CELL BILOGY AND GENETICS MANUAL (Practical 1 To 6)

CELL BILOGY AND GENETICS
MANUAL
BS106 – Semester I
Biotechnology – I
I B.Sc. Biotechnology
St. Mary’s College
(Affiliated to Osmania University)
Yousufguda, Hyderabad - 500045
Telangana, India
Complied by Dr. N. Srinath, Assistant Professor of Biotechnology, Dept. of Science, SMC Page 1
Experiment no. 1 MICROSCOPIC OBSERVATION OF CELLS: BACTERIA, FUNGI,
Date: PLANT AND ANIMAL
AIM
To microscopically observe the structure of different cells such as Bacteria, Fungi, Plant and
Animal by wet mound method
PRINCIPLE
The basic purpose of performing this experiment is to understand the cells, their structure
and observation of cell organelle at 10X and 40X magnification using wet mound method.
This would help us to identify the cell arrangements in various organisms (prokaryotes and
eukaryotes) which would help us during the learning of cell biology. Here, on using the basic
and simple dyes such as iodine, methylene blue, safranin, and lacto-phenol cotton blue for
various cells, would help us in differentiating the internal organelle under the 10X and 40X
magnifications to the basic level.
A) MICROSCOPIC OBSERVATION OF PLANT CELL – ONION CELLS
MATERIALS REQUIRED
 Onion skin
 Microscopic slide
 Cover slips
 Needle
 Scalpel blade
 Iodine stain solution
 Blotting paper
Cell Biology and Genetics

 Medicine dropper or Pasteur pipette
 Water
 Microscope with 10X and 40X magnification
PROCEDURE
1. Peel off a small layer of onion and cut the onion layer in either rectangular or square
shaped using scalpel blade.
2. Carefully remove the thin membrane using the needle from the onion layer.
3. Place a drop of water on the microscopic slide and place the thin membrane of onion
on the drop using the needle.
4. Add a drop of iodine stain solution and wait for a minute so that the thin layer would
observe the stain.
5. Place a cover slip carefully with the help of needle to avoid air bubble formation.
6. Use the blotting paper to remove the excess stain from the slide.
7. Observe the slide under the 10X and 40X magnifications under the microscope.
OBSERVATION
10X 40X
RESULT
The microscopic observation under 10X and 40X magnification of thin layer of onion
showed us the cell, its structure and the alignment of plant cell in brick arrangement model.
B) MICROSCOPIC OBSERVATION OF ANIMLA CELL: BUCCAL SMEAR

MATERIALS REQUIRED
 Tooth pick or plastic spoon
 Microscopic slide Cell Biology and Genetics
 Cover slips
 Methylene blue or Safranin stain
 Water
 Needle
 Blotting paper
PROCEDURE
1. With the help of clean tooth pick or spoon, gently scrape the buccal area or skin inside
the cheek area.
2. Place a drop of water on the microscopic slide and place the tooth pick or spoon on to
the drop with gentle mixing.
3. Add the either methylene blue or safranin stain in the mixer.
4. Place a cover slip with the help of needle carefully avoiding the air bubbles.
OBSERVATION
10X 40X
RESULT
The microscopic observation under 10X and 40X magnification of skin inside the cheek or
buccal cavity showed us the animal cell, its structure and few cell organelles stained using
either safranin or methylene blue.

C) MICROSCOPIC OBSERVATION OF BACTERIAL CELL
MATERIALS REQUIRED
 15ml Test tubes – 7 no.
 250 ml Conical Flask – 1 no.
 Watch glass, Spatula, L – Rod
 Petri dish – 3 no.
 Nutrient Agar media
 1gm Soil
 Weighing balance, Laminar Air flow chamber, Autoclave
 Micropipette and tips
 1ml glass pipette
 Cover slips
 Methylene blue or Safranin stain
 Distilled Water
 Needle
 Blotting paper
PROCEDURE
A) Isolation of Bacteria from Soil by Serial dilution method
1. With the help of spatula, collect the soil in a watch glass.
2. Weigh 1gm of soil in weighing balance and add to conical flask containing 99ml
of distilled water. Now mix well by swirling and the dilution is 10-2.
3. For serial dilution, take 7 clean 15ml tubes and add 9ml of distilled water in each
tube. Mark the tubes as 10-3, 10-4, 10-5, 10-6, 10-7, 10-8 and 10-9.
4. From the conical flask take 1ml of soil mixed water and transfer to the first tube.
Mix well and take 1ml of water from 10-3 and add to 10-4. Repeat till 10-9 tube and
from 10-9 tube take 1 ml of water and discard.
5. Take the tubes and petri plates to laminar air flow chamber. From 10-7, 10-8, and

10-9 tubes take 200 µl of serially diluted water with the help of micropipette and
add to autoclaved sterile nutrient agar plate.
6. With the help of L – rod, spread the serially diluted water and keep the labelled
petri plate in a tray at room temperature for growth.
7. Observe the petri plates after 24 hours for bacterial growth and count the number
of bacterial colonies formed.
B) Microscopic observation of Bacteria
1. Take the bacteria containing petri plates to laminar air flow chamber. With the
help of sterile loop, place a drop of water on the microscopic slide.
2. Then again with the sterile loop take the bacterial colony on to the water drop and
gently mix to prepare a thin smear.
3. Heat fix the slide over the Bunsen burner flame and add the either methylene blue
or safranin stain on the smear.
4. Place a cover slip with the help of needle carefully avoiding the air bubbles.
OBSERVATION
10X 40X
RESULT
The microscopic observation under 10X and 40X magnification of bacterial smear showed
the presence of rod or bacilli shaped bacterial cell in red or blue stained using either safranin
or methylene blue.

D) MICROSCOPIC OBSERVATION OF FUNGI
MATERIALS REQUIRED
 Fungal Mold on coconut
 Forceps
 Spirit lamp
 Laminar Air flow chamber
 Cover slips
 Lacto phenol cotton blue
 Distilled Water
 Needle
 Blotting paper
PROCEDURE
1. Take all the required material in laminar air flow chamber.
2. Add a drop of lacto phenol cotton blue on a clean microscopic slide.
3. With the help of forceps, take the fungal mold from the coconut and place on the
microscopic slide in the stain.
4. Using needle, separate the fungal mold and place a cover slip with the help of needle
carefully avoiding the air bubbles.
5. Tap with the help of pencil gently over the cover slip.
OBSERVATION
10X 40X
RESULT
The microscopic observation under 10X and 40X magnification of fungal mold showed us
the fungal hyphae with sporangium in blue stain of Lacto phenol cotton blue.
Experiment no. 2 PREPARATION OF DIFFERENT STAGES OF MITOSIS
Date: (ONION ROOT TIPS)
AIM
To study the different stages of mitosis in onion root tip squash preparation.
PRINCIPLE
All cells are produced by the division of pre-existing cells. Cell division is the process
by which a single cell divides into two cells. Mitosis is the process by which the replicated
chromosomes are equally distributed to new nuclei to form new cells. It occurs in case of
somatic cells. Mitosis helps in growth and development of the organism. It also helps in the
repair of tissues and replacement of worn out cells. The different phases of mitosis such as
prophase, metaphase, anaphase and telophase can be observed in the onion root tip by
performing squash preparation.
MATERIALS REQUIRED
Onion root tip fixative (acetic alcohol, 3:1 ratio), saffranin stain, 1 N HCl, 45% acetic
acid, slides, cover slip, compound microscope, scalpel blade, acetocarmine stain.
PROCEDURE
 The onion root tip was fixed in acetic alcohol at 50°C for 6 minutes and then washed
with distilled water repeatedly.
 The root tip was then hydrolysed in 1N HCL for ½ an hour.
 The hydrolysed material was then thoroughly washed with distilled water and then
placed in a vial containing saffranin stain or acetocarmine stain for 3-5 minutes.
 The material was then transferred to a glass slide and squashed with the help of a Cell Biology and Genetics
cover slip.
 The squash thus prepared was then observed under high power objective (40 X) of the
compound microscope.
OBSERVATION
Different stages of mitosis - the prophase, metaphase, anaphase and telophase were
observed clearly.
Prophase
 The disintegration of nuclear envelope first starts.
 The chromatids become shorten and thickened.
 The centrioles were separated and migrated towards the opposite poles of the cells.
 The cell becomes spheroid, diffractive and viscous.
 Both chromatids remain connected with each other by the centromere and both remain
closely associated along their entire length.
Metaphase
 Each chromosomes reaches equator and all the chromosomes are arranged radially at
the periphery of the spindle.
 The smaller chromosomes remain at the interior but the larger ones were found at the
periphery.
 Some of the fibres (or) microtubules of the spindle were attached with the centromere
of each chromosome and are known as chromosomal fibre.
 Certain fibres occur in between the chromosomes and are known as interzonal fibres.
Anaphase
 The centromere of each chromosome was divided into two.
 The chromatids of the chromosome were separated and two chromosomes were

formed.
 The chromosomes become shorter and thicker and migrate towards the opposite poles
of the cell.
 The migration of the chromosomes towards opposite poles was achieved by the
contraction of chromosomal fibres.
Telophase
 The chromosomes which reach the opposite pole get elongated. The cells of the DNA,
protein fibres were elongated and the chromosomes however become thread like
structure.
 The nucleolus reappears.
 The microtubules of the aster and the mitotic spindle are rearranged and disappear.
 After telophase two daughter cells are formed.
10X 40X
RESULT
The slides were prepared using onion root tips and different stages of mitosis were observed
under 10X and 40X microscope.
Experiment no. 3 PREPARATION OF DIFFERENT STAGES OF MEIOSIS
Date: (GRASSHOPPER TESTIS)
AIM
To find and isolate the testis from Grasshopper to understand the different meiosis stages.
PRINCIPLE
In this experiment, we would be studying meiosis in the testis of grasshopper is an

ideal for studying meiosis, because testis of grasshopper provides the different stages of
meiosis with the presence of haploid cells. The largest have already gone through meiosis and
produced haploid cells.
MATERIAL REQUIRED
 Acetio carmine stain
 Glass slide
 Cover slip
 Lab needle
 Sample (Grasshopper testis follicles)
 Eppendorfs microcentrifuge tubes
 Pasteur pipettes
 Watch glass
 Razor blades

 Dissection box
PROCEDURE
1. Identify the male grass hopper, which has lean and lengthy tail whereas, female has
short and blunt tail.
th
2. Turn the dorsal side of grasshopper and count the 7 segment from the tail which is
abdominal portion of the grasshopper.
th
3. Using sharp scalpel cut at the 7 segment and press gently to release a yellow fluid
clumps (which is genital organ covered with yellow fat bodies).
4. Transfer the clump carefully to the vessel containing saline for washing
5. Shake gently with lab needle so the fat bodies and follicles (white fibers in
elongated shape) get separated.
6. Transfer this follicle bunch to a Petri dish and separate it into individual follicles.
7. Take a pre-cleaned slide and transfer a follicle to the centre of the slide.
8. Add a drop of acetocarmine incubate it for 5minutes.
9. If it is dehydrated add another drop of acetocarmine and cover with cover slip.
10. Using your forefinger press gently and prepare squash carefully.
11. Remove the excess stain by blotting technique.
12. Focus at 10X and examine at 40X for sharp image using Light microscope.
13. Work together to set up slides in a series of microscopes representing as complete a

set of stages as possible.
Observation under microscope

10X 40X
RESULT
Different stages of meiotic cell division could be visible.
Meiosis stages Different stages observed

Interphase - Condensed chromosome
Prophase - Thread like chromosome structure’
Metaphase - Spindle fibers and distinct chromosome at center of the cell
Anaphase - Stretching of chromatids towards opposite poles
Telophase - cell undergoes equal division
Experiment no. 4 PREPARATION OF POLYTENE CHROMOSOME FROM
Date: DROSOPHILA SALIVARY GLAND
INTRODUCTION
Certain specialized and enlarged chromosomes are found in varied groups of animals
and plant kingdom. One of these giant chromosomes is polytene chromosome. These are
found in salivary glands, malpighian tubules, epithelial lining of gut and some fat bodies of
the larval stage of certain Dipteria. The polytene chromosomes of salivary gland of
chironomous larva can be easily studied on laboratory.
The chironomous larvae are available in plenty in fresh water ponds, pools and
ditches and even in the drains. These are red in colour due to the presence of haemoglobin in
their blood. Collect these larvae in a glass jar along with pond water. Drain off water and pick
up these larvae with forceps and now keep them in normal saline water.
MATERIALS REQUIRED
Chironomous larvae, clean slide, needles, cover glass, blotting paper, 0.7% NaCl (Saline
Water), microscope and acetocamine stain.
DISSECTION OF SALIVARY GLAND
 Put few drops of saline water on a slide and place the 3rd instar larva of chironomous
in it.
 Differentiate the anterior and posterior ends of the larva. Respiratory gills are present
at the posterior end.
 With the help of two needles separate its abdomen from thorax and first needle firmly

on the head of the larva.
 Then press the second needle in such a way that the abdomen is separated from the
head and thorax.
 Now press the thorax with a needle in your left hand and then release the pressure.
The transparent pear shaped salivary glands float out from the posterior end of thorax.
SQUASH PREPARATION
 Put a few drops of acetocarmine or acetocarcein stain on a clean slide.
 Immerse the salivary gland in this drop and leave it for 5-10 minutes.
 Then cover it with the cover glass gently. Place this slide in between 2 folds of
blotting paper and then press the cover slip gently with the tip of your finger, or press
gently with the help of the needle.
 The excess stain is absorbed by the blotting paper. Observe the material under
microscope under high magnification and study the detailed structure of polytene
chromosome.
 Draw the polytene chromosome and chironomous larva and salivary gland in the
record after observing the material and by referring the diagrams.
OBSERVATION
1. This slide presents the salivary gland chromosomes of the chironomous larva.
2. The total number of chromosomes in each group is 4 pairs
3. Polytene chromosomes of salivary gland are large size and multistranded structures
4. Each chromosome is formed of a large number of chromonomal fibers lying close

together
5. Polytene chromosomes present alternate pattern of bands and inter bands
6. The bands are darkly stained and more condensed
7. Chromocentre is present in polytene chromosomes of chironomous larva
8. Nucleolus is also not distinct

RESULTS
Experiment no. 5 MONOHYBRID AND DIHYBRID RATIO IN DROSOPHILA
Date: (SIMPLE MENDELIAN GENETICS IN DROSOPHILA)
AIM
To perform and analyse the data of Drosophila species using Monohybrid and
Dihybrid ratio based on Mendelian Genetics
INTRODUCTION
Drosophila biology
The common fruit fly is a model organism for genetic studies. The reason it is so
widely used is because it is easily cultured in the lab, has a short generation time, and can
produce many offspring. The life cycle of fruit flies is
 The life cycle (from egg to adult) takes about 10 days at room temperature.
 Eggs are laid and hatch into first instar larvae. These larvae feed voraciously on the
culture medium provided.
 You can observe this by looking at a culture bottle – you should see many tunnels in the
medium made by small white larvae (or maggots).
 These first instar larvae go through several instar stages and eventually the third instar
larvae crawl up the sides of the bottle away from the culture medium.
 There they stop and their larval cuticle hardens forming a dark brown pupa.
 Metamorphosis takes place during the pupal stage.
 Larvae tissues degenerate and reorganize forming an adult fly inside the pupal case.
 When metamorphosis is complete, the adult fly emerges from the pupal case. Cell Biology and Genetics
 After the fly emerges, the wings expand and dry, the abdomen becomes more rotund, and
the colour of the body darkens.
 The first task of the lab is to become proficient at sexing the flies.
FEMALE MALE
PRINCIPLE
Based on the Gregor Mendel’s law of Inheritance, Mendel hypothesized that allele
pairs separate randomly, or segregate, from each other during the production of gametes: egg
and sperm. Because allele pairs separate during gamete production, a sperm or egg carries
only one allele for each inherited trait. When sperm and egg unite at fertilization, each
contributes its allele, restoring the paired condition in the offspring. This is called the Law of
Segregation. Mendel also found that each pair of alleles segregates independently of the
other pairs of alleles during gamete formation. This is known as the Law of Independent
Assortment. Some alleles are dominant while others are recessive; an organism with at least
one dominant allele will display the effect of the dominant allele. This is known as the Law
of Dominance
To test the hypothesis that the observed ratio of 70:30 is the same as the expected
ratio of 3:1 in Monohybrid Cross and 9:3:3:1 in the Dihybrid cross, we can use a statistic
called the Chi square statistic.
Chi square statistic:
A chi square (X2) statistic is used to investigate whether two distributions of

categorical variables differ from each other. In statistical testing we always use a null
hypothesis that there is no difference between the distributions. (Note: Chi square tests can
only be used on actual numbers and not on percentages, proportions, means, etc.)
PROCEDURE
In our case, we can use the actual observed number of flies of each type as
our observed values. We can find the expected number of flies of each type for a 3:1 ratio in
Monohybrid and 9:3:3:1 in Dihybrid cross by using the same number of flies in both the
studies of Law of Segregation and Independent Assortment.
1. Firstly we have to consider the Null Hypothesis (H0) that if the data is similar in all cross
giving the ratio with more than 50% chance, then it is rejected otherwise it would not be
rejected based on the calculation and table value.
2. In the table, write the observed number and calculate the expected number by using the
formula, Total frequency X [x/(x+y)] and Total frequency X [y/(x+y)].
3. For each observed number in the table subtract the corresponding expected number
(O — E). This is marked as “d”.
4. Square the difference [(O —E)2 ]. This is marked as “d2”.
5. Divide the squares obtained for each cell in the table by the expected number for that cell
[ (O - E)2 / E ].
6. Sum all the values for (O - E)2/ E. This is the chi χ2 square statistic.
7. Find the Degree of Freedom which is equal to (no. of row – 1) multiplied by (no. of
column – 1).
8. Based on this find the table value using either (degree of Freedom, 0.05) or (degree of
freedom, 0.01).
9. If the table value is greater than the calculated value then the null hypothesis is failed to
be rejected. If the table value is less than the calculated value then the null hypothesis is
rejected.
CALCULATION (left hand side)
a) Monohybrid Cross Cell Biology and Genetics

Eye Colour Wing Shape
Dominant Red [Rr] Normal [Dr]
Recessive Purple [Rr+] Dumpy [Dr+]
Cross between: RrRr, DrDr X Rr+ Rr+, Dr+Dr+
F1 generation:
Female Male RrDr RrDr
Rr+Dr+ RrRr+, DrDr+ RrRr+, DrDr+
Rr+Dr+ RrRr+, DrDr+ RrRr+, DrDr+
Test Cross : RrRr+, DrDr+ X RrRr+, DrDr+
Therefore, Consider the total number of Drosophila = 360 Individual
Then, it shows that
Test Cross
Parent Rr, Dr [89] Rr+Dr+ [97]
Recombinant Rr+, Dr [94] Rr, Dr+ [80]
Ratio : 1:1
Step 1: Setting up the χ2 Table (2 X 2)
Parent Recombinant Total
Observed 186 174 360
Expected (1/2)x360 = 180 (1/2)x360 = 180 360
Step 2: The Degree of Freedom = (No. of Row – 1) x (No. of Column – 1) Cell Biology and Genetics
= (2 – 1) x (2 – 1)
=1
Step 3: Null Hypothesis (H0)
If the data is similar in all cross giving the ratio with more than 50% chance, then it is
rejected.
If the data is not similar in all cross giving the ratio with more than 50% chance, then it is not
rejected.
Step 4: Table for χ2 square Calculation
Characters Observed Expected (O-E) or d (O-E)2 or d2 (O-E)2/E or

Value (O) value (E) d2/E
Parent
Recombinant
( )
Total χ2 =
Step 5: Table value (Degree of Freedom, 0.05) = T (1, 0.05) = 3.841
Calculated value χ2 = _______________
Step 6: Therefore, T (1, 0.05) is ________________than the calculated value χ2 =

___________
Step 7: This shows that the Null hypothesis (H0) is ___________________________
b) Dihybrid Cross
Two dihybrid are crossed in a situation where complete dominance without linkage and
other complications is assumed. The F2 generation shows red eye: normal wings as 562,
yellow eye: dumpy wings as 144, red eye: dumpy wings as 194 and yellow eye: normal
wings as 81. Is this result significantly different from the expected frequencies dictated
by the genetic model?
Step 1: Based on the Hypothesis and Genetics Cell Biology and Genetics
9/16 is both dominant; 3/16 one dominant and one recessive; 3/16 one recessive and one
dominant and 1/16 is both recessive
Step 2: Computation of expected frequencies on the basis of the apriori ratio of 9:3:3:1 of the
total F2 of 981 (562+144+194+81 = 981)
9/16 x 981 = 552
3/16 x 981 =184
3/16 x 981 = 184
1/16 x 981 = 61
Step 3: Setting up χ2 Table
Red eye: Red eye: Yellow eye: Yellow eye: Total

Normal Dumpy Dumpy Normal
wings wings wings wings
Observed
Expected
Total
Step 4: H0: Degree of Freedom (D.F.) = (no. of rows – 1) x (no. of column – 1)
= (2 – 1) x (4 – 1)
=1x3 =3
Characters Observed Expected (O-E) or (O-E)2 (O-E)2/E or

Value (O) value (E) d or d2 d2/E

Red eye: Normal wings
Red eye: Dumpy wings
Yellow eye: Dumpy wings
Yellow eye: Normal wings
( )
Total χ2 =
Step 6: Table χ2 at 0.05 LS and 3 D.F. = 7. 815
Step 7: Decision: Calculated χ2 _________ is _____________than the table χ2 (7.815)
RESULT
a) Monohybrid Cross
Therefore, table χ2 (3.841) is ____________than the calculated value χ2 = ___________
This shows that the Null hypothesis (H0) is ___________________________
b) Dihybrid Cross
Calculated χ2 _________ is _____________than the table χ2 (7.815)
Experiment no. 6 MONOHYBRID AND DIHYBRID RATIO IN MAIZE
Date: (SIMPLE MENDELIAN GENETICS IN MAIZE)
AIM
To perform and analyse the data of Maize kernels using Monohybrid and Dihybrid
ratio based on Mendelian Genetics
PRINCIPLE
Based on the Gregor Mendel’s law of Inheritance, Mendel hypothesized that allele
pairs separate randomly, or segregate, from each other during the production of gametes: egg
and sperm. Because allele pairs separate during gamete production, a sperm or egg carries
only one allele for each inherited trait. When sperm and egg unite at fertilization, each
contributes its allele, restoring the paired condition in the offspring. This is called the Law of
Segregation. Mendel also found that each pair of alleles segregates independently of the
other pairs of alleles during gamete formation. This is known as the Law of Independent
Assortment. Some alleles are dominant while others are recessive; an organism with at least
one dominant allele will display the effect of the dominant allele. This is known as the Law
of Dominance
To test the hypothesis that the observed ratio of 70:30 is the same as the expected
ratio of 3:1 in Monohybrid Cross and 9:3:3:1 in the Dihybrid cross, we can use a statistic
called the Chi square statistic.
Chi square statistic:
A chi square (X2) statistic is used to investigate whether two distributions of

categorical variables differ from each other. In statistical testing we always use a null
hypothesis that there is no difference between the distributions. (Note: Chi square tests can
only be used on actual numbers and not on percentages, proportions, means, etc.)
PROCEDURE
In our case, we can use the actual observed number of maize kernels of each type as
our observed values. We can find the expected number of maize kernels of each type for a 3:1
ratio in Monohybrid and 9:3:3:1 in Dihybrid cross by using the same number of Maize
kernels in both the studies of Law of Segregation and Independent Assortment.
10. Firstly we have to consider the Null Hypothesis (H0) that if the data is similar in all cross
giving the ratio with more than 50% chance, then it is rejected otherwise it would not be
rejected based on the calculation and table value.
11. In the table, write the observed number and calculate the expected number by using the
formula, Total frequency X [x/(x+y)] and Total frequency X [y/(x+y)].
12. For each observed number in the table subtract the corresponding expected number
(O — E). This is marked as “d”.
13. Square the difference [(O —E)2 ]. This is marked as “d2”.
14. Divide the squares obtained for each cell in the table by the expected number for that cell
[ (O - E)2 / E ].
15. Sum all the values for (O - E)2/ E. This is the chi χ2 square statistic.
16. Find the Degree of Freedom which is equal to (no. of row – 1) multiplied by (no. of
column – 1).
17. Based on this find the table value using either (degree of Freedom, 0.05) or (degree of
freedom, 0.01).
18. If the table value is greater than the calculated value then the null hypothesis is failed to
be rejected. If the table value is less than the calculated value then the null hypothesis is
rejected.
CALCULATION (left hand side)
c) Monohybrid Cross
According to a genetic model, a Yellow kernel is inherited as a simple dominant trait and
Purple/Black kernels is inherited as a recessive trait. A cross between pairs of heterozygous Cell Biology and Genetics
Yellow kernel maize produces an F2 generation consisting of 124 Yellow kernels and 108
Purple/Black kernels. Does the ratio differ significantly from the expected ratio?
Step 1: Genetypes: YY – homozyogous yellow; Yy – Heterozygous Yellow; yy –

homozygous Purple/Black
Step 2: Cross between heterozygous Yellow
Yy x Yy
Female Male Y y
Y YY (Yellow) Yy (Yellow)
y Yy (Yellow) Yy(Purple/Black)
Ratio: 3/4 Yellow and 1/4 Purple/Black
Step 3: Computation of expected frequencies based on the apriori hypothesis, i.e., 3/4 of the
F2 maize should be Yellow and 1/4 should be Purple/Black
Total no. of F2 maize was 232 (124+108 = 232)
3/4 x 232 = 174
1/4 x 232 = 58
Step 4: Setting up the χ2 Table (2 X 2)
Yellow Purple/Black Total
Observed 124 108 232
Expected (3/4)x232 = 174 (1/4)x232 = 58 232
Step 5: The Degree of Freedom = (No. of Row – 1) x (No. of Column – 1)
= (2 – 1) x (2 – 1)
=1
Step 6: Null Hypothesis (H0)
If the data is similar in all cross giving the ratio with more than 50% chance, then it is Cell Biology and Genetics
rejected.
If the data is not similar in all cross giving the ratio with more than 50% chance, then it is not
rejected.
Characters Observed Expected (O-E) or d (O-E)2 or d2 (O-E)2/E or

Value (O) value (E) d2/E
Yellow
Purple/Black
( )
Total χ2 =
Step 8: Table value (Degree of Freedom, 0.05) = T (1, 0.05) = 3.841
Calculated value χ2 = _______________
Step 9: Therefore, T (1, 0.05) is ________________than the calculated value χ2 =

___________
d) Dihybrid Cross
Two dihybrid are crossed in a situation where complete dominance without linkage and
other complications is assumed. The F2 generation shows yellow: smooth as 318, yellow
wrinkled as 111, dark yellow: wrinkled as 89 and dark yellow: smooth as 71. Is this
result significantly different from the expected frequencies dictated by the genetic
model?
Step 1: Based on the Hypothesis and Genetics

9/16 is both dominant; 3/16 one dominant and one recessive; 3/16 one recessive and one
dominant and 1/16 is both recessive
Step 2: Computation of expected frequencies on the basis of the apriori ratio of 9:3:3:1 of the
total F2 of 589 (318+111+89+71 = 589)
9/16 x 589 = 331
3/16 x 589 =110
3/16 x 589 = 110
1/16 x 589 = 37
Step 3: Setting up χ2 Table
Yellow: Yellow: Dark yellow: Dark Total

smooth wrinkled wrinkled yellow:
smooth
Observed
Expected
Total
Step 4: H0: Degree of Freedom (D.F.) = (no. of rows – 1) x (no. of column – 1)
= (2 – 1) x (4 – 1)
=1x3 =3
Characters Observed Expected (O-E) or (O-E)2 (O-E)2/E or

Value (O) value (E) d or d2 d2/E
Yellow: smooth
Yellow: wrinkled
Dark yellow: wrinkled
Dark yellow: smooth Cell Biology and Genetics

( )
Total χ2 =
Step 6: Table χ2 at 0.05 LS and 3 D.F. = 7. 815
Step 7: Decision: Calculated χ2 _________ is _____________than the table χ2 (7.815)
RESULT
a) Monohybrid Cross
Therefore, table χ2 (3.841) is ____________than the calculated value χ2 = ___________
b) Dihybrid Cross
Calculated χ2 _________ is _____________than the table χ2 (7.815)

CELL BILOGY AND GENETICS MANUAL (Practical 1 To 6)

Uploaded by

Copyright:

Available Formats

CELL BILOGY AND GENETICS MANUAL (Practical 1 To 6)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

CELL BILOGY AND GENETICS MANUAL (Practical 1 To 6)

Uploaded by

Copyright:

Available Formats

CELL BILOGY AND GENETICS

Cell Biology and Genetics

B) MICROSCOPIC OBSERVATION OF ANIMLA CELL: BUCCAL SMEAR

Cell Biology and Genetics

Cell Biology and Genetics

Cell Biology and Genetics

Cell Biology and Genetics

 The root tip was then hydrolysed in 1N HCL for ½ an hour.

 The disintegration of nuclear envelope first starts.

 The chromatids become shorten and thickened.

 The cell becomes spheroid, diffractive and viscous.

 The centromere of each chromosome was divided into two.

Cell Biology and Genetics

 The nucleolus reappears.

 After telophase two daughter cells are formed.

Cell Biology and Genetics

In this experiment, we would be studying meiosis in the testis of grasshopper is an

 Acetio carmine stain

 Sample (Grasshopper testis follicles)

 Eppendorfs microcentrifuge tubes

Cell Biology and Genetics

8. Add a drop of acetocarmine incubate it for 5minutes.

11. Remove the excess stain by blotting technique.

13. Work together to set up slides in a series of microscopes representing as complete a

Observation under microscope

Cell Biology and Genetics

Different stages of meiotic cell division could be visible.

Meiosis stages Different stages observed

Cell Biology and Genetics

DISSECTION OF SALIVARY GLAND

Cell Biology and Genetics

 Put a few drops of acetocarmine or acetocarcein stain on a clean slide.

2. The total number of chromosomes in each group is 4 pairs

4. Each chromosome is formed of a large number of chromonomal fibers lying close

5. Polytene chromosomes present alternate pattern of bands and inter bands

6. The bands are darkly stained and more condensed

7. Chromocentre is present in polytene chromosomes of chironomous larva

8. Nucleolus is also not distinct

 Metamorphosis takes place during the pupal stage.

Chi square statistic:

A chi square (X2) statistic is used to investigate whether two distributions of

4. Square the difference [(O —E)2 ]. This is marked as “d2”.

CALCULATION (left hand side)

a) Monohybrid Cross Cell Biology and Genetics

Dominant Red [Rr] Normal [Dr]

Recessive Purple [Rr+] Dumpy [Dr+]

Female Male RrDr RrDr

Rr+Dr+ RrRr+, DrDr+ RrRr+, DrDr+

Rr+Dr+ RrRr+, DrDr+ RrRr+, DrDr+

Test Cross : RrRr+, DrDr+ X RrRr+, DrDr+

Therefore, Consider the total number of Drosophila = 360 Individual

Then, it shows that

Parent Rr, Dr [89] Rr+Dr+ [97]

Recombinant Rr+, Dr [94] Rr, Dr+ [80]

Step 1: Setting up the χ2 Table (2 X 2)

Parent Recombinant Total

Observed 186 174 360

Expected (1/2)x360 = 180 (1/2)x360 = 180 360

Step 3: Null Hypothesis (H0)

Step 7: Decision: Calculated χ2 _ is _____than the table χ2 (7.815)

Therefore, table χ2 (3.841) is __than the calculated value χ2 = _

Calculated χ2 _ is _____than the table χ2 (7.815)

Step 7: Decision: Calculated χ2 _ is _____than the table χ2 (7.815)

Therefore, table χ2 (3.841) is __than the calculated value χ2 = _

Calculated χ2 _ is _____than the table χ2 (7.815)