Laboratory Manual for Cell Biology

(BIO201)

Virtual University of
Pakistan
 Contents

S. No. Practical P. No.

1 Microscopy 3
2 Staining techniques 6
3 Study of Prokaryotic Cell 8
4 Study of Eukaryotic Cell 13
5 Study Plant cell 15
6 Study Animal cell 17
7 Cell structure in Staminal hair of tradescantia 20
8 Study of different types of plastids 21
9 Study of cellular reproduction 24
10 Study of Mitosis (Prepared slides) 27
11 Study of Mitosis (Prepared slides) 31
12 Mitosis: smear/squash preparation of onion roots 34

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 Practical No: 1
Microscopy

Microscope
A microscope (from the Ancient Greek: μικρός, mikrós, small and σκοπεῖν, skopeîn, to look or
see) is an instrument used to see objects that are too small to be seen by the naked eye.
Microscopy is the science of investigating small objects and structures using such an instrument.
Microorganisms are so small that they cannot be seen by naked eye. The optical instrument that
magnifies the image of these organisms and enables us to view their morphological features is a
microscope. Antony van Leeuwenhoek is often considered as the father of microscopy.
Most bacteria measure in the range of 0.5 to 4 µm. Mycoplasma and Coxiella are the shortest
among bacteria and Spirochetes the longest. Viruses are ultramicroscopic structures; they can’t
be seen by compound microscopes. They can be visualized by electron microscopes and their
measurements are in nm (nanometer).
Various types of microscopes are
There are several different types of microscopes used in light microscopy, and the four most
popular types are Compound, Stereo, Digital and the Pocket or handheld microscopes. Some
types are best suited for biological applications, where others are best for classroom or personal
hobby use.
 Simple microscope
 Compound microscope
 Dark ground microscope
 Phase contrast microscope
 Fluorescent microscope
 Interference microscope
 Electron microscope
 Atomic Force microscope
 Polarization microscope

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Fig 1.1: Detail of Microscope

A microscope is commonly used in a microbiological study of organisms. The basic modern

microscope found in schools, hospitals, and research centers is a compound microscope which
has a series of lenses to collect and focus the light transmitted through the specimen. The various
parts of a microscope with their associated function are mentioned below.
Tube:
Tube or the body tube, connects the eyepiece to the objective lenses.
Eyepiece or ocular lens:
Eyepiece is the lens, present at the top and is used to see the objects under study. Eyepiece lens
contains a magnification of 10X or 15X.
Resolving nosepiece:
It is also known as the Turret. Resolving nosepiece has holders for the different objective lenses.

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It allows the rotation of the lenses while viewing.
Objective lenses:
Generally, three or four objective lenses are found on a microscope, with ranges of 10X, 40X,
100X powers. Lenses are color coded, the shortest lens is of the lowest power, and the longest
lens is high power lens.
Diaphragm:
Diaphragm helps in controlling the amount of light that is passing through the opening of the
stage. It is helpful in the adjustment of the control of light that enters.
Coarse adjustment knob:
Used for focus on scanning. Usually the low power lens is used enabling the movement of the
tube.
Fine adjustment knob:
Used for focus on oil. It moves the body tube for focusing the high power lens.
Arm:
It supports the tube of the microscope and connects to the base of the microscope.
Stage:
A flat platform used for placing the slides under observation.
Stage clip:
Stage clips hold the slides in proper place.
Condenser:
The main function of condenser lens is focusing the light on the specimen under observation. It
gives a sharp image.
Base:
It provides basal support for the microscope.
Power switch:
It turns the illumination on or off.
Proper usage and handling the microscope:
Microscope is a delicate instrument that must be handled with care. All kinds of mechanical
shocks must be avoided. The microscope must be lifted by holding its arm in one hard and
supporting the base of the microscope with the palm of the other hand. The microscope must be
kept in dust free environment. The oil must be wiped clean using a soft tissue paper. After usage
the slide must be removed and cleaned before returning to its original place.

Practical No: 2Staining techniques

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Staining
A technique that is used to define and examine different types of microbes
Types of staining techniques
1. Simple stain techniques
Simple staining is performed with basic dyes such as crystal violet or methylene blue.
2. Differential stain techniques
Differential stain technique distinguishes two kinds of organisms. There are two types of this
technique; one is Gram stain technique while other is acid fast technique.
Gram stain technique
A most commonly used technique for study of microbes like bacteria.
Principle
This technique separates bacteria into two groups, Gram‐positive bacteria and Gram‐negative
bacteria.
Materials
 Clean glass slides
 Inoculating loop
 Bunsen burner
 Bibulous paper
 Microscope
 Lens paper and lens cleaner
 Immersion oil
 Distilled water
 18 to 24 hour cultures of organisms
Reagents
Primary Stain - Crystal Violet
Mordant - Grams Iodine
Decolourizer - Ethyl Alcohol
Secondary Stain - Safranin
Procedure
• Smear preparation
A small sample of microorganisms is placed on a slide and permitted to air dry. The smear is
heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them adhere
to the slide, and permits them to accept.
• Primary stain
Apply crystal violet as primary stain
• Mordant
Use iodine as a mordant
• Decolourizer
Wash with ethyl alcohol that acts as a decolourizing agent. Gram‐positive bacteria retain the
crystal‐violet iodine stain; however, the Gram‐negative bacteria lose the stain.
• Secondary stain

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The Gram‐negative bacteria subsequently stain with the Safranin dye, the counterstain. These
bacteria appear red under the oil‐immersion lens, while Gram‐positive bacteria appear blue or
purple, reflecting the crystal violet retained during the washing step.

Fig 2.1: Comparsion between gram positive and negative

Practical No: 3Study of Prokaryotic cell

Principle
Prokaryotic cells are cells that do not have a true nucleus or membrane-bound organelles.
Organisms within the domains Bacteria and Archaea have prokaryotic cells, while other forms of

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life are eukaryotic. However, organisms with prokaryotic cells are abundant and make up much
of Earth’s biomass.
Objective
To study the specimen preparation step for a prokaryotic cell
To study the procedure of simple staining
Light microscopic examination of the cells requires fixing and staining of the cells prior to
observation. These processes can increase the visibility and clarity of the cell under study.
Fixation
Fixation is the process by which internal and external structures of a cell are preserved. Cell
should not undergo any change in its state during the process of staining and observation. To
ensure this, it is necessary to toughen the cell and to stop any chemical reaction that might take
place within the cell that is the cell should be fixed at its current state. Based on the method of
fixation a microscopic slide might be used for longer or shorter period of time or used as
temporary or permanent slides. Even when great care is taken to fix the cells properly sometimes
artifacts might be produced. Fixation step must therefore be standardized. The procedure for
fixation varies with the fixative and the type of tissue or cell under study.
There are 2 types of fixation
Heat fixation: This helps to preserve the overall morphology of a cell. This is commonly used in
smear preparation and preparation of temporary slides.
Chemical fixation: In this method chemicals are used to preserve fine cellular substructures and
morphology of more delicate microorganism. The chemicals used for fixation are known as
fixative. A fixative might either stop any internal biochemical processes that take place within a
cell, protect the cell from external damage or might increase the mechanical strength and
stability. Chemical fixatives are commonly used in preparation of permanent slides.
Staining
The sample under examination is called the specimen. Specimen preparation is an important step
in light microscopic examination of a given sample. Light microscopy can reveal better details if
the different parts of the cell are given different color. This process of coloring a cell is called
staining and the compounds that are used to color cells are termed stains or dye.
Staining is usually done to enhance the contrast of a cell. This helps to highlight the structure
under the study of interest. Staining technique used in light microscopy will vary with the
objective of study. If the objective of the study is to observe the cell structure and size, staining
the cell with any stain that is taken up by the cell will do. But if the objective is to study a
particular cell structure, then depending on the requirement two or more stains may be used to
differentiate the structure from the surrounding cytoplasm. Staining technique that uses a single
stain is called simple staining and the one which uses more than one stain is called differential
staining.
The compounds that are used as stain contain two important features.A chromophore that gives
color to the compound and the dye that bind to the cell either through covalent bonds or ionic
bonds or through hydrophobic bonds, Ionizable dyes are classified intoBasic dye (having
positive charged groups binding to the negatively charged structure) e.g. Methylene blue and
Acidic dye (having negatively charged group binding to the positively charged cell structures).

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E.g.: Eosin, acidic fuchsine. The effectiveness of these dye vary with the pH. An example of a
dye that stains due to formation of double bond is Schiff's reagent that covalently attaches to
deoxyribose.
Staining can be done by simply immersing the sample in the stain. The actual staining process
involves immersing the sample in dye which is then followed by rinsing and observation.
Sometimes additional step such as heating might be required. Many dyes require the use of a
mordant. Mordant are chemical compounds that form an insoluble precipitate with the stain.
These chemicals are mainly used to intensify the color given by the cell.
Commonly used stains to study the prokaryotic cell
 Crystal violet: to study the cell size and morphology
 Safranin: to study the cell size and cell morphology
 Malachite green: to observe the endospore
 Methylene blue: to study the cell size and cell structure
Albert stain(toluidine blue, malachite green glacial acetic acid 95% alcohol and distilled water)-
stain metachromatic granules.
Indian ink: to observe the capsule that is seen as an unstained halo around the organism.
Steps Involved in Specimen Preparation
The major step involved in specimen preparation is bacterial smear preparation and staining. The
bacterial smear is a dried preparation of bacterial cells on a glass slide. The purpose of making
bacterial smear is to fix them onto the glass slide. A bacterial smear is prepared on a clean glass
slide and is prepared from bacteria that have grown on nutrient agar medium. These are then heat
fixed in flame so that the bacterial cells adhere to the glass slide and does not run along with
stain when excess stain is being washed. The smear should not be very thick as thicker smears
are more difficult to observe and take longer time to dry. The smear should not be too thin as
thinner smear will not contain sufficient amount of bacteria to examine under the microscope.
The smear should be an even one. Otherwise the cells will be clumped at one portion of the
smear and the other portion there will be insufficient cells for observation. There should only be
one smear per glass slide. It is easier to prepare a smear from a solid sample than from a liquid
sample. Smear adheres to the glass slide when it is heat fixed which is done by waving it over a
flame. Heat fixation should only be done after completely drying the smear in air. Too much
heating can lead to damage of the cell wall. Then the required stain is placed on the smear and
washed away after a suitable time period of exposure. Sometimes a mordant might be added to
enhance the coloring of the cell. When studying certain cell structures an additional step like
heating might be required to facilitate the entry of stain into that particular component.
The experiment illustrated here in this section is simple staining. Simple staining is one amongst
the easiest staining procedure and is mainly done to study the morphology, arrangement and
appearance of bacterial cells. The principle behind simple staining is very simple. The basic dyes
such as crystal violet, Methylene blue have a positive charge and enhance can very easily stain
the negatively charged cells. Based upon the dye used the time of exposure to the dye should be
varied.

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Methylene blue - 1 minute, Crystal violet -1 minute, Carbolfuchsin - 20 seconds. The excess
stain is then washed off, blot dried and observed under a microscope.
Prokaryotic cell Structures that are Visible through Light microscope
The maximum magnification that can be obtained from a light microscopy is 1000x or 1500x
and the maximum resolution is 0.2μm. Hence only a very few details can be observed. The use
of different staining techniques helps to identify and observe different components of the cells.
Cell size and shape can be easily determined with the help of light microscopy.
Size and morphology of bacteria:
Morphology:
Bacterial morphology is very diverse. The two major types of bacteria based on its morphology
are:
A) Coccus.
B) Bacillus.
Apart from these shapes, square shaped and rectangular shaped bacteria are also found. Some
bacteria are found in a variety of shapes and lack a single, characteristic form. These are called
pleomorphic.
Coccus:
Cocci are roughly spherical cells. They may exist as individual cells or are clustered together. In
some cases as in the genus Neisseria the cells may be found in pairs. Such a pair of Cocci is
known as diplococci.Sometimes they may be found attached to each other as in chains. This type
of morphology is shown by genus Streptococcus, Enterococcus and Lactococcus.In certain genus
like Staphylococcus the cells are clustered together like grapes.

Fig 3.1: Detail of coccus

Bacilli:
Bacilli are rod shaped bacteria. Various bacilli may vary in length to width ratio. There are
certain bacteria that are so short that they resemble coccus and are termed coccobacilli.The shape
of the rod's end may be flat, cigar-shaped or circular. Some of the bacteria as distinctive curves
and are known as vibrios.Some bacteria are shaped like long rods twisted into spirals or helices
are called spirillum.

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Fig 3.2: Detail of bacillus
Size of Bacteria:
Bacterial cells are found in a number of sizes. Smallest bacteria belonging to the genus
Mycoplasma is only 0.3μm in diameter. Most bacterial size falls in the range of 1-5 μm. The
largest bacterium is Thiomargaritanamibiensis. These are generally 0.1 x 0.3 mm (100 x 300 μm)
wide. The species was discovered by Heide N. Schulz and others in 1997, in the coastal
sediments of Walvis Bay (Namibia).
Apart from cell structure and cell morphology certain other structures of cell can be identified
and observed using light microscopy. Special staining techniques are employed to observe these
structures. Most of the time, these staining techniques are called differential staining. Some
examples of differential staining are:
Grams staining: differentiate gram negative and gram positive cells.
Endospore staining: to identify endospore. (An endospore is a non-reproductive, tough and
dormant structure produced by certain bacteria. These structures are extraordinarily resistant to
the environmental stresses such as ultraviolet radiation, gamma radiation, heat, chemical
disinfectants, and desiccation.)
Materials:
 Microscope
 Specimen
 Simple staining dyes
 Slides
 Water beaker
Procedure:
 Clean and adjust the microscope carefully.
 Prepare the specimen by simple staining technique.
 Fix the smear slide under microscope.
 Notice and write observations of respective cells examined by microscope.
Study of Prokaryotic cell under microscope
Organisms having no well-defined nucleus are called prokaryotes. Examples are Monera or
Bacteria and Archaea. Typical structure of a prokaryotic cell and its components observed under
microscope are given in figure 1.

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Figure 3.3: A typical structure of bacterial cell
Cell Wall
Provide shape and protection to cell.
Pilli
Organ for the exchange of genetic material from one bacterium to another
Capsule
Sticky projections that cover cell wall and provide protection from phagocytosis, chemicals and
dehydration
Flagellum
A “whip-like;” structure that helps in movement
Plasma Membrane
A thin, flexible asymmetrical “sac” that holds the cytoplasm and serves as a passageway for
anything that enters or leaves the cell such as nutrients and gases.
Cytoplasm
Contains organelles for different functions inside the cell
Nucleoid or Nuclear Body
Area of the cytoplasm where the DNA strand is located
Plasmids
Extra chromosomal piece of DNA,plasmids are often the site of genes that code for resistance to
antibiotics.
Ribosomes
The function of prokaryotic ribosomes widely depends on the bacteria.

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 Practical No: 4Study of Eukaryotic cell

Eukaryotic cells are cells that contain a nucleus and organelles, and are enclosed by a plasma
membrane. Organisms that have eukaryotic cells include protozoa, fungi, plants and animals.
These organisms are grouped into the biological domain Eukaryota. Eukaryotic cells are larger
and more complex than prokaryotic cells, which are found in Archaea and Bacteria, the other
two domains of life. Typical structures of Eukaryotic cells including plant and animal cell and its
components are given below in figure 2.

Figure 4.1:Representing a typical animal and plant cell

Characteristics of Eukaryotic Cells
Eukaryotic cells contain a variety of structures called organelles, which perform various
functions within the cell. Examples of organelles are ribosomes, which make proteins, the
endoplasmic reticulum, which sorts and packages the proteins, and mitochondria, which produce
the energy molecule adenosine triphosphate (ATP). They also have a true nucleus, which
contains the genetic material DNA and is surrounded by a nuclear envelope. All of the organelles
are stabilized and given physical support through the cytoskeleton, which is also involved in
sending signals from one part of the cell to the other. In eukaryotic cells, the cytoskeleton is
composed mainly of three types of filaments: microtubules, microfilaments, and intermediate
filaments. The gel-like substance that surrounds all the organelles in the cell is called cytosol.
Examples of Eukaryotic Cells
Plant Cells
Plant cells are unique among eukaryotic cells for several reasons. They have reinforced,
relatively thick cell walls that are made mostly of cellulose and help maintain structural support
in the plant. Each plant cell has a large vacuole in the center that allows it to maintain turgor
pressure, which is pressure from having a lot of water in the cell and helps keep the plant upright.
Plant cells also contain organelles called chloroplasts which contain the molecule chlorophyll.

13
This important molecule is used in the process of photosynthesis, which is when a plant makes
its own energy from sunlight, carbon dioxide, and water.
Fungal Cells
Like plant cells, fungal cells also have a cell wall, but their cell wall is made of chitin (the same
substance found in insect exoskeletons). Some fungi have septa, which are holes that allow
organelles and cytoplasm to pass between them. This makes the boundaries between different
cells less clear.
Animal Cells
Animal cells do not have cell walls. Instead, they have only a plasma membrane. The lack of a
cell wall allows animal cells to form many different shapes, and allows for the processes of
phagocytosis “cell eating” and pinocytosis “cell drinking” to occur. Animal cells differ from
plant cells in that they do not have chloroplasts and have smaller vacuoles instead of a large
central vacuole.
Protozoa
Protozoa are eukaryotic organisms that consist of a single cell. They can move around and eat,
and they digest food in vacuoles. Some protozoa have many cilia, which are small “arms” that
allow them to move around. Some also have a thin layer called a pellicle, which provides support
to the cell membrane.

Practical No: 5Study of Plant cell

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Plant cells are eukaryotic cells with a true nucleus along with specialized structures called
organelles that carry out certain specific functions.
Structure of Plant Cell
The plant cell is rectangular and comparatively larger than the animal cell. Even though plant
and animal cells are eukaryotic and share a few cell organelles, plant cells are quite distinct when
compared to animal cell as they perform different functions. Some of these differences can be
clearly understood when the cells are examined under an electron microscope.
Plant Cell Structure
Just like different organs within the body, plant cell structure includes various components
known as cell organelles that perform different functions to sustain itself. These organelles
include:
Cell Wall
It is a rigid layer which is composed of cellulose, glycoproteins, lignin, pectin, and
hemicellulose. It is located outside the cell membrane. It comprises proteins, polysaccharides,
and cellulose.
The primary function of the cell wall is to protect and provide structural support to the cell, to
protect cell against mechanical stress and to provide form and structure to the cell. It also filters
the molecules passing in and out of the cell.
Cell membrane
It is the semi-permeable membrane that is present within the cell wall. It is composed of a thin
layer of protein and fat. The cell membrane plays an important role in regulating the entry and
exit of specific substances within the cell.
Nucleus
The nucleus is a membrane-bound structure that is present only in eukaryotic cells. The vital
function of a nucleus is to store DNA or hereditary information required for cell division,
metabolism, and growth.
Mitochondria
Mitochondria are known as the powerhouses of the cell. The energy required for various
chemical activities needed for life is released by mitochondria in the form of ATP (Adenosine
triphopshate) molecules.
Plastids
Plastids are present only in plant cells. There are two types of plastids chromoplasts (colored
plastids) and leucoplasts(white or colorless plastids).
Chloroplasts Plastids containing the pigment chlorophyll are known as chloroplasts.
Chloroplasts are important for photosynthesis in plants. Chloroplasts also contain various yellow
or orange pigments in addition to chlorophyll.
Leucoplasts are primarily organelles in which materials such as starch, oils and protein granules
are stored.
Vacuoles
Vacuoles are storage sacs for solid or liquid contents. Vacuoles are small sized in animal cells
while plant cells have very large vacuoles. The central vacuole of some plant cells may occupy

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50-90% of the cell volume. In plant cells vacuoles are full of cell sap and provide turgidity and
rigidity to the cell.

Figure 5.1: Structure a typical Plant cell

Practical No: 6Study of Animal cell

Animals are a large group of diverse living organisms that make up to three-quarters of all
species on earth. With their ability to move, to respond to stimuli, respond to environmental
changes and adapt to different modes of feeding defense mechanisms and reproduction, all these

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mechanisms are enhanced by their constituent elements in the body. However, animals cannot
manufacture their own food like plants and hence they depend on plants in one way or
another.Animal cells and plant cells have similarities and differences. An animal cell does not
have a fixed shape and cell wall whereas as a plant cell have a fixed shape and surrounded by a
cell wall. Like a plant cell, an animal cell has a plasma membrane, cytoplasm and nucleus.
Animal cell size and shape
Animal cells come in all kinds of shapes and sizes, with their size ranging from a few millimeters
to micrometers. The largest animal cell is the ostrich egg which has a 5-inch diameter, weighing
about 1.2-1.4 kg and the smallest animal cells are the neurons of about 100 microns in diameter.
Animal cells are smaller than the plant cells and they are generally irregular in shape taking
various forms of shapes, due to lack of the cell wall. Some cells are round, oval, flattened or rod-
shaped, spherical, concave, rectangular.
Animal cell structure

Fig 6.1: Detail of animal cell organelles

Nucleus
The nucleus contains all the genetic material in a cell. This genetic information is called
deoxyribonucleic acid (DNA). DNA contains all the instructions for making proteins, which
control all of the body’s activities. Therefore, the nucleus is like the manager’s office of the cell.
Ribosomes
Ribosomes are organelles found in both prokaryotic and eukaryotic cells. They are like mini
machines that synthesize all the proteins in the cell. In any single animal cell, there can be as
many as 10 million ribosomes.
Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a network of flattened, membrane-bound sacs that are
involved in the production, processing, and transport of proteins that have been synthesized by
ribosomes.There are two kinds of endoplasmic reticulum: smooth and rough. The rough ER has
ribosomes attached to the surface of the sacs. Smooth ER does not have ribosomes attached and
has functions in storage, synthesizing lipids, removing toxic substances.

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Golgi apparatus
The Golgi apparatus also called the Golgi complex or Golgi body, receives proteins from the ER
and folds, sorts, and packages these proteins into vesicles.
Lysosomes
Lysosomes are a type of vesicle. Vesicles are spheres surrounded by a membrane that excludes
their contents from the rest of the cytoplasm.
Mitochondria
Mitochondria are the energy-producing organelles, commonly known as “the powerhouse of the
cell.” The process of cellular respiration occurs in the mitochondria. During this process, sugars
and fats are broken down through a series of chemical reactions, releasing energy in the form of
adenosine triphosphate (ATP).
Cytoplasm
The cytosol is the gel-like liquid contained within cells. The cytosol and all the organelles within
it – except for the nucleus – are collectively referred to as the cell’s cytoplasm.
Cytoskeleton
The cytoskeleton is a network of filaments and tubules found throughout the cytoplasm of the
cell. It has many functions: it gives the cell shape, provides strength, stabilizes tissues, anchors
organelles within the cell, and has a role in cell signaling.
Cell Membrane
The cell membrane surrounds the entire cell and separates its components from the outer
environment. The cell membrane is a double layer made up of phospholipids (called the
phospholipid bilayer). Phospholipids are molecules with a phosphate group head attached to
glycerol and two fatty acid tails. They spontaneously form double membranes in water due to the
hydrophilic properties of the head and hydrophobic properties of the tails.
Experiment
Objective:
 To prepare slides of animal cell (Human epidermal cell)
 The inner part of your cheek is covered with skin cells called epidermal cells. These
surfaces cells can be easily obtained for study
Procedure
 Gently rub the soft end of a cotton swab on the inner surface of your cheeks.
 Rub the end of the swab onto a clean glass slide
 Add a very small drop of methylene blue dye
 Allow the preparation to stand for one minute
 Cover the preparation with a cover slip
 Examine under low power, then medium and finally high power.
Observations
Draw what you have seen in the microscope as diagram and label cell membrane, nucleus and
cytoplasm.
Table 6.1:Similarities and dissimilarities in plant and animal cell
Organelles Plant Cell Animal Cell

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 Cell wall made of cellulose is present in
Cell Wall Cell wall is absent
almost all cells
Plastids like leucoplasts, chloroplast and
Plastids No plastids found
chromoplasts are present
Plants cells have chloroplasts to prepare
Chloroplasts Chloroplasts completely absent
their own food
Vacuoles are usually absent or
Vacuoles Cell sap containing vacuoles are present
one or more small vacuoles are
Lysosomes Lysosomes not evident Lysosomes occur in cytoplasm
Due to the presence of the vacuole at the
Nucleus is usually located
Nucleus center of the cell, nucleus may be located
centrally
at the edge of the cell
Animal cells have a single highly
Plant cells have many simpler units of
Golgi bodies
Golgi complex, called dictyosomes
elaborate Golgi complex
Endoplasmic
Present Present
reticulum
Ribosomes Present Present
Mitochondria Present Present
Centrioles Present only in lower plant forms. Present

Practical No: 7Cell structure in staminal hair of tradescantia

Tradescantia (a spiderwort) is an invasive species and is well known to show cytoplasmic

movements. In an attempt to study the phenomenon of cyclosis, the purple-heart species
(Tradescantia pallida) was selected.

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Presence of staminal hair emerging from the base of the filaments is a characteristic feature of
the plant. The staminal hair (also referred to as trichomes) had oval shaped cells arranged in a
uniseriate manner. A screen-shot image of a magnified cell appeared amazing. The cells
appeared like rosary beads. We tried to capture slight movement in the cells (on zooming),
though not very clear.
To study cyclosis from Tradescantia stamina hair
Requirements: Tradescantia flowers, Microscope, Cover slip, Glass slide, brush and forceps
Theory:Cyclosis is automatic movement of protoplasm of a cell. This movement may be due to
the movement of organelles like chloroplast in response to changing intensity of light. It is
believed that cyclosis occurs due to Sol-gel interconversions or microfibrils.
Procedure:
1. Take fresh tradescantia flowers.
2. Staminal hairs remove and keep on clean glass slide with a drop of water.
3. Place a coverslip over sample and observe under microscope.
Precautions:
1. Flowers should be fresh
2. There should not be any air bubble

Fig7.1: Study of cell structure in staminal hair of Tradescantia

Practical No: 8Study of different types of Plastids

Introduction
Plastids are double membrane bound organelles found inside plants and some algae, which are
primarily responsible for activities related to making and storing food. Many plastids are

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photosynthetic, but some are not. Some of the most common plastids include: chloroplasts,
chromoplasts, gerontoplasts, and leucoplasts.

Fig8.1: Some of the major types of plastids

Types and Functions
The chloroplasts are probably for the photosynthesis. The chloroplast most is known of filled
with the plastids. These are responsible thylakoids and chlorophyll, where photosynthesis occurs.

Fig 8.2: The basic structure of the chloroplast

Chromoplasts
These are found in flowering plants, fruits, and aging leaves. The chloroplasts actually convert
over to chromoplasts. There are carotenoid pigments here that allow for the different colors you
see in fruits and the fall leaves. One of the main reasons for these structures and the colors is to
attract pollinators.
Gerontoplasts

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They are basically chloroplasts that are going through the aging process. These are chloroplasts
of the leaves that are beginning to convert into different organelles or are being repurposed since
the leaf is no longer utilizing photosynthesis (such as in the fall months).
Leucoplasts
They are the non-pigmented organelles. Unlike the others we have talked about, leucoplasts have
no color at all. They are found in the non-photosynthetic parts of the plant, such as the roots.
Depending on what the plant needs, they may become essentially just storage sheds for starches,
lipids, and proteins. They are more readily used for synthesizing amino acids and fatty acids.
Leucoplasts are further subdivided into three different plastids: amyloplasts, proteinoplasts, and
elaioplasts. Amyloplasts are the largest of the three and are charged with storing starch. Then
there are the proteinoplasts that help to store the proteins that a plant needs and are typically
found in seeds. Finally, the elaioplasts are used to store fats and oils that are needed by the plant
especially in seeds.
Objective:
Observation of amyloplast from potato tuber
Observation of chromoplast from rose petal.
Observation of amyloplast from potato tuber
Materials:
 Potato tuber
 Razor blade
 Iodine stain
 Slides with cover slips
 Microscope
Procedure:
 Make a thin section of potato tuber with the help of razor blade.
 Stain with iodine stain for a few seconds.
 Add cover slips on slides.
 Observe it under microscope
 The intensely stained structures in the cells are amyloplasts, a type of plastids that store
starch.
Observation of chromoplast from rose petal
Materials:
 Rose petal
 Razor blade
 Water beaker
 Slides with cover slips
 Microscope
Procedure:
 Make a thin section of red pepper with the help of razor blade.
 Place the section in a drop of water on glass slide and add a cover slip.

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 Examine it under the microscope.
 The tiny pinpoint orange organelles are chromoplasts, a type of plastid containing pigments
other than chlorophyll.

Practical No: 9Study of cellular reproduction

Introduction
Cell division is fundamental to all living organisms and required for growth and development.
As an essential means of reproduction for all living things, cell division allows organisms to

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transfer their genetic material to their offspring. For a unicellular organism, cellular division
generates a completely new organism. For multicellular organisms, cellular division produces
new cells for the general development of the organism, as well as produces healthy cells to
replace damaged cells from injury. Reproduction of multicellular organisms requires cell
division to create reproductive cells.
Cell cycle
About 78% of a cell’s life is spent in Interphase, growing and preparing itself for cell division.
Interphase itself contains three sub phases. Immediately after cell division, a newly-formed cell
enters the Gap 1 or G1 portion of Interphase. During G1, cells actively grow and, in many cases,
differentiate to perform specific functions. At this stage, the cell is sensitive to internal and
external signals to determine whether to divide or not. Some cells, such as neurons do not
proceed to cell division after differentiation and remain in G1 until they die. Once the cell meets
criteria to proceed to cell division, it enters the synthesis or S stage of Interphase, during which
the cell replicates its entire genome. Next, the cell enters the Gap 2 or G2 phase to synthesize all
the necessary proteins for cell division. This is followed by the cell division phase.
Types of Cell Division in Eukaryotes
There are two types of cell division in eukaryotic cells—mitosis and meiosis.
Mitosis
In single-cell organisms, mitosis is the only form of cellular reproduction. One round of mitosis
yields two genetically identical cells. In bacteria, this process results in an entirely new,
independent organism. This is classified as asexual reproduction because it does not require sex
for the creation of new organisms. In multi-cellular organisms, mitosis only occurs in somatic
cells, which comprise all cells in an organism excluding germ cells.
Cells that undergo mitosis duplicate their chromosomes, resulting in cells with two times their
normal haploid or diploid numbers (4Nchromosomes). Newly-synthesized chromosomes remain
closely associated with their like-chromosome. These two identical chromosomes are called
sister chromatids. Once duplicated, sister chromatids separate such that one copy of each
chromosome lines up on opposite ends of the cell. The cell then pinches in the center until it
breaks into two different cells. A nucleus then forms around the chromosomes in each cell to
yield two cells with the same original number of chromosomes as the preexisting cell.

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Fig 9.1.: Events of mitosis
Meiosis
There are two major differences between mitosis and meiosis. First, meiosis involves not one,
but two cell divisions. Second, meiosis leads to the production of germ cells, which are cells that
give rise to gametes. Germ cells are different from somatic cells in a critical way. Whereas
somatic cells are diploid, meaning they have two copies of each chromosome, germ cells are
haploid. The haploid nature of germ cells is vital to the process of sexual reproduction.
There are two different sex cells or gametes: sperm and eggs. Males produce sperm and females
produce eggs. Because they are produced from germ cells, gametes are likewise haploid. In order
to create a new individual via sexual reproduction, a sperm cell needs to activate an egg by
joining it in a fertilization process. When these two haploid cells unite, a diploid cell resulted.
This specialized cell can then develop into a new individual. The sexual reproductive process
just described ensures that the resulting offspring will have an equal maternal and paternal
genetic contribution.
As we mentioned earlier, higher-order cells contain homologous pairs of chromosomes--one
from the father and the other from the mother. In meiosis, as in mitosis, the maternal and paternal
homologues are replicated during DNA replication yielding two pairs of sister chromatids. After
the first cell division, each of the resulting cells contains a pair of sister chromatids—-one
maternal pair and the other paternal. Unlike mitosis, meiosis does not end after one division; it
continues with a second cell division. In this division, the sister chromatids are separated
yielding four total haploid cells.

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Figure 9.2: Events of meiosis

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 Practical No: 10Study of Mitosis

In cell biology, mitosis is a part of the cell cycle when replicated chromosomes are separated into
two new nuclei. Cell division gives rise to genetically identical cells in which the number of
chromosomes is maintained. In general, mitosis (division of the nucleus) is preceded by the S
stage of interphase (during which the DNA is replicated) and is often accompanied or followed
by cytokinesis, which divides the cytoplasm, organelles and cell membrane into two new cells
containing roughly equal shares of these cellular components. Mitosis and cytokinesis together
define the mitotic (M) phase of an animal cell cycle—the division of the mother cell into two
daughter cells genetically identical to each other.
In mitosis, the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the
splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief
stages:
Cytokinesis – Cytoplasm division
Karyokinesis – Nucleus division
Stages of Mitosis
The various stages of mitosis are:
1. Prophase
The process of mitosis is initiated at this stage wherein coiling and thickening of the
chromosomes occurs. Shrinking and hence the disappearance of the nucleolus and nuclear
membrane takes place. The stage reaches its final state when a cluster of fibers organizes to form
the spindle fibers.
2. Metaphase
Chromosomes turn thick in this phase. The two chromatids from each of the chromosomes
appear distinct. Each of the chromosomes is fastened to the spindle fibers located on its
controller. Chromosomes align at the centerline of the cell
3. Anaphase
Each of the chromatid pair detaches from the centromere and approaches the other end of the cell
through the spindle fiber. At this stage, compressing of the cell membrane at the center takes
place
4. Telophase
Chromatids have reached the other end of the cell. The disappearance of the spindles. Chromatin
fibers are formed as a result of uncoiling of daughter chromosomes. The appearance of two
daughter nuclei at the opposing ends due to the reformation of the nucleolus and nuclear
membrane. At this phase, splitting of the cell or cytokinesis may also occur.Post mitosis, the next
stage is referred to as interphase which is part of the cell cycle that is non-dividing and between
two consecutive cell divisions. A cell spends most of its life in the interphase. It comprises the
G1, S and G2 stages.

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Fig 10.2: Detail of animal cell cycle

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Study of the Stages of Mitosis from Permanent Slides
Objectives
In today’s lab, you will first observe prepared slides animal cells in different stages of cell
division. In the second part of the lab, you will prepare your own slides of onion root tips and
examine the tissue for both interphase and mitotic cells. By the end of today’s lab, you should be
able to:
 Use a microscope to identify cells in interphase and different stages of mitosis from prepared
slides.
 Prepare your own temporary mounts of onion root tips and examine them under the
microscope for different phases of the cell cycle in practical 12.
Materials Required
 Permanent slides of mitosis
 Compound Microscope
Study
The teacher will place some permanent slides on the stage of compound microscope and ask the
students to observe and identify the stages of mitosis.
1. Prophase: In this slide some chromosomes are seen; the chromosomes are long and scattered.
No spindle fiber is seen. Therefore the stage is Prophase of Mitosis.
2. Pro-Metaphase: In this slide some chromosomes are seen. Spindleapparatus is also seen. The
chromosomes are seemed to moving towards equatorial plane. The chromosomes are divided
into chromatids. Therefore this is the Pro-Metaphase of Mitosis.

Fig 10.2: Slides detail of cell cycle

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3. Metaphase: Some chromosomes are seen in this slide. Spindle apparatus is seen here. The
chromosomes are situated on the equatorial zone. The chromosomes are divided into chromatids.
Therefore this is the Metaphaseof Mitosis.
4. Anaphase: In this slide two sets of chromosomes are seen. Two sets arepresent near the two
poles. Therefore it is the Anaphase of Mitosis.

Fig 10.3: Slides detail of cell cycle

5. Telophase: In this slide two sets of chromosomes are seen. Two sets ofchromosomes are
present at two poles. No spindle apparatus is seen. Nuclear membrane is present surrounding the
chromosomes in each pole. Therefore it is the Telophase of Mitosis.

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 Practical No: 11Study of Meiosis

Meiosis is a process where a single cell divides twice to produce four cells containing half the
original amount of genetic information. These cells are our sex cells – sperm in males, eggs in
females. During meiosis one cell divides twice to form four daughter cells.These four daughter
cells only have half the number of chromosomes of the parent cell – they are haploid.Meiosis
produces our sex cells or gametes (eggs in females and sperm in males).
Meiosis can be divided into nine stages. These are divided between the first time the cell divides
(meiosis I) and the second time it divides (meiosis II):
Meiosis I
1. Interphase
The DNA in the cell is copied resulting in two identical full sets of chromosomes.
Outside of the nucleus are two centrosomes, each containing a pair of centrioles, these structures
are critical for the process of cell division.
During interphase, microtubules extend from these centrosomes.
2. Prophase I
The copied chromosomes condense into X-shaped structures that can be easily seen under a
microscope.Each chromosome is composed of two sister chromatids containing identical genetic
information.The chromosomes pair up so that both copies of chromosome 1 are together; both
copies of chromosome 2 are together, and so on.The pairs of chromosomes may then exchange
bits of DNA in a process called recombination or crossing over.At the end of Prophase I the
membrane around the nucleus in the cell dissolves away, releasing the chromosomes.The meiotic
spindle, consisting of microtubules and other proteins, extends across the cell between the
centrioles.
3. Metaphase I
The chromosome pairs line up next to each other along the center (equator) of the cell.
The centrioles are now at opposites poles of the cell with the meiotic spindles extending from
them.The meiotic spindle fibers attach to one chromosome of each pair.
4. Anaphase I
The pair of chromosomes is then pulled apart by the meiotic spindle, which pulls one
chromosome to one pole of the cell and the other chromosome to the opposite pole.
In meiosis I the sister chromatids stay together. This is different to what happens in mitosis and
meiosis II.
5. Telophase I and cytokinesis
The chromosomes complete their move to the opposite poles of the cell.
At each pole of the cell a full set of chromosomes gather together.
A membrane forms around each set of chromosomes to create two new nuclei.
The single cell then pinches in the middle to form two separate daughter cells each containing a
full set of chromosomes within a nucleus. This process is known as cytokinesis.

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Fig 11.1: Stages of Meiosis I
Meiosis II
6. Prophase II
Now there are two daughter cells, each with 23 chromosomes (23 pairs of chromatids).In each of
the two daughter cells the chromosomes condense again into visible X-shaped structures that can
be easily seen under a microscope.The membrane around the nucleus in each daughter cell
dissolves away releasing the chromosomes.The centrioles duplicate.The meiotic spindle forms
again.
7. Metaphase II
In each of the two daughter cells the chromosomes (pair of sister chromatids) line up end-to-end
along the equator of the cell.The centrioles are now at opposites poles in each of the daughter
cells.Meiotic spindle fibres at each pole of the cell attach to each of the sister chromatids.
8. Anaphase II
The sister chromatids are then pulled to opposite poles due to the action of the meiotic
spindle.The separated chromatids are now individual chromosomes.
9. Telophase II and cytokinesis
The chromosomes complete their move to the opposite poles of the cell.At each pole of the cell a
full set of chromosomes gather together.A membrane forms around each set of chromosomes to
create two new cell nuclei.This is the last phase of meiosis, however cell division is not complete
without another round of cytokinesis.Once cytokinesis is complete there are four granddaughter
cells, each with half a set of chromosomes (haploid):in males, these four cells are all sperm
cellsin females, one of the cells is an egg cell while the other three are polar bodies (small cells
that do not develop into eggs).

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Fig 11.2: Stages of Meiosis II

Study of the Stages of Meiosis from Permanent Slides

Objectives
In today’s lab, you will first observe prepared slides animal cells in different stages of cell
division. In the second part of the lab, you will prepare your own slides of onion root tips and
examine the tissue for both interphase and meiotic cells. By the end of today’s lab, you should be
able to:
 Use a microscope to identify cells in interphase and different stages of meiosis from
prepared slides.
Materials Required
 Permanent slides of meiosis
 Compound Microscope

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 Practical No: 12Mitosis: smear/squash preparation ofonion roots

A quick overview of cell division

The genetic information of plants, animals and other eukaryotic organisms resides in several (or
many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46
chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate
their DNA when dividing. During DNA replication, the two strands of the DNA double helix
separate, and foreach 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 aregion called the centromere.DNA replication in eukaryotes is
followed by the process called mitosis which assures that eachdaughter 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 andtelophase. 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 ofthe
replicated chromosomes prior to cytokinesis.
Objective
Better understand the process and stages of mitosis.
Prepare your own specimens of onion root in which you can visualize all of the stages of mitosis.
Observation of mitosis in onion root tips
Roots and stems grow from their tips. Actively dividing tissues such as meristem are good
sources of mitotically dividing cells. You will prepare squashes of onion root tip (Allium cepa)
and observe cells in various stages of mitosis.
Why use onion roots for viewing mitosis?
 The roots are easy to grow in large numbers.
 The cells at the tip of the roots are actively dividing, and thus many cells will be in stages of
mitosis.
 The tips can be prepared in a way that allows them to be flattened on microscopes slide
(“squashed”) so that the chromosomes of individual cells can be observed.
 The chromosomes can be stained to make them more easily observable.
Regions of Onion Root tips
 There are three cellular regions near the tip of an onion root.
 The root cap contains cells that cover and protect the underlying growth region as the root
pushed through the soil.
 The region of cell division (or meristem) is where cells are actively dividing but not
increasing significantly in size.
 In the region of cell elongation, cell are increasing in size, but not dividing.

Materials:

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 Onion plant with root
 Acetocarmine stain
 Scissors
 Forceps
 Razor blade
 Pasture pipette
 1.5 ml Centrifuge tubes
 Dissection probe with wooden back
 Microscopic slides and cover slips
 Water bath
 Light Microscope
Procedure for preparing root tip squashes
While actively growing onions are present in the lab for you to observe, you will be provided
with rootsthat have been previously harvested and treated with a fixative to stabilize the cells.
You will work in groups for this lab exercise.
The first step will be to ‘soften’ the roots so that they later can be spread on amicroscope slide.
1. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into a plasticmicro-tube.
(One of the rots will be an extra one.)
2. Fill the tube about 2/3 full with 1N HCl from a dropper bottle.
Caution: Work with the HCl carefully, it is a strong acid.
3. Place the tube in a 60OC water bath, and allow the roots to incubate for 12 minutes.
4. After the 12 minute incubation period, remove the tube from the water bath.
Rinse the roots in H2O
1. Using forceps, carefully transfer the root tips to a small petri plate.
2. Using a plastic ‘squeeze’ pipet, carefully remove the HCl from the micro-tube and transfer it
tothe “discard flask”.
3. Rinse the root tips 3 times with water from the dropper bottle, disposing of the rinses in
thediscard flask.
Staining the chromosomes
1. After removing the water from the third rinse, cover the root with the Feulgen stain. Caution:
Although the Feulgen stain does not appear colored,it will strongly stain skin and clothing.
2. Incubate the roots in the stain for 12 minutes. During this time the very tipof the root will
begin to turn red as the DNA stains the numerous smallactively dividing cells at the tip.
Remove the stain and again rinse the roots
1. Using a plastic ‘squeeze’ pipet, carefully remove the Feulgen stain anddiscard it in the discard
flask.
2. Again, rinse the root tips 3 times with water.
Preparing the root tip squash
1. Transfer a root to the center of a clean microscope slide and add a drop ofwater.
2. Using a razor blade cut off most of the unstained part of the root, anddiscards it.

35
3. Cover the root tip with a cover slip, and then carefully push down on thecover slide with the
wooden end of a dissecting probe. Push hard, but donot twist or push the cover slide sideways.
The root tip should spread out toa diameter about 0.5 – 1 cm.
Observations of onion root tip squash:
Scan the microscope under the 10x objective. Look for the region that has large nuclei relative to
the size of the cell; among these cells will be found cells displaying stages of mitosis. Examples
are shown in the figure to the right. Switch to the 40X objective to make closer observations.
Since prophase and Prometaphase are difficult to distinguish, classify all these cells as prophase.
Record your observations in the table provided.

Fig 12.1: Detail of Mitosis

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References:
1. http://www.tutorvista.com/biology/microscope-parts-and-functions
2. http://www.microrao.com/simple_staining.htm
3. https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/
microscopy-3/light-microscopy-29/general-staining-methods-243-4284/
4. http://www.cliffsnotes.com/study-guides/biology/microbiology/microscopy/staining-
techniques.
5. http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2
6. http://www.microscopemaster.com/prokaryotes.html
7. http://biology.tutorvista.com/animal-and-plant-cells.html
8. http://www.cliffsnotes.com/study-guides/biology/biology/the-biology-of-cells/prokaryote
–and-eukaryote-cell-structure
9. http://w3.marietta.edu/~biol/introlab/Onion%20root%20mitosis.pdf
10. Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings,
San Francisco, CA.
11. http://www.biology4friends.org/cell-cycle-timing-lab1.html
12. http://vlab.amrita.edu/?sub=3&brch=188&sim=1102&cnt=2
13. http://sites.fas.harvard.edu/~bs50/Mit%20Mei%20Notebook.pdf
14. http://www.sparknotes.com/biology/cellreproduction/intro/section2.rhtml
15. http://vlab.amrita.edu/?sub=3&brch=188&sim=1102&cnt=2
16. http://study.com/academy/lesson/plastids-definition-structure-types-functions.html
17. http://faculty.valenciacollege.edu/tklenk/botany/labs/The%20Cell.htm
18. http://www.uq.edu.au/_School_Science_Lessons/UNBiol1.html#9.9.1

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