lab manual fm sem ii - welcome to dav college jalandhardavjalandhar.com/dbt/botany/sop-lab...

Department of Botany, DAV College, Jalandhar (PB.)

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BOTANY

Lab Manual

BSc.-I Medical Semester II


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Syllabus

1. To study cell structure from onion leaf peels; demonstration of standing and mounting

methods.

2. Comparative study of cell structure in onion cells, Hydrilla and Spirogyra. Study of cyclosis

in Tradescantia Staminal Cells.

3. Study of plastids to examine pigment distribution in plants (e.g. Cassia, Lycopersicon and

Capsicum).

4. Examination of electron micrographs of eukaryotic cells with special reference to organelles.

5. Study of electron micrographs of viruses, bacteria, cyanobacteria and eukaryotic cells for

comparative cellular organization.

6. Examination of various stages of mitosis and mitosis using appropriate plants material

(e.g. onion root tips, onion flower buds).

7. Preparation of karyotypes from dividing root tip cells and pollen grains.

8. Cytological examination of special types of chromosomes: bar body, lampbrush and polytene

chromosomes.

9. Working out the laws of inheritance using seed mixtures.

10. Working out the mode of inheritance of linked genes from test cross and/or F2 data.


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AIM 1. Demonstration of Staining and Mounting Methods

A. Staining is a technique used in microscopy to enhance contrast in a microscopic image.

Stains and dyes are frequently used to highlight structures in microbes for viewing, often with

the aid of different microscopes. Staining may be single or double.

1. Single Stain: Safranin or fast green is used to stain filaments of lagae, fungi, sections of

bryophytes, spores of pteridophytes, pollen grains of angiosperms etc. Aniline blue or safranin is

usually used for single staining methods.

Procedure: - Keep material in watch glass

- Few drops of stain are added to the water to another watch glass to dilute the stain.

- The diluted stain is added to the material.

- After the stain is taken up by sample, the extra stain is washed with water.

- Washing is repeated till the stain stops coming out.

- The stained material is now ready for mounting.

2. Combined Stain: In this technique, two or more stains are used. Generally contrasting colours

are used. The following combinations are commonly employed.

• Haematoxylin and Safranin

• Safranin and fast green

• Safranin and Aniline blue

• Safranin and Crystal violet

• Crystal violet and Erythrosine

Procedure: - The section is first stained with principle stain.

- Section is passed through a graded series of alcohol for degradation.

- Section is transferred to watch glass containing required amount of alcohol and water.

- Watch glass should be covered by larger watch glass to avoid alcohol evaporation.

- Now the counter stain is employed for few seconds.

- The section is transferred to absolute alcohol for 5-7 min for complete dehydration.

- Section is finally immersed in xylene. If dehydration is not complete, pure xylene

turns white or turbid.

- Now the section is ready for mounting.

B. Mounting is necessary for proper positioning of objects for clear view. Lactophenol, glycerin

and glycerine jelly are used for temporary mounting while Canada balsam is used for permanent

mounting.

Mounting Media: Following are some of the common media:


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- Canada Balsam: It is resin obtained from conifer. It is most suited for permanent slide

preparation.

- Lactophenol: It is a mixture of equal parts of phenol crystals, lactic acids, glycerine and

distilled water.

- Glycerine: Pure glycerine diluted to 15-25% is widely used for semipermanent and temporary

preparations.

- Glycerine Jelly: Jelly is used for mounting. It is made up of gelatin.

Many other mounting media like cedar oil, balsam, venetiane, turpentine, synthetic resin

etc are also used.


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AIM 2. Comparative study of cell structure in onion cells, Hydrilla and Spirogyra.

Requirement: Onion cells, Hydrilla, Spirogyra, Tradescantia flowers, watch glass, forecep,

slide, coverslip, microscope.

Procedure:

1. A thick onion scale was taken out and teared it from concave

side so as to get transparent thin and membrane onion peel.

2. Placed the peel in watch glass containing water and 2-3 drops of

methylene blue.

3.Cut a small portion of peel and placed it on cleansed glass slide

with a drop of glycerine.

4. Put a cover slip over it and observe under the microscope.

Onion Peel Cell

1. A cleansed glass slide was taken.

2. Mount the material on it with a drop of glycerine.

3. Cover it with watch glass and observe it under microscope.

Hydrilla Cell

1. A cleansed glass slide was taken.

2. Mount the material on it with a drop of glycerine.

3. Cover it with watch glass and observe it under microscope.

Spirogyra Cell

Department of Botany, DAV College, Jalandhar (

AIM 3: To study cyclosis from Tradescantia stamina hair.

Requirements: Tradescantia flowers, Microscope, Cover

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

Procedure: 1. Fresh tradescantia flowers were taken .

2. Staminal hairs were removed and kept on clean glass slide with a drop of water.

3. Placed a coverslip over sample and observed under microscope.

Precautions: 1. Flowers should be

2. There should not be any air bubble.

Enlarged view of Staminal Hair

Jalandhar (PB.)

study cyclosis from Tradescantia stamina hair. (e-Reource generated)

Tradescantia flowers, Microscope, Cover slip, Glass slide, brush and forceps

Cyclosis is automatic movement of protoplasm of a cell. This movement may be due to


believed that cyclosis occurs due to Sol-gel interconversions or microfibrils.

1. Fresh tradescantia flowers were taken .


3. Placed a coverslip over sample and observed under microscope.

1. Flowers should be fresh.

2. There should not be any air bubble.

Filament with stamina hairs

Enlarged view of Staminal Hair

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Reource generated)

slip, Glass slide, brush and forceps.

Cyclosis is automatic movement of protoplasm of a cell. This movement may be due to




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AIM 4: Examination of electron micrographs of eukaryotic cells with special reference to

organelles.

Plants Eukaryotic Cell

It has a cell wall, plasma membrane, cytoplasm

having various organelles like nucleus,

vacuole, chloroplast, mitochondria, golgi

bodies, ribosomes and fat granules embedded

in it.

Nucleus

1. It is a specialized double membrane bound

protoplasmic body.

2.It is divided into 5 main parts like nuclear

envelop, nucleoplsm, nuclear matrix,

chromatin and nucleolus.

3. The nuclear membrane is interconnected

with endoplsmic reticulum.

Mitochondria

1. It is double membrane bound power house

of the cell.

2. Its outer membrane is smooth and inner

membrane is highly folded inwards.

3. It contains its own DNA called mtDNA.

4. It is actively involved in oxidative

phosphorelation in aerobic eukaryotes.

Chloroplast

1. It is greenish plastid which possesses

photosynthetic pigments.

2. It takes part in synthesis of food from

inorganic raw material in presence of sun light.

3. It possess stroma and thylakoids.

4. It is a double membrane organelle.


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Endoplasmic reticulum

1. Endoplasmic reticulum is double membrane

bound cell organelle.

2. It contains 3 main components like

cisternae, vesicles and tubules.

3. ER is of two types i.e., rough endoplasmic

reticulum (RER) and smooth endoplasmic

reticulum (SER).

4. RER is bound by ribosomes.

Golgi body

1. It is an important part of endomembrane

systems.

2. It has a cis and trans face.

3. Cis face is formative face and trans face is

productive face.

4. It induces glycosylation of proteins.

Vacuole

1. These are non cytoplasmic spaces present

inside the cytoplasm.

2. They can be sap vacuoles, contractile

vacuoles, food vacuoles and air vacuoles.

3. These vacuoles are membrane bound. The

membrane is called tonoplast.


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AIM 5: Study of electron micrographs of viruses, bacteria, cyanobacteria and eukaryotic

cells for comparative cellular organization.

Bacteriphage

1. A bacteriophage is a virus that infects and

replicates within a bacterium.

2. Bacteriophages are composed

of proteins that encapsulate a DNAor RNA genome.

3. Bacteriophages may have a lytic cycle or

a lysogenic cycle, and a few viruses are capable of

carrying out both.

4. To enter a host cell, bacteriophages attach to

specific receptors on the surface of bacteria.

Bacterial cell

1. Bacteria constitute a large domain of prokaryotic

microorganisms.

2. Bacterial cells are about one-tenth the size of

eukaryotic cells and are typically 0.5–

5.0 micrometres in length.

3. Bacteria do not have a membrane-bound nucleus,

and their genetic material is typically a single

circular DNA chromosome located in the

cytoplasm.

Cyanobacteria

1. Cyanobacteria also known as Cyanophyta, is

a phylum of bacteria that obtain their energy

throughphotosynthesis.

2. Cyanobacteria are a photosynthetic nitrogen fixing

group that survives in wide variety of habitats, soils,

and water.

3. Their thalli vary from unicellular to filamentous.

4. Many cyanobacteria form motile filaments of

cells, called hormogonia.


AIM 6: Examination of vario

material (e.g. onion root tips, onion flower buds).

Requirements: Onion root tips, onion flower bulbs, slides, watch glass, distilled water,

acetocarmine/acetooricine, burner, forecep, needle, coverslip.

Interphase

1. This stage is prior to actual mitotic cycle.

2. This cell appears to be inactive.

3. Nuclear membrane and nucleolus are very

distinct.

4. Chromosome appear double stran

Early Prophase

1. Nuclear membrane appears distinct.

2. Nucleolus is also clear.

3. Chromosome become clear and shortened.

Late Prophase

1. Nucleolus and nuclear membrane partially

disappear.

2. Chromosomes are condensed.

3. Spindle fibres also begin to appear.

Jalandhar (PB.)

Examination of various stages of mitosis and meiosis using appropriate plants

(e.g. onion root tips, onion flower buds).

Onion root tips, onion flower bulbs, slides, watch glass, distilled water,

acetocarmine/acetooricine, burner, forecep, needle, coverslip.

A. Mitosis

1. This stage is prior to actual mitotic cycle.

2. This cell appears to be inactive.

3. Nuclear membrane and nucleolus are very

4. Chromosome appear double stranded.

1. Nuclear membrane appears distinct.

3. Chromosome become clear and shortened.

1. Nucleolus and nuclear membrane partially

begin to appear.

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using appropriate plants

Onion root tips, onion flower bulbs, slides, watch glass, distilled water,


Metaphase

1. Nuclear membrane and nucleolus

disappears.

2. Centromere get arranged on equatorial plate

and are attached to spindle fibres.

3. Spindle is made up of fibres only.

Anaphase

1. Chromatids are pulled toward opposite

poles.

2. Each chromosome appear in characteristic

shape.

Telophase

1. Chromosomes are present at both ends.

2.Chromosomes increase in l;ength and

become thread like.

3. Nuclear membrane and nucleolus reappear.

4. Spindle fibres appear to deassemble.

Jalandhar (PB.)


2. Centromere get arranged on equatorial plate

and are attached to spindle fibres.

3. Spindle is made up of fibres only.

1. Chromatids are pulled toward opposite

2. Each chromosome appear in characteristic

1. Chromosomes are present at both ends.

2.Chromosomes increase in l;ength and


4. Spindle fibres appear to deassemble.

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Cytokinesis

1. Cytoplasm divides into two. It results in two

daughter cells.

2. Cell plate formation begins at the centre of

plant cell and progress towards the periphery.

Interphase Prophase Metaphase Anaphase Telophase & Cytokinesis


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B. Meiosis

Pr

op

ha

se

(

I)

Leptotene (Prophase I)

1. Nuclear membrane and nucleolus are

intact.

2. Chromosomes are long thread like.

3. This stage is also called bouquet stage.

4. All the chromosomes finally move towards

one part of the nucleus.

Zygotene (Prophase I)

1. Nucleolus and nuclear membrane are still

very clear.

2. Synapsis of homologous chromosomes

occur.

3. Synaptonemal complex is formed.

Pachytene (Prophase I)

1. Nucleus and nuclear membrane are

distinct.

2. Chromosomes are thickened, coiled and

thread like.

3. Crossing over occurs.

Diplotene (Prophase I)

1. The nucleolus and nuclear membrane is

disappearing.

2. Homologous chromosomes are still intact.

3. Chromosomes are shortened and

thickened.

Diakinesis (Prophase I)


completely disappear.

2. Chromosomes start separating from

centromere.

3. The chromosomes appear circular due to

contraction.


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Metaphase (I)

1. Nuclear membrane and nucleolus completely

absent.

2. Spindle is distinct.

3. Bivalents are arranged on equatorial plate.

Anaphase (I)

1. Nuclear membrane and nucleolus completely

absent.

2. Chromosomes separate out of the pair.

3. Results in haploid set of chromosomes at each

pole.

Telophase (I)


2. There are two nuclei on each pole.

Meiosis (II)

Prophase (II)

1. Nuclear membrane and nucleolus are distinct in

early stages.

2. Chromosome are short and thick.

3. Each chromosome is made up of two

chromatids.

Metaphase (II)

1. Nuclear membrane and nucleolus are absent.

2. Soindle is quite visible.

3. Chromosomes lie at equater.


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Anaphase (II)

1. Nuclear membrane and nucleolus are absent.

2. Spindle fibres contract and chromosomes are

pulled to opposite poles.

3. Chromatids show characteristic shape.

Telophase (II)

1. Chromosomes occur in group at end of parent

cell.

2. Nuclear membrane and nucleolus start

reapperaring.

3. Four haploid daughter cells are formed.


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AIM 7: Preparation of karyotypes from dividing root tip cells and pollen grains.

Procedure: 1.Karyotype refers to the phenotypic appearance of the chromosome at somatic

metaphase. Ideogram is the diagrammatic representation of karyotype.

2. Study the number and morphology of chromosome from well spread out mitotic preparation.

3. Draw well spread out mitotic metaphase plate.

4. Mark the positions of centromere carefully.

5. Measure the arm length of various chromosomes and plot their relative length on graph as

histograms.

6. The chromosomes should be arranged in decreasing order of their total length with the larger

arms towards the bottom.

7. Uniform gaps should be used to indicate primary and secondary constrictions.

8. Compare the karyotypes and idiograms of different species of different varieties of same

species.

Karyotype of Allium cepa

Chromosomes of Vicia faba


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AIM 8: Cytological examination of special types of chromosomes: bar body, lampbrush

and polytene chromosomes.

Polytene Chromosome

1. Polytene chromosomes are over-sized

chromosomes which have developed from

standard chromosomes and are commonly

found in the salivary glands of Drosophila

melanogaster.

2. Cells undergo repeated rounds of DNA

replication without cell division.

3. Polytene cells may also have a metabolic

advantage as multiple copies of genes permits

a high level of gene expression.

4. Polytene chromosomes have characteristic

light and dark banding patterns.

5. Polytene chromosomes have characteristic

light and dark banding patterns.

6. Balbiani ring is a large chromosome puff

.

Lampbrush Chromosome

1. Lampbrush chromosomes are a special

form of chromosome found in the growing

oocytes (immature eggs) of most animals,

except mammals.

2. Chromosomes transform into the

lampbrush form during the diplotene stage of

meioticprophase I due to an active

transcription of many genes.

3. Chromosomes have loops projecting in

pairs.

4. These pairs give lampbrush chromosome

its characteristic appearance.


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Bar Body

1. Rinse your mouth thoroughly with tap

water and followed with distilled water.

2. Scrap the mucosa gently from the deeper

layer of your mouth with the help of

cleanlywashed spoon or spatula.

3. Spread the sample over a small area on a

clean dry slide and allow it for air drying.

4. Add 95% Alcohol and allow it to settle for

5-6 min.

5. Stain the slide with Carbol fuchsin and

allow it to settle for 15-20 min.

6. Wash the slide with distilled water and

immediately observe the slide under the

microscope.

Phorograph of Cheek Cells showing Barr Body


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AIM 9: Working out the laws of inheritance using seed mixtures.

1. Law of Segregation: During gamete formation, the alleles for each gene segregate from each

other so that each gamete carries only one allele for each gene.

Procedure: 1. Count 50 yellow and 50 red buttons and put them in box A.

2. Similarly put count 50 blue and 50 green buttons and put them in box B.

3. Now randomly pick one button from each box, until the boxes are empty. Record each

reading.

4. Find out if the genotypic ration comes out to be 1: 2: 1 and phenotypic ratio as 3:1.


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2. Law of Independent Assortment: Genes for different traits can segregate independently

during the formation of gametes.

Procedure: 1. Count 40 buttons of two different colours and put them in beaker A.

2. Repeat it for beaker B with 40 buttons of two different colours.

3. Now randly draw one capsule from each beaker .

4. Continue this until beakers are empty.

5. Note down the readings and analyse if you get ratio of 9:3:3:1.


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AIM 10: Working out the mode of inheritance of linked genes from test cross and/or F2

data.

Explanation: The coexistence of two or more genes in the same chromosome is called

linkage. If genes are present on same chromosomes, then the test cross ratio comes out to be 1:1

while if the genes are present on different chromosomes the test ratio comes out to be 1:1:1:1.

Procedure: 1.Assume two genes A and B where a and b are recessive alleles.

2. A cross between AABB and aabb would give rise to F1 progeny as AaBb.

3. Depending upon the distance between any 2 genes which is inversely proportional to the

strength of linkage, non cross overs will vary from 50 to 100 %.

4. Thus cross over will vary from 0 – 50% and will never exceed 50%.

5. The easiest way to find out the proportion of non cross overs and cross overs is make a test

cross.

6. From phenotypic ratio of test cross the relative proportion of crossing over and non cross

overs can be easily determined.

Complete Linkage Incomplete Likage

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