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SCHOOL

LABORATORY
MANUAL
BIOLOGY CBSE
CLASS-XII
TERM-1
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 SECTION-A
EXPERIMENT-1
AIM OF THE EXPERIMENT
To isolate DNA from plant materials such as spinach, green peas, papaya and any other available
plant material.

NECESSARY MATERIALS & APPARATUS

 Any available plant materials
 Mortar and pestle
 Test tubes
 Beakers
 Ethanol
 Spool
 Enzymes (Cellulase, ribonuclease, lipases, protease)

THEORY
1. Rupture or lysis of the cell wall and cellular membranes with the help of mechanical or
non-mechanical methods.

2. In the mechanical method, force is applied to the cell wall to open and spill the contents.
In the non-mechanical method, enzymes or chemicals that specifically break down cell
wall components are and with additional mechanical force. The advantage of mechanical
disruption over the non-mechanical is that no chemicals are introduced in the cell
solution that might interfere with the extracted cellular substance.

3. After lysis, small cracks are formed in the cell membrane for the accessibility of
detergents. Chemical detergents break down the cell membranes due to their
amphipathic. The DNA is then precipitated from the protein with ethanol. The clean
DNA is now suspended in a 1xTE buffer or distilled H2O.

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PROCEDURE

 Take the available plant material and grind it in the mortar.

 Treat the material with cellulase to break down the cell wall of the plant cells.
 Next, treat it with protease to hydrolyze the peptide bonds of proteins in the plant
material. In other words, the enzyme removes the histone proteins which are intertwined
with the DNA.
 Dissolve RNA with ribonuclease
 Use lipase to dissolve lipids.
 Add chilled ethanol to enable the precipitation of the DNA. It essentially increases DNA
concentration.
 Use spooling to extract the precipitated DNA. Spooling involves winding the fine threads
of DNA on to a reel.

OBSERVATION
The DNA appears as white precipitates of fine thread on the spool.

PRECAUTIONS:
1. Prevent any wastage of extract during the process of the extraction of DNA.
2. Always use fresh plant material for extraction of DNA.
3. Use the reagents of correct mentioned strength.

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 EXPERIMENT-2
AIM OF THE EXPERIMENT:

TO study and calculate the percentage of pollen on a slide.

REQUIREMENTS:

Mature pollens of china rose,cavity glass slides, coverslips, microscope, distilled water, beaker,
weighting machine, filter paper, glass rod, boric acid, sucrose, calcium nitrate, brushes and
dropper.

THEORY:
POLLEN GRAINS
Pollen grains are male reproductive structures of an angiospermic flower which contain male
gametes. Each pollen grain consists of a mass of protoplast surrounded by a thick. Double
layered wall. The protoplasm also contains a single haploid nucleus, which divides to form two
nuclei, i.e. the tube nucleus and the generative nucleus. The outer wall of pollen or exine, is
usually thick, cuticularised and smooth but may be thin at certain places. These thin regions are
termed as germ pore.The inner layer of wall or intine is thin with cellulose and pectin as its chief
constituents. It is hydrophilic in nature and can easily absorb water and swell.

POLLEN GRAIN GERMINATION

In nature, pollen grains germinate on the compatible stigma of the carpel. This process is called
pollen-pistle interaction. It is known to be an essential step in fertilization of angiosperms which
determines the compatibility and incompatibility of both pollen and pistil. This dynamic process
involves pollen recognition followed by inhibition or promotion of pollen germination.

PROCEDURE:
 Firstly, prepare a pollen germination medium by dissolving 10 g sucrose, 30 mg calcium
nitrate and 10 mg boric acid in 100 ml of distilled water. Alternatively, 10% sucrose
solution can also be used as the medium.
 Put a drop of this prepared medium or 10% sucrose solution on cover slip with the help
of a dropper in the cavity of the slide.
 Dust the pollen on the medium on same slide with the help of a drawing brush.
 Now, cover pollens with the cover slip.
 Observe the slide containing pollen under the microscope after 10-15 minutes.
 Finally, count the total number of non-germinating pollen and germinating pollen grains.

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OBSERVATION/TABULATION:

Count the germinated and total no. of pollens per microscopic field and tabulate your readings in
the following table:

S.NO NAME OF THE FLOWER NO.OF POLLEN TOTAL NO OF % OF POLLEN

SHOWING POLLENS SEEN(b) GERMINATION
GERMINATION(a) =𝑎/𝑏 × 100
1 China rose

CALCULATION:

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Percentage of pollen germination=𝑎/𝑏 × 100

where,a=Number of pollens showing germination,b=Total number of pollens

RESULT:

All the pollens of different species in the medium germinate with different percentage. Also,
there is great variation in time taken for the germination of pollen of each species.

PREACUTION:
 Only few pollen grains should be dusted in the nutrient solution in order to avoid the
overcrowding.
 The nutrient solution should be made carefully otherwise pollen grains might fail to
germinate.
 While placing covers slip, air bubbles should be avoided else they might interfere with
observation and counting of pollens.
 The entire process should be carried out at room temperature.

SECTION-B
EXPERIMENT-1
AIM OF THE EXPERIMENT: To study the flowers adopted to pollination by different
agencies (wind, insect, birds)

ANEM
OPHIL
Y
(POLLI
NATIO
N BY
WIND)

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IDENTIFYING CHARACTERS

1. Maize plant bears male and female flowers separately on the same plant. Male
flowers are terminal where as, female flowers are axillary in position.
2. Flowers are small, inconspicuous, colourless, odourless and nectarless.
3. Both stigma and anthers hang outside the perianth (exerted in position).
4. Pollens are light, small and dusty. They are produced in large numbers.
5. Stigma is hairy, feathery, branched and sticky to catch wind dispersed pollens.
IDENTIFICATION-

Due to the above characters’ the give specimen is anemophily (pollination by wind)

ENTOMOPHILY (POLLINATION BY INSECT):

IDENTIFYING CHARACTERS

1. flowers are showy, brightly coloured and attract insects like honeybees.
2. They are borne on verticellaster inflorescence to become conspicuous.
3. Corolla is gamopetalous and two-lipped (bilabiate). The lower lip provides.landing platform
for the insects.
4. Each flower has two epipetalous stamens in which stamen is attached to the petals by filamets.
5. Stamens have special modifications which help insects to pollinate flowers.
6. One of the anther lobes is sterile and the other is fertile. Both lobes are separated apart by a
long connective tissue.

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7. The upper arm of connective tissue bears fertile anther and lower bears sterile anthers.
8. When an insect lands on lower lip of flower to collect nectar, the sterile anther lobe is pushed
down, which brings fertile lobe down in contact with the body of the insect thus, dusting pollen
on it.
9. In older flowers, the style brings the stigma in such a position that it brushes against the back
of insect and collects pollen grains brought by the insect from a young flower.

IDENTIFICATION-

Due to the above characters’ the give specimen is Entomophily (Pollination by Insect)

ORNITHOPHILY (BIRDS POLLINATED FLOWER)

IDENTIFYING CHARACTERS

1. The flowers are big and brightly coloured red, orange, yellow or blue.
2. The flower parts are thick and leathery especially corolla.
3. The flowers produce abundant nectar and may also have certain edible parts. Birds
visit flowers for feeding on this nectar.
4. The flowers are usually odourless (without any fragrance or scent).
IDENTIFICATION-

Due to the above characters’ the give specimen is ORNITHOPHILY (BIRDS POLLINATED
FLOWER)

EXPERIMENT-2
AIM OF THE EXPERIMENT: To identify, study and comments on stages of gametes
development. From T.S. of testis

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IDENTIFICATION CHARACTERISTICS:
1. The somniferous tubules are easily seen in cross-section of testis.
2. The presence of larger cells called Sertoli cell is the characteristic feature of this slide.
3. The presence of interstitial cells or Leydig’s cell is evident.
GENERAL CHARACTERISTICS:
1. The transverse section of testis shows many semniferous tubules embedded in interstitial
tissues. These appear as coiled, circular structures.
2. Each semniferous tubule is lined on its inside by germinal epithelium which divides
mitotically to produce male germ cells called spermatogonia.
3. Various stages of development of sperm from spermatogonia are seen from periphery to
the lumen of semniferous tubules. The sequence of stages of its development is as
follows: Spermatogonia(2n) Primary spermatocyte(2n)meosis I
Secondary spermatocyte Spermatids meosis II Spermatozoa(n)
4. All these stages are clearly visible in a transverse section of testis. The mature sperms are
present in the centre and primary spermatocyte in the middle layer.
5. Sertoli cells are prominent pyramid-shaped cells found in between the germinal cells.
These cells provide nutrition to the developing spermatozoa.
6. Leydig cells are seen in the interstitial tissue. They produce the hormone called
testosterone responsible for development of male secondary sexual characters.

IDENTIFICATION:
Due to presence of above characterstics the given slide is identified as T.S of testis of mammal.

AIM OF THE EXPERIMENT: To identify, study and comments on stages of gametes

development. From T.S. of ovary

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 T.S OF MAMMALIAN OVARY
IDENTIFICATION CHARACTERISTICS:
1. The presence of Graffian follicle is characteristic feature of mammalian ovary.
2. Each Graafian follicle contains one ovum and a cavity called antrum.
GENERAL CHARACTERISTICS
1. The section of mammalian ovary shows the mass of tissues lined up by germinal
epithelium.
2. The stroma is made up of connective tissue, blood vessels and nerve fibres.
3. Various circular, structures seen in stroma are Graffian follicles in their various
developmental stages like primary and secondary oocytes.
4. In the later stages of follicular development, a fluid-filled cavity called antrum appears
which separates the ovum and the cells around it except at one point.
5. As this cavity enlarges, Graffian follicle (mature follicle) becomes ready to release ovum.
6. The mature follicle ruptures and releases ovum (ovulation). The ruptured follicles form
corpus luteum (which is an endocrine tissue) and release progesterone (the pregnancy
hormone).
7. All stages of oogenesis cannot be seen at a same time, however, they can be seen when
an ovary is observed for entire duration of menstrual cycle.

IDENTIFICATION:
Due to presence of above characterstics the given slide is identified as T.S of Ovary of mammal.

EXPERIMENT-3
AIM OF THE EXPERIMENT: To study and comments on T.S. of blastula

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IDENTIFICATION CHARACTERISTICS:
1. It is a spherical mass of cells.
2. The outermost layer, i.e. zonapellucida followed by a layer of trophoblasts is clearly
seen.
3. Within the envelope, a fluid filled cavity called blastocoel is commonly found.
4. Mass of cells inner to the trophoblast is called inner cell mass.
5. Inner mass cells form the embryo.
6. The blastocyst has embryonic or animal pole which is found opposite to the embryonic
pole.

IDENTIFICATION:
Due to presence of above characterstics the given slide is identified as T.S of Blastula of
mammalian embryo.

EXPERIMENT-4
AIM OF THE EXPERIMENT: To study the meiosis using permanent slides.
MEIOSIS-l (First Nuclear Division)
Prophase-I
It is a complex phase characterised by number of events, It is divided into five main sub-stages:
(a) Leptotene (leptos — slender; tene — band or thread)
1. Nuclear membrane and nucleolus are not clearly visible.
2. Fine network of thin chromatin threads is seen. These are chromatin fibres in condensed form
called chromosomes.

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(b) Zygotene(Zygon — paired)
1. Pairing of homologous chromosomes occurs in this stage and they form bivalents.
2. Each pair has two chromosomes similar in their length and their centromere position.
3. Chromosomes become more distinct as they become much shorter than before.

(Zygotene stage)

(c) Pachytene(Pachy- thick)

1. The homologous pairs are clearly visible.
2. Each chromosome has two chromatids and thus each bivalent consists of four chromatids.
Hence, the chromosomes exhibit tetrad configuration.
3. Crossing over, i.e. exchange of chromatid segments takes place between non-sister chromatids
of homologous chromosomes.

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 (Pachytene stage)

(d) Diplotene (diplos — double)

1. Diplos means double, so each homologous chromosome has two chromatids that show distinct
separation from each other except at some points.
2. The attachment points of two homologous chromosomes are called chiasmata (sing. chiasma).
3. These chiasmata represent the site of crossing over.

(Diplotene stage)

(e) Diakinesis (dia — opposite; kinesis — separation or movement)

1. Bivalent condense further during this stage and appear to be more shortened, thick and more
prominent than before.
2. Chiasmata are clearly visible.
3. All homologous pairs appear in a scattered form within the cell.
4. Nuclear membrane and nucleolus have disappeared completely.
5. Spindle formation can be seen in its early stages.

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 (Diakinesis stage)

Metaphase-I
1. The homologous chromosomes are still in pairs and are arranged along the equatorial plane of
the cell.
2. At this stage, number of bivalents can be counted.
3. Chiasmata may still be seen in few bivalents.

4. The spindle fibres attach themselves to the centromosome of each chromosome pair.

(METAPHASE -1 )

Anaphase-I

1. As a result of shortening of spindle fibres, the paired chromosomes start separating.

2. At the end of anaphase-l, the chromosomes assemble at two poles.
3. This results into the reduction of chromosomes number to half.
4. Each chromosome has two chromatids at this stage.

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 (ANAPHASE-I)

Telophase-I

1. Chromosomes present at the two poles appear decondensed.

2. The nuclear membrane is formed around the two new daughter nuclei.
3. Nucleolus also reappears.
4. Thus, each nucleus formed has half number of chromosomes as compared to the nucleus of the
parent cell.

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 3. Nucleolus also reappears.
4. Thus, each nucleus formed has half number of chromosomes as compared to the
nucleus of the parent cell.

MEIOSIS-Il (Second Nuclear Division)

Melosis-II is similar to mitosis without duplication of DNA. It is divided into following stages:

Prophase-Il
1. The chromosomes reappear as distinct rod-shaped or thread-like chromatin fibres.
2. Each chromosome has two chromatids.
3.Nuclear membrane and nucleolus start disappearing.
4.. The chromosomes become short by coiling and condensation.

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Metaphase-Il

1. This phase similar to that of mitotic division.

2. The chromosomes having two chromatids attached at the centromere are observed arranged at
the equatorial plane of the cell.
Metophase-Il can be differentiated from metaphase-I by the following features:
1. Every chromosome of metaphase-Il has two chromatids while in meta phase-I, paired
homologous chromosomes each having two chromatids form tetrad.
2. In metaphase-I a few chiasmata are observed, while no chiasmata are observed during
metaphase-II.
Anaphase-Il
1. The centromere of each chromosome divides into two, so that each chromatid gets its
2. S of the spindle fibres occurs so that chromatids are pulled apart towards their respective
poles.
3. The two chromatids of each chromosome after separation appear to lie at the two poles of the
cell.

ANAPHASE II
The following are the events occurring in Anaphase II
 The sister chromatids separate from one another
 The sister chromatids are pulled to opposite poles due to contraction of spindle
fibres
 The chromatids after reaching the poles are called chromosomes

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TELOPHASE II
The following are the events occurring in Telophase II
 Reconstitution of nuclei takes place
 The chromosomes begin to uncoil and become thin.
 Nucleolus and nuclear membrane reappear at each pole.

EXPERIMENT-5
AIM OF THE EXPERIMENT:

To study of prepared pedigree charts of genetic traits such as rolling of tongue, blood groups,
widow’s peak and colour blindness.

REQUIREMENTS

Questionnaire about a particular disorder, a family with genetic disorder for more than one
generation, paper, pencil, etc.

THEORY/PRINCIPLE

The principle of inheritance of traits which was given by Mendel were applicable to plants,
animals and human beings. But the type of crossing done on plants and animals cannot be
performed on humans. So, a record of inheritance of certain genetic traits for two or more

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generations in the form of a diagram or a family tree called pedigree chart is prepared. The
Mendelian concept of dominance of genes and segregation of characters in subsequent
generation can be studied by this method. Few internationally approved symbols used in this
analytical study are as follows:

PROCEDURE

1. Select a family with a monogenetic trait, such as rolling tongue, widow’s peak, colour
blindness, blood group, hitch-hiker’s thumb, hypertrichosis and of ear dimpling of cheeks, etc.

2. Ask persons exhibiting the trait to tell in which of his/her parents, grandparents, their children
and grandchildren, the trait in question is present.

3. Among the surviving individuals the trait may also be examined.

4. The information made available is the basis for the preparation of pedigree chart using
appropriate symbols.

5. The careful examination of chart would suggest whether the gene for the character is

(i) Autosome-linked dominant or recessive.

(ii) X-chromosome linked dominant or recessive.

(iii) Y-chromosome linked or not.

Autosome Linked Dominant Traits

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1. The encoding gene of this trait is present on any one of the autosomes.

2. The mutant allele is dominant and the wild type allele is recessive for such traits.

3. In this pedigree chart, the female being interviewed is exhibiting the trait and is indicated by
an arrow mark in the chart.

4. Transmission of trait occurs from any of the parent.

5. Male and female are equally affected.

6. The trait is marked to be present in each of the generation, i.e. pedigree chart is vertical.

7. Multiple generations are characteristically affected.

8. Common examples are brachydactyly, polydactyly, widow’s peak and dimple in the cheek.

Widow’s Peak

It is a V-shaped hairline across the forehead. It is a dominant trait. Thus, both homozygous
dominant (AA) and heterozygous (Aa) individuals have widow’s peak, while homozygous
recessive (aa) individuals have a straight hairline.

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 Autosome Recessive Traits

1. In this, mutant allele is recessive to its wild type allele.

2. These traits occur in equal proportions in male and female siblings, whose parents are carriers
of the allele, i.e. they have only a single copy of the allele and therefore they are normal.

3. The siblings are homozygous for the defective allele, but their parents, though some may
appear normal are obviously heterozygous, i.e. are merely carriers of the trait.

4. The marriage between man and woman genetically related with each other, occasionly results
in the appearance of such traits, e.g. albinism and rolling of tongue.

Albinism

Pedigree analysis of albinism

1. The dominant allele A’ produces pigment melanin while, recessive ‘a’ fails to produce it.

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2. The female (our subject in third generation) is therefore of genotype aa’ so, it is showing
albinism.

3. She must have received each of her ‘a’ allele from both the parents (second generation) who
are therefore, normal but are definitely of genotype Aa’ and are carriers of the trait.

4. The allele a’ must also have been present in her grandparents too, in heterozygous condition to
make them carriers (first generation).

5. Albinism in the subject’s children (fourth generation) suggest her husband to be of genotype
Aa’, a carrier.

6. The marriage of her albino daughter to an albino man is bound to produce all her
grandchildren albino (fifth generation).

The Rolling of Tongue

Some persons are able to roll their tongue in U-shape. The inability to roll the tongue is caused
by autosomal recessive allele a’. Thus, both homozygous dominant (AA) and heterozygous (Aa)

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Haemophilia

It is also a sex-linked disorder in which the patient lacks blood clotting factor and continues to
bleed even from minor cut. This disease is caused due to a recessive sex-linked gene h carried by
X-chromosome.

A female having only one allele for haemophilia (XX’ is a carrier. A female becomes
haemophilic only when both its X-chromosomes carry the gene (XhXh). In males, a single gene
for the defect is able to express itself as the Y-chromosome is devoid of any corresponding allele
(X’ The pedigree analysis for haemophilia can be done in a similar manner as that of colour
blindness.

Blood Group Inheritance

Blood group inheritance was first studied by Karl Landsteiner.

1. Blood group inheritance is independent of sex of the organisms.

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2. The ABO blood groups are controlled by gene I which have three alleles i.e. IA and IB&IO.

OBSERVATIONS:

The interrogated families showed the characteristic symptoms of the genetic disorders infered in
the studies undertaken for the pedigree analysis.

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