difference in chemical composition
of bacterial cell wall. Gram positive bacteria have a thick peptidoglycan layer while gram
negative cell is much thinner and surrounded by another lipid containing layer.
Materials required
Glass side, bacterial culture, gram stain, microscope, Bunsen burner, Inoculation chamber,
Inoculation loop, distilled water.
Reagents:
Primary stain- Crystal violet is used first and stains all cells purple.
Mordant- Gram iodine serves as a mordant, the resulting crystal violet complex serves to
intensity color of cells.
Decolorizing agents- Ethanol (95%) serves as a dual function as a protein dehydrating agent and
as a lipid solvent agent. In gram negative cell, the alcohol increases the priority of the cell wall
by dissolving the lipid in outer area. Thus CVI complex can be easily removed from thinner and
less highly cross-linked peptidoglycan layer.
Saffranin- It is a final reagent used to stain those cells which are previously decolored. The
gram negative cell undergoes decolorization. Because the thin peptidoglycan layer was
surrounded by a lipid layer.
Procedure
(i) A clean glass slide was taken
(ii) Using sterilization techniques a smear of each organism was prepared. A smear consisting of
a mixture of Staphylococcus and E. coli was prepared on another slide.
(iii) The smear was allowed to air dry and it was heat fixed in the usual manner.
(iv) The smear was gently flood with crystal violet and left for one minute and washed under tap
water.
(v) Grams iodine was added and other two allow to stand for one minute. It was washed under
tap water.
(vi) The smear was flood with decolorizing agents (95% ethanol) and it was washed under tap
water.
(vii) The smear was then flood with a counter stain (saffranin) for 45 seconds.
(viii) It is washed under tap water.
(ix) Blot dried with tissue paper and examined under oil immersion.
Observation
The stain was observed to show pink color when observed under microscope. Thus the bacterial
culture was identified as gram negative.
Result
Thus the given bacteria were identified as gram negative bacteria.
Exp. No.5 Date:
STAIN AND DIFFERENTIATE NUCLEATED AND NON-NUCLEATED BLOOD
CELLS

Aim
To observe nucleated and non-nucleated blood cells by using Leishman Staining procedure.
Principle
Blood cells absorb different dye substance, appears indistinct appearance of different cells when
Leishman stain is used. The RBC, platelets, Easinophils, is getting differentiable and could be
observed by using microscope. This helps in clinical biochemistry diagnosis of hematology.
Leishman stain consists of 2 dyes, Eosin (acidic dye) and Methylene blue (Basic dye). When the
blood cells stained with Leishman stain, Easinophils having basic nucleus and granules uptake
Eosin dye and give red colour. Basophils having acidic nucleus and granules uptake Methylene
blue gives blue colour. Neutrophils will partially uptake both dyes.
Materials required
Clean glass slide and cover slip, blood sample [fresh or EDTA added stored blood], Leishman
stain powder, methanol, phosphate buffer pH 6.8, microscope alcohol, sterilized needle for skin
puncture.
Preparation of stains
Leishman stain preparation
Weigh exactly 0.15g of Leishman powder, measure about 500ml of methanol, dissolve the above
stain powder completely in glass bottle. Store in a glass stopped bottle and use after 12 hours
period.
Preparation of phosphate buffer (Diluted solution)
Solution I
Weigh about 8gm of sodium hydroxide. Measure about 1000ml of distilled water in a glass
bottle and dilute the above solution in NaOH completely.
Solution II
Measure exactly 27.2gm of potassium dihydrogen phosphate. Take 1000ml of distilled water and
dissolve the above completely in a glass beaker.
Mixing
Mix 23.7ml of solution I and 50ml of solution II from the above. Take 20 ml from this and make
up to 1000ml using distilled water. The pH of this mixed solution should be 6.8.
Procedure
1. Take clean glass slide. Wipe with sterilized cotton using alcohol and keep on the table.

2. Make a skin puncture and collect a drop of blood and place on one end (2/3 length) of
glass slide.

3. Take glass slide and place in front of the blood drop at an angle of about 30 o to the slide
and move it backwards to make contact with the drop with a steady movement of band.
Spread the drop of blood along the slide quickly and confirm the thin film.

4. Dry the blood film on the glass slide in the air.

5. Add Leishman stain to the blood film on the glass slide and allow the stain for 3 minutes.

6. Then add phosphate buffer and allow it for 10-12 minutes.

7. Wash the excess stain with slowly running tap water

8. Allow these slide to dry completely in air

9. Observe the slide by using microscope in 10X, 40X and 100X oil immersion and record
the observation

Observation
The cells appeared in microscope are observed and identified. The following cells were
observed.
Basophil- purple black colour (irregular nucleus)
RBC- deep pink colour
Easinophil- reddish orange (two lobbed nucleus)

Result
Thus the blood cell differentiation was done by using Leishman staining.
Exp. No.6 Date:
STAINING METHOD TO IDENTIFY BLOOD PARASITES

Aim
To identify the morphology of blood cells from blood sample by using Giemsa staining
Principle
This blood smear stained with Giemsa stain absorbs the dye and exhibits its cell morphology.
Giemsa stain enables the evolution of WBC, RBC and platelets morphology. The presence of
parasites or any foreign cells in blood can be identified based on morphology. Giemsa stain
consists of 3 dyes, Eosin (acidic dye) and Methylene blue (Basic dye) and Azure B(Basic dye).
When the blood cells stained with Geimsa stain, Easinophils having basic nucleus and granules
uptake Eosin dye and give red colour. Basophils having acidic nucleus and granules uptake basic
dyes Methylene blue and Azure B gives blue colour. Neutrophils will partially uptake both dyes.

Materials required
Blood sample, Giemsa stain, ethanol, clean glass slide, microscope, alcohol, sterilized cotton,
sterilized needle for skin puncture.
Stain preparation
Giemsa stain powder - 0.5g
Glycerine - 33ml
Absolute alcohol - 33ml
Mix the above contents well and keep it for further use.
Working solution
Stock solution- 1 ml
Distilled water- 50ml (pH 6.5)
Procedure
1. Take clean glass slide. Wipe with sterilized cotton using alcohol and keep on the table.

2. Make a skin puncture and collect a drop of blood and place it on the end of slide leaving
about 1.5cm.

3. Take another clean glass slide and place it in front of the blood drop at an angle of about
30o to the bottom slide and move it backwards to make contact with the drop. With a
steady movement of the hand spread the drop of blood along the slide quickly and
confirm the thin film.

4. Dry the blood film on the glass slide in the air

 5. Fix the above in methanol for 2-3 minutes at room temperature

6. Add few drops of Giemsa stain on dried blood film to cover completely and allow
staining for 2-5mins.

7. Gently wash with running tap water and leave for 3-4 minutes in distilled water

8. Allow to dry in the air

9. Examine under microscope with higher magnification

Observation
Different blood cells like monocytes, easinophils, RBC, and neutrophils were observed. Apart
from that the parasites and foreign cells also could be observed
Result
Thus the blood cells were differentiated by using Giemsa staining and identified under the
microscope.
Exp. No.7 Date:
CELL LYSIS THROUGH OSMOSIS AND TONICITY

Aim
To demonstrate the principles of cell lysis through osmosis using the natural semipermeable
membrane (potato cells)
Principle
Osmosis may be defined as the movement of particles of water molecules from a solution where
the concentration is higher to another solution where the concentration is lower.
Osmosis works on the principle of molecule transport in various life processes. Semipermeable
membrane is plasma membrane of living cells.
Materials required
Conc. NaCl solution, whole potato, beaker, distilled water, knife, measuring jar.
Procedure
1. Peel the potato and wash it with distilled water
2. Cut the potato and make a well
3. Pour about 10ml of NaCl solution in the well of potato
4. Keep the potato partly immersed with water in the beaker.
5. Allow the set up for sixty minutes.
6. Observe the quantity of solution in the well of potato.
7. Calculate for the concentration.
8. Observe the osmosis process.
Observation
The volume of concentrated NaCl solution (10%) poured into the potato well was observed to
increase.
Result
Thus osmosis was performed through semipermeable membrane.
Exp. No.8 Date:
PERIPHERAL BLOOD MONONUCLEAR CELL SEPARATION FROM BLOOD
Aim
To separate mononuclear cells from blood

Principle
Peripheral blood is the primary source of lymphoid cells for investigations of the human immune
system. Its use is facilitated by Ficoll-Hypaque density gradient centrifugation— a simple and
rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of
the density differences between mononuclear cells and other elements found in the blood sample.
Mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer because they have a
lower density; in contrast, red blood cells (RBC) and granulocytes have a higher density than
Ficoll-Hypaque and collect at the bottom of the Ficoll-Hypaque layer.

Materials required
Blood sample, microscope, EDTA, test tube, haemocytometre, tryphan blue, syringe,
micropipette, phosphate buffer solution, ficol hypaque solution.

Procedure
1. Blood is drawn using sterile syringe and transferred into a tube containing EDTA. The
contents were mixed thoroughly.
2. To blood sample, equal volume of ficol hypaque solution was overlaid without mixing.
3. This was centrifuged at 2000 rpm for 30 minutes at room temperature
4. Four layers were obtained. The lowest layer contains RBC.
5. The next layer contains ficol hypaque with granulocytes.
6. The third layer contains mononuclear cells, the upper layer contains plasma.
7. The plasma layer was carefully removed with a micropipette and discarded.
8. The layer containing mononuclear cells was transferred to a sterile tube. Then wash with
phosphate buffer saline 3 times by centrifugation at 2000 rpm for 20 minutes.
9. The clear pellet was collected
10. 1µl of mononuclear cells were mixed with 10 µl of tryphan blue and viable cells were
counted in Haemocytometer.

Observation
Mononuclear cells appear as water bubble against blue back ground, the dead cells
appear as deep blue colour. Mononuclear cells were observed between plasma and ficol hypaque
layer.

Result
Thus the mononuclear cells were separated from blood cells.
Exp. No.9 Date:
GROWING ROOT TIPS OF Allium cepa AND STUDY DIFFERENT STAGES OF
MITOSIS
Aim
To observe different stages of mitotic cell division occurring in onion by using onion root tip
with staining procedure.
Principle
The genetic information of plants, animals and other eukaryotic organisms resides in several
individual DNA molecules, or chromosomes. All cells must replicate their DNA when dividing.
During DNA replication, the two strands of the DNA double helix separate, and for each original
strand a new complementary strand is produced, yielding two identical DNA molecules.
DNA replication yields an identical pair of DNA molecules (called sister chromatids)
attached at a region called the centromere. DNA replication in eukaryotes is followed by the
process called mitosis which assures that each daughter cell receives one copy of each of the
replicated chromosomes. During the process of mitosis, the chromosomes pass through several
stages known as prophase, metaphase, anaphase and telophase. The actual division of the
cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages,
particular events occur that contribute to the orderly distribution of the replicated chromosomes
prior to cytokinesis.
A nuclear stain (Safranin) is used for staining onion root tips which required preparation
and treatment with chemicals and heating.
Materials required
Onion root tips (young stage), Safranin, Glacial acetic acid, distilled water, glass slide, cover
slip, dissection needle, microscope.
Procedure
1. Onion root tips are cut into small pieces and kept on a clean glass slide
2. Add few drops of glacial acetic acid, water and dye.
3. Slightly heat the specimen
4. Cover with cover slip and gently press it
5. Absorb excess stain and Neat slide is prepared
6. Observe the slide under microscope
7. Record the stages of cell division

Result
Thus the different stages of mitotic cell division occurred in onion by using onion root tip with
staining procedure was observed.
Exp. No.10 Date:
STUDY OF MEIOTIC CELL DIVISION USING PERMANENT SLIDES

Aim:
To perform Meiotic cell division in the given sample (testis follicles)
Materials Required:
Acetocarmine, Glass slide, Cover slip, Lab needle, Sample (Grasshopper testis follicles),
Eppendorfs, Pasteur pipettes, Watch glass, Razor blades, Dissection box
Procedure:
1. Identify the male grass hopper, which has lean and lengthy tail
where as, female has short and blunt tail.
2. Turn the dorsal side of grasshopper and count the 7th segment from the tail which is
abdominal portion of the grasshopper.
3. Using sharp scalpel cut at the 7th segment and press gently to release a yellow fluid
clumps (which is genital organ covered with yellow fat bodies).
4. Transfer the clump carefully to the vessel containing saline for washing.
5. Shake gently with lab needle so the fat bodies and follicles (white fibers in
elongated shape) get separated.
6. Transfer this follicle bunch to a Petri dish and separate it into individual follicles.
7. Take a pre-cleaned slide and transfer a follicle to the centre of the slide.
8. Add a drop of acetocarmine and incubate it for 5minutes.
9. If it is dehydrated add another drop of acetocarmine and cover with cover slip.
10. Using your forefinger press gently and prepare squash carefully.
11. Remove the excess stain by blotting technique.
12. Focus at 10x and examine at 40x for sharp image using Light microscope.

Result:
Different stages of meiotic cell division could be visible.
Interphase - Condensed chromosome.
Prophase - Thread like chromosome structure.
Metaphase - Spindle fibers and distinct chromosome at center of the cell.
Anaphase - Stretching of chromatids towards opposite poles.
Telophase - cell undergoes equal division.
Exp. No.11 Date:
THIN LAYER CHROMATOGRAPHY
Aim
To separate the amino acids by using thin layer chromatography.
Principle
Amino acids are the basic units of the polypeptide chain. They are high molecular weight
compounds which can be separated by using thin layer of silica gel coating based on the
molecular weight. Silica gel act as the stationary phase and the solvent mixture acts as mobile
phase which carry the amino acid molecule based on their molecular weight. The distance
travelled by the amino acid molecule in the stationary phase is taken in the calculation. Based on
this technique individual amino acids of unknown origin are separated and identified using
known sample value.
Materials required
Thin layer plates of silica gel, Chromatogram chamber, solvent mixture (butanol, Glacial acetic
acid, water in ratio 4:1:1), micropipettes, known amino acid sample, unknown amino acid
sample, Ninhydrin solution.
Procedure
1. Clean the glass slide with ethanol.
2. Keeps the slides on the table
3. Take a measured small quantity of silica gel and mix with distilled water in the ratio of
1:2.
4. Pour the mixed aqueous slurry of gel on the glass slide
5. Spread the silica gel slurry uniformly on the glass slide to obtain a thin surface of 250mm
thickness.
6. Air dry the above silica gel coated glass slides for 10 to 15 minutes
7. Now activate the coated silica gel plates by heating at 90 to 100 oC for one hour and
allowed to cool.
8. Spot out a drop of amino acid over the slide and make a mark which should be near end
of the slide leaving about 1 to 1.5 cm distance.
9. In the same fashion, spot out an unknown mixture of amino acids on a glass slide.
10. The sample spotted TLC plates should be kept in a Chromatogram chamber containing
solvent mixture.
11. Allow the Chromatogram to develop, ie, till the solvent front is reached on the top of the
plate.
 12. Stop the Chromatogram and remove the developed plate by leaving 1cm distance on the
edge of the plate
13. Air dry the developed TLC plate for 10 to 15 minutes.
14. Now apply Ninhydrin solution by spraying on the developed TLC plate.
15. Allow the slide to dry in the air and heat it slightly on the hot plate
Observation
Observe the colour spot on the developed silica gel plate. Measure the distance of solvent front
reached on the silica gel plate and then record the value. Measure the distance of sample spot
moved on the silica gel plate and record the value.
Calculate the Rf values of the sample spot and solvent front.

Rf value = Distance travelled by solute

Distance travelled by solvent

Result
The Rf value of methionine sample
Exp. No.12 Date:
CELL VIABILITY: TRYPHAN BLUE ASSAY

Aim:
To calculate the percentage viability of cell by using Tryphan Blue Assay.
Principle:
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. In this test, a cell
suspension is mixed with dye and then visually examined to determine whether cells take up or
exclude dye.