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Yashwantrao Chavan
Maharashtra
Open University
Post Graduate Degree Programme (Bio-Technology)
SBT085: Lab Course M. Sc. (BioTechnology)
Semester 8 Lab
Workbook
Yashwantrao Chavan Maharashtra Open University, Nashik
Vice-Chancellor-Dr. Rajan Welukar
Expert Advisory Committee
Mr. Manoj Killedar
Director, School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Chetana Kamlaskar
School of Science & Technology, Y.C.M. Open University, Nashik
Dr. Sunil Ganatra
135, Krushnakunj, Toata Colony, Lakadganj, Nagpur
Prof. Indira Ghosh
Bio-Informatics Center, University of Pune, Pune
Prof. Urmila Kulkarni-Kale
Bio-Informatics Center, University of Pune, Pune
Prof. Dr. Piyali Kar
Maharashtra Education Foundation, CBD Belapur, Navi Mumbai
Course Writer
Mr. Pravinkumar Domde
G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345,
Kingsway, Nagpur
Course Editor
Dr. Suchitra Godbole
G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345,
Kingsway, Nagpur
Course Coordinator and IT Editor
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
E-Production
Manoj Killedar, Director, School of Architecture, Science & Technology
E-Version available at
http://www.ycmou.com=>Architecture, Science and Technology=>Master
of Science (Bio-Technology)=>Learning Resource for Semester 8
© Yashwantrao Chavan Maharashtra Open University, Nashik
Printed & Published by: Dr. Rajendra Vadnere, Registrar, YCMOU, Nashik
SBT085_Lab Manual
Page 2
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Page 3
List of practicals of MSc Biotechnology Course SBT 085
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Titles of the practical
P
age
r
N
o.
N
o.
Isolation of single colony on solid media: streak plate and
spread plate method
Staining Techniques - simple staining, gram staining, acid fast
staining, endospore staining
To perform hanging drop technique for demonstration of
motility of bacteria.
Anaerobic cultivation of bacteria
Determination of antibiotic resistance of bacteria - Test for
antibiotic sensitivity by Disc method (Kirby Bauer Method)
Demonstration of oligodynamic action
Determination of phenol coefficient of disinfectants.
Effect of salt, sugar and pH on microbial growth
Sub-cellular fractionation of animal tissues by homogenization
method
0
1
2
3
Preparation of temporary slides of mitosis from onion root tips
Preparation of temporary slides of Meiosis
Histology of animal tissues
I) Epidermal cells (skin)
II) Nerve cells
III) Connective tissue-blood, cartilage muscular tissue
striated, non-striated cardiac muscles
IV) Epithelilal Tissue
Histology of plant tissue and Preparation of Microscope Slides
of
a) monocot/dicot -stem/ root
b) Leaf isobilateral, dorsiventral
Study of permanent slide of Polytene chromosome
4
Determination of monohybrid ratio
5
6
Determination
Assortment}
of
Dihybrid
Ratio
{Law
of
Independent
Karyotype analysis
7
Human pedigree analysis
8
9
Computer aided visualization of amino acid sequence of
protein and its 3D structure using RCSB PDB (Protein Data
Bank).
Retrieval of metabolic pathway using internet
0
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1
Retrieval and study of nucleic acid sequence databanks in
Gene Bank
Homology searching using BLAST
2
Computer aided survey of scientific literature
3
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Experiment No. 1
Aim: Isolation of single colony on solid media: streak plate and spread plate
method
Streak Plate Method
Principle:
In order to study microbiology systematically, it is necessary to examine characteristic
behavior of one kind of organisms at a time. Several different methods are used for the
isolation of pure culture of microorganisms. These isolation methods usually involve
separating microorganisms on a solid medium into individual cells that are then allowed to
reproduce clones.
A pure culture, one containing a single kind of microbes, is required in order to
study its varied characteristics like, growth, physiology, metabolism, pathogenicity etc.
Generally, bacteria exist in mixed population. It is very rare to get a single and
pure form. The streak plate method offers a most practical method of obtaining isolated
colonies and pure culture.
In
this method a sterilized loop or transfer needle is dipped into a dilute
suspension of organism which is then streaked on the surface of an agar plate to make
a parallel series , non-overlapping streaks . The
aim of this method is to
obtain
colonies of microorganisms that are pure.
Requirements:
Petri plate,
Nutrient agar,
Bacterial culture,
Inoculating needle.
Procedure
1. Take sterilized Petri plate and pour cooled nutrient agar in Petri plate
under aseptic condition
2. Allow it to solidify.
3. Transfer a loop full of culture near one edge of the agar surfaces
and perform four way streaking on agar plate under aseptic
condition .
4. Incubate the petri dishes in an inverted position at 37°C for 24-48 hrs
Photographs of petriplates showing isolation of bacterial cultures by streak
method
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plate
Observation
By streaking, dilution gradient gets established. This is the main principle of this
method. Because of this dilution gradient, confluent growth ( mixed or crowed)
occurs on part of the surface where cells are not sufficiently separated. On the
other hand, individual isolated colonies develop in the other part of the agar
surface where only few bacterial cell get deposited
( due to dilution) . These
isolated colonies can be removed and inoculated into fresh medium and maintained
as pure culture.
After incubation at 37°C for 24 hrs isolated colonies can be seen on the media Petri
plate .
Note: Incubation temperature and time varies from bacterial species to species.
Result:
Isolated colonies of the ----------- culture were obtained by streak plate method
Spread plate method.
Principle
This is another way of obtaining pure culture by developing colonies on agar plate. In this
method the diluted source sample is inoculated in small volume (0.1ml) and spread
throughout the agar surface of the plate using a sterile spreading rod. As a result the cells in
the inoculum get separated and grow as individual colonies on incubation. By counting
colonies and multiplying with dilution factor and volume plated, the viable count of bacterial
cells in source sample can be calculated.
When inoculums is transferred into Petridish containing nutrient agar it is then
spread over solidified agar medium with the help of L-shaped glass tube called spreader.
Requirements
Petri plates
Nutrient agar
Spreader
Bacterial culture
Pipettes
Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.
Prepare the serial dilutions of source (mixed culture) sample in sterile
distilled water.
Take sterilized Petri plates and pour cooled nutrient agar under aseptic
condition.
Allow it to solidify.
Add 0.1ml of mixed culture from each dilution with the help of pipette on
the solidified nutrient agar plate.
With the help of sterile spreader spread the inoculums immediately on
agar plate.
Each dilution should be plated in at least three plates.
Incubate the plate in inverted position at optimum temperature in
incubator.
Observe the growth of colonies in plates after 24-48 hrs incubation
Find out the viable count of bacterial cells in the given sample.
Observation
•
•
color.
•
•
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Sketch the appearance of streak plates
Describe the colonies-diameter, appearance, margin, elevation and
Count the number of colonies per plate in pour / spread plates.
Calculate the viable count in the given sample.
Page 8
After spread plate method of the given sample following observation has been obtained
No. of colonies on the plate-----------Diameters of colonies ---------------Appearance of colonies --------------Margin of colonies ---------------------Elevation and color of colonies-------Viable count -------------------Result
On the basis of above observation by spread plate method it has been concluded that the
given sample contain (pure or mixed) population of the -------------- culture.
Experiment No. 2
Aim: Staining Techniques - simple staining, gram staining, acid fast staining, endospore
staining
Simple Staining
Principle
This technique is recommended to study the morphology and arrangement of bacterial cells.
When a single dye is used, the process is referred to as ‘simple staining’ or ‘monochrome
staining’ since only one staining solution is employed for colorization of bacterial smear. In this
case basic stains with positively charged chromogen ( dye) binds with negatively charged cell
wall components ( slightly acidic in nature). Carbol fuchsin, crystal violet and methylene blue are
commonly employed for simple staining.
Requirements
Inoculating loop
Bunsen burner
Staining tray
Microscope
Glass slide
Methylene blue (0.5%)
Crystal violet (1%)
Carbol fuchsin ( diluted 10 times before use)
Procedure
1)
Fix the smear on the slide by gentle warming over the flame of spirit
lamp.
2)
Flood the smear with a solution of a basic dye such as carbol fuchsin,
crystal violet and methylene blue. The carbol fuchsin requires 15-20 seconds, crystal
violet 2-60 seconds and methylene blue stains with within 1-2 minutes.
3)
Then gently rinse with tap water and dry with blotting paper.
4)
Examine the smear under oil immersion objective.
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Staining
Result
Bacteria are observed blue, violet or red depending upon the stain used against the colorless
background.
Gram Staining
Principle
Bacteria are chemically different from their environment and thus can be stained, in a contrasting
fashion, for visualization. This procedure was first developed in 1884 by the Danish Bacteriologist
Christian Gram, who discovered that all bacteria could be divided into two major groups –Gram
positive and Gram negative, based on their staining reaction. This technique is called differential
staining since it allows the microbiologist to highlight the differences between the cell types. It is
perhaps the most powerful staining protocol employed in microbiology.
Staining requires four solutions, used in a sequence, a basic dye (initial stain), a mordant,
a decolorizing agent and a counter stain. A mordant is a chemical substance that enhances the
affinity between the cells and the dye making it much harder to remove the stain. The
decolorizing agent removes excess dye from the stained bacterial cells. The gram stain is based
on the capacity for some bacteria to decolorize at a faster rate than other. The counter stain is a
basic dye of different color from the initial one and is used to give the decolorized cell a different
color than that of the initial stain. Bacteria that do not decolorize hold the initial basic stain color,
whereas those that decolorize will have the color of the counter stain. Safranin is the most
preferred counter stain as it provides the greatest contrast and makes the cells more visible.
Specifically in the Gram stain, fixed cells are stained with crystal violet (basic dye), next
exposed to Gram’s Iodine (mordant) and then treated with alcohol (decolorizing agent). Bacteria
retaining the basic dye after decolorization are termed Gram positive, whereas bacteria that are
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decolorized are Gram negative and can be restained with safranin (counter stain) which has a
red/pink color.
Requirements
Materials
Clean glass slides
Bunsen burner/spirit lamp
Inoculating loop or needle
Microscope
Dropping bottle
Bacterial cultures ( Gram +ve and Gram –ve), mixed culture.
Reagents
Crystal violet stain
Gram’s iodine
95% Alcohol
Safranin solution
Procedure
•
•
•
•
•
•
•
•
•
•
Prepare and fix the smears of each culture on the slide by gentle
warming over the flame of spirit lamp.
Flood the smear with crystal violet stain and let stand for 1-2 min.
Wash gently with drops of water.
Flood the smear with Gram’s iodine solution and allow it to remain for 12 min.
Wash gently with drops of water
Decolorize with 95% alcohol for about 10 -20 seconds, until only faint
violet color remains in the solvent after the proper time interval. Caution: do not
over decolorize.
Wash gently with drops of water and blot dry
Counter stain by flooding the slide with safranin for 1-2 min.
Wash gently with drops of water and blot dry
Examine the slide under low/ high power and oil immersion objectives
and determine morphology, Gram reaction (color) and arrangement of each culture.
Gram Staining of bacteria
isolated on Nutrient agar
plate.
Long gram positive chains are
in violet., Gram –ve rods in
red.
Observation/Result
•
Gram +ve bacteria stain blue whereas Gram –ve bacteria red.
In the mixed culure staining if used, both types could be observed.
•
Make drawing of the bacterial cell.
•
Describe the nature of the cells according to the their
morphology and arrangement.
•
Describe the color of the stained cells
•
Classify the organisms as to the Gram +ve/ -ve
Result
The given bacterial culture has been found to be ---------------------( Gram –ve/ Gram +ve)
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Acid-Fast Staining
Principle
Certain bacteria have special chemical structures in their cell wall which do not allow the entry of
routinely used dyes when stained and do not get destained on decolorization. One such
bacterium is the Mycobacterium sp which is stained with special method of treating the cells. This
is due to the presence of specific fatty acids, like mycolic acid in their cell wall structure. Due to
this they resist mild acid environment. Therefore the cells are subjected to staining at high
temperature in wet condition. Subsequently they are treated with acid alcohol which makes other
organisms to get destained and they appear with counter (methylene blue) stain color. Whereas
mycobacteria retain the initial stain ( carbol fuschin) color, making it easy for their identification.
Requirements
Cultures : 72 hrs broth cultures of Mycobacterium or
Sputum smears of tuberculosis patient
Reagents
Acid alcohol
Carbol fuschin
Methylene blue
Equipments
Bunsen burner
Staining tray
Lens paper
Inoculating loop
Glass light
Microscope
Other routine requirements
Procedure
•
•
•
•
•
•
•
•
•
Observation
•
•
•
•
Prepare the smear of the culture or sputum sample of TB patient
carefully by using aseptic technique, air dry and heat fix.
Flood the slide with carbol fuschin and heat till the stain starts boiling
in steam.
Allow the slide to stain for two to three minutes, heating
intermittently to keep the stain hot.
Wash the slide thus prepared with drop of water gently.
Now de-colorize the slide with acid alcohol adding the reagent drop
by drop until carbol fuschin fails to wash from smear.
Wash with drops of water.
Counter stain with methylene blue for 2 min.
Wash the smear gently with drops of water.
Blot dry the smear in coarse filter paper and examine under
microscope, first under low power followed by high magnification.
fast
Make drawings of the organism
Describe the cells according to their shapes and arrangement
Describe the color of the stained cells
Describe the organisms as to the reaction-acid –fast or non –acid –
Result
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The given bacterial culture had been found to be------------------------------------------- (acid –
fast or non –acid –fast)
Endospore staining
Principle
Endospores are the most resistant structure of certain bacteria and are know for their resistance
to high temperatures, radiation, desiccation and chemical disinfection hence play major role in
the perpetuation of bacteria forming them. The spore formation is primarily observed in two
major genera- Bacillus (aerobic) and clostridium(anaerobic), and also noted in certain Sarcnia.
The endospores are resistant to chemical stains. However they are stained by subjecting
to heat treatment at which the cells become permeable. Once stained, the spore cells do not
loose color at normal temperature. By using two different colored dyes for staining, spores and
vegetative cells can be differentiated where in spores retain initial color and vegetative cells get
the counter stain color. The Malachite green staining method employs hot malachite green as
intense stain, which is not removed from the endospore by washing and safranin as a counter
stain. Thus the endospore stains green, but the remainder of the cell ( or a cell without
endospore) stains light red.
Requirements
Materials
Sporulating bacterial culture- Bacillus sp. Or Clostridium sp.
Water bath
Spirit lamp/Bunsen burner
Clean glass slides
Wire gauge
Blotting/filter paper
Reagents
Malachite green solution
Safranin solution
Microscope
Immersion oil
Procedure
•
•
•
•
•
•
•
•
Prepare smears of bacterial cultures on grease-free slides, air dry and
heat fix in the usual manner.
Place the slide on staining rack above boiling water bath.
Cove the smear with small piece of filter paper towel, keep it saturated
with malachite green (aqueous solution) and continue heating for 5 minutes.
Do not allow the filter paper to dry keep it saturated all through the
heating
Remove the paper towel and wash the slides gently with drops of water.
Counter stain with safranin for 1-2 min.
Wash with drops of water and blot dry.
Observe under low / high power and oil – immersion objectives and
make drawings.
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Endospore stain B. subtilis
Observation
• Endospore appear green and remainder of the cell without endospore i.e. vegetative
cells appear light red.
• Make drawing of the cells with spores observed.
• Describe the nature and location of the spores within the cell, as being central,
subterminal or terminal
• Indicate the color of the spore and the vegetative cell.
Result
Endopore staining of the given bacteria sample has resulted in the -------------------------presence /a0bsence) of endospore.
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Experiment No.3
Aim: To perform hanging drop technique for
bacteria.
demonstration of motility of
Principle
The simplest method for examining living microorganisms is to suspend them in a fluid
(water, saline or broth) and prepare a hanging drop using cover glass and cavity slide.
The slide is ground with a concave well in the centre, the cover glass holds a drop of
suspension when the cover glass is inverted over the well of the slide, the drop hangs
from the glass in the hollow concavity of the slide. This technique is specially used to
determine the ability of organisms to move independently in liquid cultures. It is very
essential to distinguish between true motility and the Brownian movement. In true
motility the organism changes its position in the field and Brownian movement is false
movement which is an oscillatory movement of very small non motile which are
suspended in the field.
Many bacteria are motile due to the presence of one or more very fine thread like
filamentous appendages called “flagella”. Bacteria show four types of flagellation pattern.
I. Monotrichous – having single flagellum at one end.
II. Lophotrichous – having many flagella at one end.
III. Amphitrichous - having flagella at both the ends.
IV. Peritrichous
- having flagella all over the surface.
Hanging drop method is an indirect method for demonstrating the presence of
flagella.
Requirement
Cavity slide,
Cover slip,
Culture,
Inoculating needle.
Procedure
1. Make suspensions of overnight slant cultures in broth and incubate for 2-3 hrs
to have actively growing cells
2. Place a little amount of grease with the help of match stick at the four corners
of a clean cover slip.
3. Place small drop of suspension on coverslip and invert the slide over cover
slip to adhere to the grease and turn it upside down quickly so that the cover
slip is up and drop is hanging in the cavity.
4. Drop should be hanging from the coverslip in the middle of the concavity.
5. Do not let the drop fall or touch the bottom of the cavity.
6. Examine the hanging drop under the microscope.
7. First locate edge of the drop in the centre of the microscope field with low
power objective and reduce the light to create dim background.
8. You will see the edge as bright wavy line against the background.
9. Now turn the high power objective into position and focus the edge of the
drop with fine adjustment.
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10. The bacteria within the drop will dome into focus and appear as transparent
particles with fine adjustment. Observe the movement.
Observation
•
•
•
Draw the type of organisms seen and indicate their nature of motility
observed
Differentiate the motile and non-motile bacterial cells
Discuss the advantage of method
The motility of organism from given ----------- culture was observed under microscope.
Result
Organisms from
the given culture were found to be ------------ (motile /nonmotile).
Experiment No.4
Aim - Anaerobic cultivation of bacteria.
Principle
Bacteria show wide variation in the oxygen requirement. An organism that requires Oxygen is
called as ‘aerobic’ while the one whose growth is inhibited by Oxygen is called as ‘anaerobic’.
Bacteria that can grow under both aerobic and anaerobic conditions are termed as ‘facultative
anaerobes’.
‘Microaerophilic’ organisms are those that do require, but at a very low
concentration. The effect of atmospheric Oxygen on microbial growth is closely related to the
oxidation-reduction potential of the culture medium. There are several methods for the
cultivation of anaerobic organisms such as eliminating the Oxygen from the tube, cultivation of
organism in butt or by placing oil on inoculated growth.
Requirements
Test tubes
Mineral oil
Nutrient agar
Nutrient broth
Bacterial culture
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Procedure
I)
Anaerobic cultivation of bacteria by using mineral oil
1.
Prepare nutrient broth and sterilize it.
2.
Distribute sterilized nutrient broth in test tubes.
3.
Inoculate bacterial culture, take 0.1-0.5 ml culture and suspend into the
broth aseptically.
4.
Shake the tube gently for the uniform distribution of organism.
5.
Pour mineral oil in the tube side-wise so that oil cannot enter the broth.
6.
Keep tube for incubation at 37˚C for 24 hours.
II) Anaerobic cultivation of bacteria by preparing butt
1.
Prepare nutrient agar, sterilize it.
2.
Take sterilized test tubes and pour nutrient agar to it. Bring it to lower
temperature, taking care that it does not solidify.
3.
Take 0.1-0.5 ml of culture and suspend it into the nutrient agar tube
and gently shake it to mix the culture in nutrient agar.
4.
Allow it to solidify.
5.
Keep the tubes for incubation at 37˚C for 24 hours.
Observation
Microbial growth was observed in both nutrient broth as well as butt after incubation.
Result
Anaerobic organisms can be cultivated by using oil and butt method.
Experiment No. 5
Aim: Determination of antibiotic resistance of bacteria - Test for anitibiotic sensitivity
by Disc method (Kirby-Bauer Method)
Principle
The main drugs used in the medical sciences include antibiotics, sulphonamides and
chemotherapeutics. All are called antimicrobics in nature. The antimicrobics i.e antibiotic
sensitivity is quite significant due to development of resistance among various microorganisms.
The sensitivity of the drug helps in selecting the appropriate line of treatment. The effectiveness
is based on size of inhibition zone. However, zone may vary due to diffusibility of drug, size of
inoculum, type of medium etc.
Requirements
Agar plate
Swab
Bacterial culture
Incubator
Forceps
Procedure
• Plate the culture on the entire surface of the agar plate swabbed with organism to be
tested or the bacterial lawn is prepared on the plate.
• Use the ready made antibiotic disc in cartridges to dispense individual disc on the plate
• If the cartridge of antibiotic is not available, prepare solution of known concentration
of an antibiotic in sterile distilled water and dip discs (0.5 mm diameter) of whatman
filter paper no.1.
• Place only 4 to 6 discs on one plate and incubate for 12-24 hours at 37oC
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Page 17
•
Examine the plates and measure the diameter of the clearing zones to the nearest
millimeters.
The faint growth of tiny colonies in the clearing zone may appear due to resistant nature
of some bacteria. Avoid such growth.
Nutrient agar plates showing zone of inhibition by different antibiotic discs on
bacterial lawn
Results
Clear zone around the discs shows inhibitory nature of the drug/antibiotic
Experiment No. 6
Aim: Demonstration of oligodynamic action
Principle
The oligodynamic action (oligo = small, dynamic = power) is the effect of small amounts of
heavy metals of bacteria. This effect is due to high affinity of heavy metals with the cellular
proteins of the bacteria. The bacterial cells die due to cumulative effects of metal ions within the
cell. The oligodynamic action of different metals on bacteria can be compared.
Requirements
Nutrient agar tube
Water bath
Culture of E.coli or S. aureus
Petri dishes
Copper or aluminum coins
Detergents
Water
Scale
Incubator
Procedure
• Prepare culture tube of nutrient agar or liquefy nutrient agar in a tube.
• Cool to 50oC and inoculate with E. coli or S. aureu.
• Pour half of the medium into sterile Petri plate and leave the other half on waterbath.
• Leave the plates to solidify.
• Mean while take some kind of coins ( copper and aluminium), clean them with
detergent and water. As soon as they are cleaned place them on the surface of agar.
• Pour the remaining medium seeded with bactera over the metal coins and incubate the
plate at 73oC for 48 hrs.
• Bacterial growth occurs. The clearing zone may be visible around the coins.
• Measure the diameter of the clearing zone of inhibition and compare the effect of both
metallic coins on growth of the bacteria.
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Result
The clearing zone surrounding the coin indicates no growth, whereas there is a narrow zone
showing heavy bacterial growth called stimulatory growth as low amount of heavy metal coins
induced bacterial growth. Rest of the area contains normal growth. Such phenomenon confirms
oligodynamic acition.
Experiment No. 7
Aim: Determination of phenol coefficient of disinfectants.
Principle
There are many liquid chemicals that are used for cleaning of materials to lower the number of
microorganisms. Such chemicals are called as disinfectants e.g. Lysol, phenol, hypochlorite
(bleach), etc. however the effectiveness of different disinfectants varies against the given
microorganism. Therefore phenol coefficient of these disinfectants by using a standard bacterial
culture ( e. g.Staphylococcus aureus ) is determined. Hence, effectiveness of phenolics (phenol
and its derivatives) is determined by comparing with phenol. The phenolic compound kill bacteria
by inactivating plasma membrane and enzymes, and denaturing the protein. The phenol
coefficient is determined only of such chemicals which are bactericidal ( but not bacteriostatic) in
nature.
Requirements
Phenol dilution (1:10, 1:20, 1:30, ……..1:90, 1:100)
Phenol derivative’s dilution (1:100, 1:150, 1:200,…..1:400, 1: 450, 1:500)
Sterile nutrient broth tubes (20)
Broth culture of staphylococcus aureus
Sterile pipette (1ml)
Inoculation loop
Bunsen burner
Test tube stand
Procedure
•
•
•
•
•
•
Take the test tube stand each for phenol and phenolic compound to be tested.
Place a test tube of each concentration of each phenol and phenolic compound
separately in the test tube stand.
Similarly, take equal number of sterile nutrient broth tubes ( in other test tube stand)
for each dilution of phenol and phenolic compounds. Lable them according to dilution
of disinfectant(s). take one sterile nutrient broth tube as control.
After 5, 10 and 15 minutes of intervals, transfer aseptically one loopful bacterial
culture from each nutrient broth tube appropriately labeled with the same dilution.
Similarly, also transfer one loopful fresh bacterial culture in control broth tube.
Incubate all the broth tubes inoculated with Staphylococcus aureus at 37 C for 48 hrs.
Results
Observe the growth of Staphylococcus aureus in all the broth tubes with phenolic compounds and
arrange the result as given in table. Put mark on the highest dilution of phenol and the phenolic
compounds tested that kill the bacteria in 10 min but not in 5 minutes and calculate the phenol
coefficient as given below:
Phenol coefficient of the test chemicals = Reciprocal of the test chemical dilution marked
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Reciprocal of the phenol dilution marked
Suppose test chemical shows no growth at 10 minutes in 1: 400 dilution, but growth at 5
minutes, and phenol shows no growth at 10 minutes in 1:80 dilution but no growth at 5 minutes,
the phenol coefficient of test chemical will be as below:
Phenol coefficient of test chemical = 1/400 = 5
1/80
Effect of phenol and phenol derivatives on Staphylococcus aureus treated for different
time intervals
Disinfectant
chemicals
Dilution
Phenol
1:10 – 1:60
1:70
1:80
1:90
1:100
Phenolic
derivatives
1:100 – 1:350
1:400
1:450
1:500
SBT085_Lab Manual
Exposure time (min)
5
10
+
+
+
+
+
+
+
+
+
+
15
-
Page 20
Experiment No.8
Aim: Effect of salt, sugar and pH on microbial growth
To determine the effect of sugar on microbial growth
Principle
Bacteria vary widely in their osmotic requirements. Thus the principal of osmotic pressure can be
used as an effective antimicrobial agent. Hyper tonicity, as a food preservation technique,
employs addition of sugar to the food, increasing its osmotic pressure thereby making it
impossible for most organisms to grow. However, some micro organisms such as yeast and
moulds are capable of growing at high osmotic pressure and hence termed as ‘osmophilic’. Some
organisms grow only at high osmotic pressure.
This experiment tests the degree if inhibition of micro organisms that results with
media containing different concentrations of glucose.
Requirements
Culture of yeast, E.coli, B.cereus, and S.aureus.
Nutrient agar plates containing each of the following glucose concentrations- 0.5%,
5%, 10%, 15%, 20%, 30% and 50%.
Bunsen burner, inoculating loop and marker pen.
Procedure
• Divide the bottom of each nutrient agar plate in four segments.
• Label each of the four segments of each plate with the code of name of the micro
organisms to be inoculated, here 1- Yeast, 2- E.coli, 3- B.cereus, and 4- S.aureus.
• Inoculate each of the four micro organisms into the appropriate section on each of the
7 nutrient agar plates by making a single line loop inoculation.
• Repeat step 3 for the three cultures.
• Incubate all 7 inoculated plates at 37˚C in an inverted position for 24 hours.
Observation
Growth of the micro organism in varying proportions were observed in all nutrient agar plates,
which indicate the degree of growth for each micro organism with respect to the sugar
concentration on the plate.
Result
Yeast can resist high concentration of sugar while E.coli and B.cereus moderately and S.aureus
least.
To determine the effect of different salt concentrations on microbial growth.
Principle
Micro organisms vary widely in their salt tolerance. In a hyper tonic environment, all cells loose
water by osmosis and shrink. This phenomenon is called as ‘plasmolysis’. Its effect on cell
reproduction is inhibited. In a hypotonic environment water is taken by the cell and it swells. This
phenomenon is called as ‘plasmoptysis’. There is no harmful effect on micro organisms by hypo
tonic environments, while in such environments animal cells undergo lysis, which cause their
death. Most natural environments of high osmolarity contain high concentration of salts
particularly sodium chloride. Micro organisms which grow in this type of environment are called
as ‘halophiles’.
Bacteria can be divided into 4 groups depending upon their ability to grow at
various sodium chloride concentrations as non-halophilic; halophilic can be divided further into 3
types as slightly halophilic, moderately halophilic and extremely halophilic.
This experiment tests the degree if inhibition of micro organisms that results with
the media containing different concentrations of sodium chloride.
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Requirements
Cultures of yeast, E.coli, B.cereus, and S.aureus.
Nutrient agar plates containing each of the following salt concentrations- 0.5%, 5%,
10%, 15%, 20%, 30% and 40%.
Bunsen burner, inoculating loop and marker pen.
Procedure
1.
2.
Divide the bottom of each nutrient agar plate in four segments.
Label each of the four segments of each plate with the name of the
micro organism to be inoculated.
3.
Inoculate each of the four micro organisms into their appropriate section
on each of the 7 nutrient agar plates.
4.
Repeat step 3 for the remaining three cultures.
5.
Incubate all 7 inoculated plates at 37˚C in an inverted position for 24
hours.
Observation
Organism Concentration
0.5
5
10
15
20
30
40
1
2
3
4
0
0
0
0
0
0
0
0
Result
Only B.cereus and E.coli were able to tolerate small salt concentration up to
inhibited.
0.5%, rest were
To determine the effect of pH on microbial growth
The hydrogen ion concentration of an organism’s environment has the maximum influence on
bacterial growth. It limits the synthesis of bacterial enzymes responsible for synthesizing the new
protoplasm. Similarly to temperature, each microorganism has its optimum pH. These
parameters work only when other factors remain constant. If the other factors such as the media
composition temperature or osmotic pressure vary, the pH will also vary.
Requirements
Nutrient broth
pH meter
E.coli
Photocolorimeter
Incubator
Graph paper
Pencil
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Glass maker
Procedure
• To perform this experiment, prepare the nutrient broth of varying pH values viz, 5, 7 and
9
• Inoculate each tube with a loopful of E.coli in each tube and allow to incubate at 37oC for
48 hrs.
• Measure the turbidity of each tube at different intervals by using the photo-colorimeter at
610 nm.
• Plot a graph between incubation time and pH. If the nutrient broth is prepared in buffer,
the pH may not change during the course of growth .
Observation
Observe each tube at different time interval and take O.D. for growth determination.
Result
The pH optima can be seen by observing maximum optical density.
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Experiment No. 9
Aim: Sub-cellular fractionation of animal tissues by homogenization method
Cell Fractionation
Cell Structure
Basic Cellular Architecture
All living material is composed of cells and their products. The size and shape as well as function
of these cells vary widely so that, in one sense there is no such thing as a typical cell although a
great many cells have a number of features in common.
Under the light microscope, two distinct regions of cell are visible, namely the nucleus and
the cytoplasm which appears empty apart from a number of small particles. However the electron
microscope shows that the cytoplasm contains a number of quite distinct structures. The diagram
shows certain basic features of cellular architecture common to a great many cells.
Plasma Membrane: Under the light microscope, the plasma membrane appears as a skin
stretched over the cell with surface folds directed towards the interior (vesicles) and the exterior
(microvilli). The overall thickness of the membrane is about 8nm.
Endoplasmic Reticulum: Many cells have a three-dimensional network of membranes known as
the endoplasmic reticulum. Two types of membranes can be seen: the so called smooth
endoplasmic reticulum and rough endoplasmic reticulum named after their appearance
under the electron microscope. The rough endoplasmic reticulum has small electron-dense
particles called ribosomes attached to its surface, while the smooth form is free of ribosomes.
The endoplasmic reticulum appears to be connected to the external nuclear membrane and the
Golgi complex, the site of synthesis of a number of membranes.
Nucleus: Nearly all cells apart from bacteria contains a nucleus, a large structure of about 6µm
diameter which is clearly visible under the light microscope, Virtually all of the DNA of the cell is
present in the nucleus complexed with histones in the form of nucleohistones. When the cell is
not dividing, the DNA is distributed throughout the nucleus as chromatin, but during cell division,
chromatin becomes organized into distinct linear structures.
Nucleolus: Within the nucleus there maybe one or more distinct bodies of about 1µm diameter
which contains the bulk of nuclear RNA.
Mitochondria: Mitochondria which are the particles responsible for most of the oxidative
metabolism in the cell, are just visible by light microscopy. The organelles from animal cells,
plant cells and algae have a similar type of structure and under electron microscope appear as
cylindrical or spherical particles with a double membrane with inner membrane invaginating into
cell to form cristae.
The size and number of mitochondria in a cell vary, but in rat liver there are about 800 of
them occupying about 20 percent of the cell volume. The size of mitochondria from different
sources is very similar and those found in rat liver are from 1 to 2 µm long and have a diameter
of 0.5 µm.
Lysosomes: Lysosomes are spherical particles about 0.5 – 1.0 µm diameter bound by a single
membrane. There are no obvious internal structures visible within the particles but they contain a
whole range of hydrolytic enzymes which are involved in the digestion of exogenous and
endogenous material.
Peroxisomes: Another group of membrane bound particles about the same size of lysosomes
contain the oxidative enzymes, catalase, urate oxidase and D- amino acid oxidase. These
particles are known as microbodies or peroxisomes. The interior of these particles contains
enzyme crystals which can occupy almost the whole of the internal volume.
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Ribosomes: The ribosomes which are the site of protein synthesis in the cell, occur free in the
cytoplasm or attached to the endoplasmic reticulum. They are much smaller than the other
organelles with a diameter of only 0.01-0.02 µm. Ribosomes from bacteria are 70S particles
while those from higher organisms are a slightly larger with a sedimentation constant of 80S.
Ribosomes are made up of protein and RNA in about equal quantities and contain about 60% of
total RNA in the cell. Each ribosome is about two oblate spheroids of unequal size which can be
separated under low Mg2+ concentration.
Practical Cell Fractionation
The metabolic function of cell can be investigated to some extent by using histochemical means
for detecting regions that contain a high activity of a particular enzyme. This approach has its
limitations and it is more convenient to separate organelles and examine the properties of the
isolated particles.
When cells are subjected to high shear, the cell membrane ruptures and the contents are
released into the medium. By carefully controlling the conditions of homogenization, it is possible
to avoid damaging the cell organelles, which can then be separated from each other by
centrifugation. All steps must be carried out at 0ºC to avoid damaging the particles.
Homogenization: A coaxial homogenizer consisting of a glass mortar and a hard Teflon pestle is
very convenient.
The pestle is attached to an electric motor and small pieces of tissue suspended in the
medium are placed in the glass mortar. The two parts of the homogenizer are brought together
and the mortar is slowly moved up and down for about six to eight complete strokes while the
pestle rotates at a controlled speed of about 2000 rev/min. As the homogenate is forced between
the stationary wall of the mortar and the rotating pestle, the tissue is subjected to a shearing
force which is sufficient to rupture the cells but not the organelles. These conditions are quite
effective for the liver but tougher tissues such as kidney and skeletal muscle may need first to be
freed of connective tissue by forcing the tissue through a steel plate with holes under pressure
before homogenization.
The clearance between the pestle and mortar, the speed of rotation of the pestle and
number of strokes all affect the preparation and must therefore be carefully defined, and thus
one set of conditions which are suitable for a particular tissue cannot automatically be used for
another tissue.
Suspending Medium: The homogenizing medium should be cheap, uncharged and metabolically
inert and for these reasons sucrose is the compound most frequently employed. For rat liver, a
slightly hypotonic solution of sucrose (0.25 mol/litre) buffered with 20 mmol/litre tris to pH 7 has
been found to be quite suitable. Ethylene diamine tetra acetic acid (EDTA) adjusted to pH 7 is
sometimes incorporated into the medium at a concentration of 0.1 mmol/litre. This chelates
calcium and other divalent ions which if present in even trace amounts can cause extensive
swelling of mitochondria. On the other hand, EDTA renders the mitochondrial membrane more
permeable to monovalent ions so that some workers prefer to use sucrose alone.
Differential Centrifugation: After homogenization, the suspension is separated into a number
of fractions by centrifugation at various g values. The intracellular particles then sediment at
different rates according to their mass.
The actual conditions of the fractionation depend on the tissue studied and those for the
separation of rat liver mitochondria are not necessarily the same as those for the isolation of
mitochondria from other rat tissue. Also, some fractions which are more or less homogenous for
one tissue may be very heterogeneous in others.
Density Gradient Centrifugation: Subcellular particles can also be separated by using
differences in their density rather than mass. To do this, the homogenate is placed on top of a
discontinuous gradient formed by layering a series of different sucrose concentrations on top of
each other. The tubes are then centrifuged and, at equilibrium, particles will be found as a band
in that concentration of sucrose whose density is close to that of organelles. This technique has
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been particularly useful in fractionating brain tissue when nerve endings and myelin can be
isolated in a more or less homogeneous condition.
The alternative to a discontinuous density gradient is a continuous one, and relatively
large quantities of material can be fractionated on such a gradient set up in a hollow centrifuge
rotor, a technique known as zonal centrifuge.
Experiment: The Fractionation of Rat Liver.
Principle
Rat liver has probably been subjected to fractionation more times than any other material, and a
more or less standard scheme for separating subcellular particles is now available.
Centrifugation Conditions
g value
Time (mins)
500
5
8000
10
15000
10
100000
60
Final supernatant
Major components in
fraction
Nuclei,
whole
cells,
debris
Mitochondria,
some
lysosomes
Lysosomes,
some
mitochondria
Microsomes (membrane
fragments,
largely
endoplasmic reticulum)
and ribosomes.
Soluble components of
the cell.
Materials
Isolation Medium (0.25 mol/litre sucrose; 5mmol/litre tris-HCl buffer, pH 7.4;
0.1mmol/litre EDTA)
Rats
Coaxial homogenizers
Ice baths
Ultracentrifuges
Method
Kill a rat, exsanguinate it and rapidly remove the liver. Wash the tissue free of blood in ice-cold
sucrose, lightly blot and place in a tared beaker to weigh. Cut the liver into small fragments and
homogenize in sucrose (20g/100ml) at 2000 rev/min by moving the mortar relative to the pestle
for 8-10 complete strokes. Centrifuge the suspension in a refrigerated centrifuge according to the
schemes shown in the below figure
Liver homogenate (20% in ice cold 0.25 mole/liter sucrose)
10 min at 600g
Supernatant
Pellet (resuspended in sucrose)
10 min at 600g
Supernatant
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Pellet
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Nuclei & Cell debris
10 min at 8000g
Supernatant
Pellet
Mitochondria
10 min at 15000g
Supernatant
Pellet
Lysosomes
60 min at 100000g
Supernatant
Pellet
Microsomes
Fig: The Fractionation of Rat Liver
Ideally each fraction should be resuspended in sucrose and the washings combined with the
supernatants. This has the advantage of producing purer fractions, but the disadvantage of
introducing an increasing dilution of cellular components.
Carefully resuspend the pellet in about 10ml of sucrose and store on ice until
required.
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Experiment No 10
Aim: Preparation of temporary slides of mitosis from onion root tips.
Mitosis
Definition:
The process of cell division where by the chromosome are duplicated and distributed
equally to the daughter cells is called mitosis.
The mitotic cycle is divided into many phases Prophase, Metaphase, Anaphase and
Telophase. The period between the two mitotic cycles is called interphase.
Interphase: When the cell is not in division its nucleus is said to be in interphase. In this phase
the nucleus has a definite nuclear wall. The karyo-lymph (nuclear sap) is very dense with one or
more nucleoli and inconspicuous chromatin network in it.
Prophase: At the beginning of prophase chromosomes appears as thin, filamentous, uncoiled
structure. Soon it becomes coiled shortened and more distinct. During prophase there is
longitudinal splitting of each chromosomes into two sister chromatids attached only at
centromere (or kinetochore). Soon after the nuclear membrane disappears in late prophase.
Similarly nucleolus also disappears before the cell enters the meiotic metaphase.
Metaphase: At early metaphase spindle tubules start appearing. These tubules get attached to
chromosomes on the centre or at equatorial plate or metaphasic plate.
Anaphase: the chromosomes are arranged on the equatorial plate for a period only. The
centromere of the chromosome divide simultaneously as anaphase commences and the
chromatids of each pair is separated. They move towards opposite poles of spindle. This
movement is due to repulsion between centromeres and contraction of spindles fibres which help
the movement.
Telophase: Telophase begins when the two sets of daughter chromosome reach opposite poles
of the cells. The spindle disappears. A new nuclear membrane is formed around each set of
chromosome. Nucleolus reappears at constriction called the nucleolar organization in one pair of
chromosome. The chromosomes gradually uncoil and become less compact. They eventually lose
there ability to stain.
Cytokinesis: Division of one nucleus into two is often called karyokinesis and is followed by
cytokinesis, which divides cytoplasm into two cells.
In case of plant cell a more rigid cell wall is present. In such cases a cell
plate is usually initiated at centre and complete towards periphery. After the cell plate is laid
down primary walls are deposited on either side. The thick secondary cell wall by cellulose may
be laid down later on.
Requirement
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Fixative – methyl alcohol: acetic acid (3:1)
1N HCl
Stain –Acetocarmin or Aceto orcin
Onion root tips
Various phases of mitosis in plant cells
Procedure
Cut the fresh growing root tips of onion and keep it in the fixative for about 24 hrs (can be
used freshly without fixing). Wash the root tips in distilled water. Hydrolyse the tissue in 1N HCl
at 60ºC for 10 mins.
Stain the root tips in any of the two stains acetocarmin or aceto orcin for 10 mins. Transfer
the stained material on the clean slide .Put drop of stain on it to keep the material wet. Then put
the coverslip and gently squash the material by pressing the coverslip. Observe under the
microscope.
For preparation of the slide process take dried material on slide with:
1. n-butanol I
2. n-butanol II
3. absolute alcohol
4. Xylol I
5. Xylol II
6. Mount the material in DPX
Observation
Observe the slide under microscope and note down various phases of mitosis in plant cells.
Result
After observing the slide under microscope following phases of mitosis have been observed in the
plant cell
1) -------------------------
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2) -----------------------3) -----------------------4) ------------------------5) -------------------------
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Experiment No 11
Aim: Preparation of temporary slides of Meiosis.
Principle
The process, during which the germ cells are generated, is called meiosis. It represents nature's
solution of the problem of chromosome doubling that would occur, if two diploid cells, i.e. two
cells with a double set of chromosomes would fuse. Accordingly meiosis produces haploid germ
cells, with maternal and paternal germ cell fusing at fertilization and thus generating a diploid
fusion product, the zygote. Meiosis is made up by two subsequent processes, both of which
resemble mitosis. In the first process the homologous chromosomes are separated. It has an
unusually long prophase that is subdivided into different stages: leptotene, zygotene,
pachytene, diplotene and diakinesis. They are followed by metaphase, anaphase and
telophase.
Mitosis is a process by which a cell can reproduce itself and the number of chromosomes and the
nature of the DNA will be identical to the original parent cell. Very few species will grow or live
indefinitely, so there must be some way to ensure the continuity of the species. Reproduction is
the only way a species can be perpetuated, without perpetuation the species will become extinct.
Reproduction can occur in several ways as vegetative propagation, such as in the development of
runners in strawberry plants, or by special cells called vegetative spores which are products of
mitosis. In these processes, the ‘offspring’ have identical cells and identical chromosomes to the
parent cells and thus the processes are called asexual reproduction—a means without, so without
sex reproduction.
Most plants, however, will undergo sexual reproduction which involves the production and
recombining of sex cells called gametes. In flowering and cone-bearing plants this involves the
production of seeds. The gametes produced are male and female, and are called sperm cells and
egg cells, correspondingly. When the gametes combine together, the cells fuse and form a single
cell called a zygote. It is the zygote that will go on to become the plant embryo and eventually a
mature, adult plant.
However, in thinking about this process, what would happen if both gametes had the same
number of chromosomes as the rest of the cells in the organism? When they fused to become a
zygote, they would have two times the number of chromosomes as the rest of the cells in the
organism. The number of chromosomes would increase exponentially through the generations if
this occurred. This is where meiosis comes in to play. Meiosis is the process by which gametes,
sex cells, are formed. It is unique because gametes have exactly half of the total number of
chromosomes as the rest of the cells in the parent organism. When two gametes, each with half
the number of chromosomes, get together they are able to restore the chromosome number to
the same as the rest of the cells in the parent organism. When the zygote develops into a plant
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embryo and eventually a mature plant, it will have the exact number of chromosome specific to
the species. Note that the processes and steps in meiosis are very similar to mitosis, so make
certain you have a good understanding of mitosis so that you will be able to compare the two
processes.
Before we get into the nitty-gritty of meiosis, keep in mind that all living cells have two sets of
chromosomes—one from a male and one set from a female parent. The genes in the
chromosomes may control the same characteristics but in contrasting ways—for example: genes
for plant height, genes for plant color, genes for fruit color, etc—the female gamete might code
for short plants, while the male gamete might code for tall plants. That is more of a genetics
topic though. But the chromosomes that code for the same characteristics are called homologous
chromosomes.
Phases of Meiosis:
The end result of one round of meiosis will be four cells with half the number of chromosomes as
the parent cell. The daughter cells are rarely, if ever, identical to each other or the parent cell
depending on the organism involved. There are two successive divisions in meiosis, which in
plants occur without a pause. Mitosis takes roughly 24 hours, while meiosis takes up to two
weeks. In some organisms, meiosis takes weeks or years depending on the organism.
Meiosis is a process which consists of two cell divisions, in which four cells are formed; possess
half the chromosome number (haploid) of the parent cell (diploid number).
During the two cell division of meiosis, the chromosome and centromere divide
only once. The two cell divisions are called the first and the second meiotic division and between
them is a period called as interkinesis. The various stages of meiosis are as follows:
First Meiotic Division:
Division I –Reduction division—the chromosome number is reduced to half the parent cell
chromosome number. End result of division one is two cells.
Prophase I—Main features:
1. Chromosomes coil, becoming shorter and thicker, the two-stranded nature becomes
apparent, two strands are called a chromatid and chromosomes are aligned in pairs. Each
pair of chromosomes has four chromatids and they have a centromere attached in the
center holding the four strands together.
2. Nucleolus disassociates and nuclear envelope dissolves.
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3. Segments of the closely associated pairs of chromatids may be exchanged with each other
(between the pair members) this is called crossing-over. Each chromatid contains the
original amount of DNA but now may have “traded” genetic material.
4. The chromosomes separate. Some spindle fibers are forming and some are attaching to
the centromeres of the chromosomes. The fibers extend from each pole of the cell.
Prophase of meiosis is prolonged and differs from that of mitosis.
I) Proleptotene Stage:
The nucleus increases in volume, chromosome are thin and indistinct but are actually double
because of DNA replication in interphase.
II) Leptotene:
Chromosomes become distinct. They appear as long, thin, optically single (but double) threads.
III) Zygotene:
Chromosomes contract further and become shorter. Homologous maternal and paternal
chromosomes, attract each other and undergo lengthwise pairing, zero synapsis through
proteinaceous synaptonemal complex to form bivalence.
IV) Pachytene:
The bivalent shorter and thicker, each chromosome now appears optically double and consists of
two chromatids. Each pair of chromatid is united by a centromere. A transverse break occurs in
two chromatids belonging to two different homologous chromosomes. Interchange or fusion of
broken ends (crossing over)now takes place. These points of fusion are called as chaismata.
V) Diplotene:
The homologous chromosomes separate. Each homologous chromosome now contains same part
of other.
VI) Diakinesis:
Chromosomes contract further.
Metaphase I—Main features:
1. In pairs, the chromosomes align at the equator of the cell, with the centromeres and
spindle fibers apparent.
2. The two chromatids, from each chromosome, function as a single unit.
Anaphase I—Main features:
1. One entire chromosome, consisting of two chromatids, migrates from the equator to a
pole. The chromosomes do not separate from each other and retain both chromatids when
the reach their pole. At each pole, there will be half the chromosome number. If crossing
over occurred in prophase then the chromosomes will consist of original DNA and DNA
from a homologous chromosome—now at the opposite pole.
2. The centromere remains intact in each pair of chromatids.
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Telophase I—Main features:
1. What occurs in this step, depends on the species involved, as they may revert to
interphase or proceed directly to division II.
2. If they revert to interphase, they will only do so partially and the chromosomes will
become longer and thinner.
3. nuclear envelopes will not form, but the nucleoli will generally recluster.
4. Telophase is over when the original cell becomes two cells or two nuclei.
Interkinesis I
The period between 1st meiotic and 2nd meiotic division is called as interkinesis. In interkinesis no
replication takes place.
Second Meiotic Division:
Division II—Equational division—the chromosome number stays the same, the cells replicate
and result in four cells. The events closely resemble the events in mitosis, except that there is no
duplication of DNA during the interphase that may or may not occur between the two divisions.
Prophase II—Main features:
Chromosomes of both nuclei become shorter and thicker. The two-stranded nature becomes
apparent once again.
Metaphase II—Main features:
1. Chromosomes align their centromeres along the equator.
2. Spindle fibers form and attach to each centromere, extending from one pole to the other.
Anaphase II—Main features:
The centromeres and chromatids of each chromosome separate and begin their migration to the
opposite poles.
Telophase II—Main features:
1. The coils of chromatids—now called chromosomes again—relax and the chromosomes
become longer and thinner.
2. Nuclear envelopes and nucleoli reform for each group of chromosomes.
3. New cell walls form between the four groups of chromosomes.
4. Each set of chromosomes in the four new cells, has exactly half of the chromosome
number of the original number.
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Procedure:
1. Take a slide.
2. Arrange bud to there size in ascending order
3. Measure the length of all stamens.
4. Then take one anther of stamen.
5. Open the anther and take out the spores on the slide.
6. Then stain the anthers with acetocarmine and heat for few minutes.
7. Then observe under the microscope.
Observation
Observe the slide prepared as above for the presence of various meiotic stages
Result
The meiosis stages of ------------ plants have been successfully observed.
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Experiment No. 12
Aim: Histology of animal tissues
I) Epidermal cells (skin)
II) Nerve cells
III) Connective tissue-blood, cartilage muscular
tissue striated, non-striated cardiac muscles
IV) Epithelilal Tissue
To study the microscopic structure of human tissues
Prior Concepts
Tissue: It is the group of cells having similar structure and functions.
Histology: It is the study of tissues which deals with the study of tissues, its structure, location
and functions.
NEW CONCEPTS:
Proposition 1:
Microscopic study of tissues.
Study of structure, location and functions.
Proposition 2:
To understand the function of the tissues.
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General Concept Structure
Types of Tissues
Muscular Tissue
Connective Tissue
Nervous Tissue
All over the
body.
Binding structure
between two
tissues.
Brain
Locomotion and
movement of
body binding
support.
Binding Support
Co-ordination and
Control
Epithelial Tissue
Lining the
membrane and
covers the free
surface.
Protection,
Absorption,
Secretion &
Excretion
Learning Objectives:
1. Intellectual skills:
Identify the tissue by observing microscopic structure.
2. Motor skills:
Ability to adjust the microscope to see the clear structure of tissues.
Apparatus and Materials:
•
Apparatus:
Microscope with mechanical stage.
•
Material:
1. Tissue slides.
2. Xylol
3. Muslin cloth
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Diagrams
Fig:
Columnar
Fig: Stratified squamous epithelium
Fig: Cuboidal epithelium
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epithelium
Cilia
Fig: Ciliated columnar epithelium
Fig: Stratified columnar epithelium
Cardiac muscle
Intercalated disc
Nucleus of cardiac muscle
Striation
Fig: Cardiac muscle Fibres
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Nucleus
Skeletal Muscle Fibres
Fig: Skeletal Muscle Fibres
Nucleus
Fig: Smooth Muscle Fibres
White Blood Cell
(leucocyte)
Blood Plasma
Red Blood Cell
Platelet
Fig: Blood Smear
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Nucleus in cell body
Dendrite
Axon
Nucleus in cell body
Fig: Neuron
White Blood cells
Reticular Fibres
Reticular Cell
Lymph Spaces
Fig: Lymphoid Tissue
Fig: Adipose Tissue
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Adipocytes
Collagen Fibres
Fibroblast
Elastic Fibres
Fig: Loose (Areolar) Connective Tissue
Stepwise Procedure:
1.
2.
3.
4.
5.
Clean microscope with xylol.
Clean and dry with muslin cloth.
Adjust microscope on 10X.
Light source was adjusted to get clear structure of the tissue.
Keep Tissue slide on stage of microscope and adjust to get a clear
microscopic image of the culture.
6.
Observe carefully. Identify the following tissues. Note its
characteristics, staining etc.
A. Epithelilal Tissue:
1. Simple Epithelium
a. Squamous epithelium
b. Cuboidal epithelium
c. Columnar Epithelium
d. Cilliated columnar epithelium
e. Compound Epithelium
f. Stratified Epithelium
g. Transitional Epithelium.
B. Muscular Tissue:
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a. Striated, Voluntary, Skeletal muscle tissue.
b. Smooth, non-striated, involuntary muscle tissue.
c. Cardiac muscle.
C. Connective Tissue:
A.
B.
C.
D.
E.
Blood
Areolar Tissue
Adipose Tissue
Bone
Cartilage
I.
II.
III.
Hyaline cartilage
White fibro cartilage
Yellow elastic fibro cartilage
D. Nervous Tissue:
It consists of cyton and nerve fibre neuron is the structural and functional unit of nervous
system.
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Observations:
SR NO.
TYPE OF TISSUE
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LOCATIONS
STRUCTURAL
FEATURES
FUNCTIONS
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Experiment No. 13
Aim: Histology of plant tissue
Preparation of Microscope Slides of
a) Monocot/dicot -stem/ root
b) Leaf isobilateral, dorsiventral
Section Cutting
In order to reveal the cellular structure of plant material sections are being cut in various planes.
1. Cross section:
Here the section passes at right angle to the material. It is of two types.
a) Transverse section: Here the section is cut at right angle to vertical axis of
the plant such as stem and roots.
b) Vertical section: In case of thallus, leaf, etc., the section is cut in transverse
plane and is known as vertical section. It is generally applied to the dorsiventral
leaf and thallus growing prostrate.
2. Longitudinal Section:
The section is cut at right angles to the transverse axis. It is of two types:
a)
Radial longitudinal section (R.L.S): It is the section that
passes through the radius.
b)
Tangential longitudinal section (T.L.S): It is the section
that passes through the tangent, this does not pass through the central region,
it is transverse through the medullary ray.
Method
To cut this section suitable pith material should be used. In case of thalli and leaves, etc., the
pith is first divided longitudinally into two equal halves with the help of a scalpel and the material
is placed inbetween the two halves of the pith. In case of stem, roots, etc., a hole is made in the
pith and the material is being fitted in the hole.
During the process of section cutting the students should be cautious so that the material does
not dry up in the whole operation of section cutting. To avoid this a few drops of water may be
put now and then on the material. For cutting the sections, the pith is being held in between the
fingers and thumb of the left hand and the razor in the right hand. The first two fingers remain at
the back of the razor and the thumb remains pressed against the milled surface of thick shank of
the blade. The cutting edge of the razor blade should be kept always in the horizontal position so
that the uniform and the thin sections are cut. The sections should be cut by sliding the razor
over the material repeatedly. This process should be repeated several times and then the
sections thus cut are transferred with the help of camel’s hair brush to a watch glass containing
water. With the help of camel’s hair brush, the thinnest and perfect sections are being selected
for staining.
Microscope Slide Preparation
Whole Mounts:
As the name indicates, whole mounts are preparations of the object entire. Many interesting
mounts may be made without cutting sections, and without staining. Fibers of cotton, fern
sporangia, the fruiting heads of mosses (if dried first) and pine pollen, may be dropped directly
into xylol, placed in a drop of balsam on a slide and covered.
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Free-hand Sections:
Free-hand sections of cork, pith and the stems and roots of many of the common plants may be
cut. For this purpose the razor must be sharp and free from nicks. Only thinnest sections should
be used and they maybe placed into 95% alcohol as cut. Subsequent treatment of sections is as
follows. Handling should be as gentle as possible, using section lifter and camel’s hair brush.
1. 95% alcohol
- ½ hour
2. 50% alcohol
– 5 minutes
3. Water
– 5 minutes
4. Safranin
– 4 hours (1% solution in water)
5. Water to rinse.
6. 50% alcohol
– 5 minutes
7. 95% alcohol
– 5minutes
8. Absolute alcohol
– 5 minutes
9. 95% alcohol containing 1% light green 1-3 minutes
10. Absolute alcohol
– 3-5 minutes
11. Clove oil
– 5 minutes
12. Xylol
– indefinite
13. Mount in balsam: cover with cover glass.
The time values given are approximate and will vary somewhat with the kind of section being
studied. Practice on a few sections will teach whether to hasten or retard the process.
Maceration Technique
The various parts of the plants consist of various types of tissues. Such tissues can be studied
well by a special technique known as maceration. This process involves the separation of a
particular cell from a mass of cells. This type of dissociation is brought about by chemical
treatment of the plant organ that dissolves the middle lamella. And the cells are separated
from each other.
Usually three under mentioned techniques are used for the purpose:
1. Jeffrey’s Method:
Take the dried or fresh plant material and make very thin slices of it. Boil the material in
water by keeping it in a test-tube. After sometime when the material becomes air free and
settles down in the bottom of the test tube, it is macerated in a solution. The maceration
solution may be prepared as follows:
i. 10% Nitric acid (i.e., 90ml water + 10ml nitric acid)
ii. 10% Chromic acid (i.e., 90ml water + 10ml chromic acid)
Mix (i) and (ii) acids.
Now take the material in this maceration solution in a test tube. Heat this, solution and
separate the material in small pieces by piercing a needle to the material. Stop heating as
soon as the material becomes soft and pulp like. Now transfer this pulpy material to a watch
glass. Drain out all the maceration fluid. Wash the material several times with water so that
the acid traces are removed completely. Now strain the material with aqueous safranin and
mount it in glycerine or glycerine jelly. The material may also be passed through alcohol
series for making permanent slides.
2. Harlow’s Method:
Treat the slice and boiled material with chlorine water for two hours. Now wash the
material with water. After proper washing boil the water in sodium sulphite for fifteen
minutes. Now transfer this fluid to a watch glass. Drain out the water. Tease the material
with the help of a needle for the separation of tissues. Prepare temporary or permanent
mounts.
3. Schultze’s Method:
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Make thin slices of the material and boil it in water in a test tube. Now fill the test tube
with concentrated nitric acid, and add a few crystals of potassium chlorate to it. Heat it
gently till material becomes white. Now transfer the fluid through the watch glass and
drain out the liquid leaving material alone. Wash the material with water. Tease the
material with the help of a needle, so that the cells maybe separated from each other.
Stain the material and make temporary or permanent mounts.
Staining
The tissue differentiation is possible only by staining the different tissues with different stains.
The stains are the chemical dyes having different reactions with the cell wall of the tissue and
thus giving a particular stain to a particular tissue. For example, the acid dye stains unlignified
tissue while the basic ones stain lignified tissue.
Single Staining:
The plant materials having no differentiation of tissues (e.g. Algae, fungi, bryophytes) are stained
by this process.
Double Staining:
The plant material having highly differentiated tissues (e.g. Pteridophyta, gymnosperms,
angiosperms, etc.) are stained by this process. Double staining involves the use of two dyes, one
acidic and other basic. The acidic dye stains unlignified tissues while the basic one stains lignified
tissues. Some important dyes are as follows:
1. Safranin:
Alcoholic:
Safranin
- 1gm
Alcohol 95%
- 50 c.c.
Distilled water
- 50 c.c.
Aqueous:
Safranin
- 1gm
Distilled water
- 10 c.c.
This is mainly used to stain lignified tissues.
2. Crystal violet or Gentian violet:
Crystal violet
- 1gm
Distilled water
- 100 c.c.
It is basic violet dye and stains lignified tissues.
3. Aniline Blue:
Aniline Blue
Alcohol (95%) of
Distilled water
It stains cellulose walls.
- 1gm
-100 c.c.
4. Erythrosine:
Erythrosine
- 1gm
Absolute Alcohol
- 5 c.c.
Clove oil
- 95 c.c.
It is used to stain gelatinous sheath, eg. Algae.
5. Eosin:
Eosin
Water
It stains cytoplasm.
6. Light Green:
Light Green
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- 1gm
- 100 c.c.
- 0.5 gm
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Alcohol 95%
- 100 c.c.
OR
Light Green
Alcohol 95%
Clove oil
It is used to stain cellulose and
7. Fast Green:
Fast Green
Alcohol 95%
- 1 gm
- 2.5 c.c.
- 75 c.c.
lignified cell walls.
- 0.5 gm
- 100 c.c.
OR
Fast Green
Alcohol 95%
Clove oil
- 0.5gm
- 2.5 c.c.
- 75 c.c.
8. Cotton Blue:
Aniline Blue
- 0.1gm
Phenol
- 25gm
Glycerine
- 25 c.c.
Lactic acid
- 25 c.c.
Distilled water
- 25 c.c.
This is used for staining various fungi.
9. Basic Fuchsin:
Solution A
Basic Fuchsin
- 0.3 gm
Alcohol 95%
- 10 c.c.
Solution B
Phenol (melted)
- 5gms
Distilled water
- 95 c.c.
Mix solution A and B
This is a nuclear stain and also used to stain bacteria.
10.Hematoxylins:
Heidenhain’s hematoxylin
Hematoxylin
- 0.5 gm
Warm distilled water
- 100 c.c.
Store in a dark place to ripen atleast for four days before use.
Delafield’s hematoxylin
i. Saturated aqueous solution (100 c.c.) of ferric ammonium sulphate
ii. 1 gm hematoxylin + 6 c.c. absolute alcohol.
iii. Mix i and ii solutions.
The prepared solution is kept for sufficient time and then used. The solution becomes
dark red in color. It is good nuclear stain.
11. Acetocarmine:
Carmine
- 1gm
Glacial acetic acid
- 45 c.c.
Dissolve 1gm of carmine in 100 c.c. of boiling 45% acetic acid. Cool this mixture and
decant. Add few drops of ferric acetate aqueous solution in the mixture. Cool the mixture
abruptly by keeping it in ice. Filter the mixture and stock it in a cool place.
Mounting Media and Mounting
Mounting Media for Temporary Preparations:
1. Glycerine:
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Prepare the mounting medium as follows- 30 ml pure glycerine + 70 ml water. This medium
is quite good for the mounting purpose of algae and stained sections.
2. Glycerine Jelly:
It is prepared by dissolving one part of gelatin with six parts of water by boiling. Add to it
seven parts of glycerine. Add also 10% phenol which acts as preservative.
This is a good mounting medium for algae. The slide becomes semipermanent and can be
kept for long.
3. Lacto Phenol:
This mounting medium can be prepared by adding equal amounts of phenol, lactic acid,
glycerine and distilled water. This makes a good mounting medium to mount various fungi.
Mounting Media for Permanent Preparations:
1. Canada balsam:
This medium is used for permanent slide preparations. According to need this may be
diluted by adding xylene to it. This medium is somewhat yellowish in color.
2. D.P.X. Mountant:
This is a good mounting medium for permanent slide preparations. This is perfectly
transparent and gives good results.
Method of Mounting:
The stained sections or materials are generally mounted in any one of the media mentioned
above. While mounting one should be watchful that the object is being mounted on the centre of
the slide. Put a drop of the mounting medium in the centre of the slide with the help of a dropper
and the material is being transferred in this drop of medium with the help of a fine brush. With
the help of forceps or needle the cover glass is kept on the material in such a way that air
bubbles are avoided. The mounting fluid should not flow outside the coverslip. The extra amount
of fluid can be removed with the help of a blotting paper.
Labeling:
After making the preparation neat and clean, it should be properly labeled. The labels should be
uniformly pasted on the left side of the slide. Usually the generic and specific names, name of the
part and plane of section are being written on the label.
Ringing:
Usually the temporary and semi-permanent slides are sealed by cementing material with the help
of a ringing machine. By this method only those slides can be sealed which possess round cover
glasses.
Anatomy of a Typical Monocot Stem
Example: Zea mays (Maize)
A transverse section passing through the stem of Maize reveals the following details.
Epidermis
Epidermis is the outermost covering of the stem represented by a single layer of compactly
arranged, barrel-shaped parenchyma cells. Intercellular spaces are absent. Trichomes are
absent. A cuticle is present. The epidermis contains numerous minute openings called stomata.
Hypodermis
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Hypodermis is a region that lies immediately below the epidermis. It is represented by a few
layers of compactly arranged sclerenchyma cells.
Ground Tissue
Ground tissue is a major component of the stem. It is undifferentiated. The ground tissue is
represented by several layers of loosely arranged parenchyma cells enclosing prominent
intercellular spaces. The ground tissue is meant for storage of food.
Vascular Bundles
They are found irregularly scattered in the ground tissue. Towards the periphery, the bundles are
smaller in size while towards the centre, they are larger in size. The smaller bundles are younger,
while the larger ones are older. Hence, the arrangement is described as centrifugal. Each
vascular bundle has a covering called bundle sheath formed by a single layer of sclerenchyma
cells. The vascular bundle encloses both xylem and phloem. Xylem is found towards the inner
surface and phloem towards the outer surface. Cambium is absent. Hence the vascular bundles
are described as conjoint, collateral and closed. In the xylem, there are two metaxylem and two
protoxylem vessels arranged in 'the shape of 'Y'. The lower protoxylem vessel is non functional
and remains as a water filled cavity called lisigenous cavity or protoxylem cavity. Xylem is
described as endarch. In the phloem, only sieve tubes, companion cells and phloem fibres are
present. Phloem parenchyma is absent.
TS section of monocot stem
Cross section of monocot stem
Diagnostic Features of Monocot Stem
Absence of tricomes
Presence of stomata
Presence of a hypodermis made up of sclerenchyma
Presence of undifferentiated ground tissue
Presence of numerous vascular bundles irregularly scattered with
centrifugal arrangement
Vascular bundles are conjoint and closed with endarched xylem.
Presence of only two protoxylem and metaxylem in each bundle
Presence of bundle sheath made up of sclerenchyma
Anatomy of monocot root
Example: Maize
A transverse section passing through the Maize root reveals the following details
Epiblema
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Epiblema is the outermost covering of the root formed by a single layer of compactly arranged,
barrel-shaped parenchyma cells. The cells are characteristically thin-walled since they are
involved in absorption of water. A cuticle and stomata are absent. Some of the epiblema cells are
produced into long unicellular projections called root hairs. Hence, epiblema is also known as
piliferous layer.
T.S. of monocot root
Cross-Section of a Monocot Root
Cortex
Cortex is a major component of the ground tissue of root. It is represented by several layers of
loosely arranged parenchyma cells. Intercellular spaces are prominent. The cortex is mainly
meant for storage of water. The cells also allow a free movement of water into the xylem vessels
Endodermis
It is the innermost layer of cortex formed by compactly arranged barrel-shaped cells. Some of
the cells in the endodermis are thin-walled and are known as passage cells. The passage cells
allow water to pass into the xylem vessels. The remaining cells in the endodermis are
characterised by the presence of thickening on their radial walls. These thickenings are known as
casparian thickenings. They are formed by the deposition of a waxy substance called suberin. The
casparian thickenings play an important role in creating and maintaining a physical force called
root pressure.
Stele
Stele is the central cylinder of the root consisting of pericycle, conjunctive tissue, pith and
vascular bundles
Pericycle
Pericycle is the outermost covering of the stele represented by a single layer of parenchyma cells.
Conjunctive tissue
It is represented by loosely arranged parenchyma cells found in between the vascular bundles.
The cells are specialized for storage of water.
Pith
Pith is the innermost region of the root representing the central axis. It is composed of few
loosely arranged parenchyma cells.
Vascular bundles
Vascular bundles are radial in arrangement. There are eight bundles each of xylem and phloem.
Hence, the condition is described as polyarch. Xylem is described as exarch.
Diagnostic Features of a Monocot Root
Presence of thin walled cells in the epiblema
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Absence of cuticle and stomata
Presence of unicellular root hairs
Presence of passage cells and casparian thickening in the
endodermis
Presence of parenchyma cells in the pericycle.
Presence of conjuctive tissue
Presence of distinct pith
Presence of radial vascular bundles with polyarch condition and an
exarch xylem
Plant Anatomy - Anatomy of a Typical Young Dicot Stem
Example: Helianthus annus (Sunflower)
A transverse section taken through the young stem of Sun-flower reveals the following details.
Epidermis
Epidermis is the outermost covering of the stem. It is represented by a single layer of compactly
arranged, barrel-shaped parenchyma cells. Intercellular spaces are absent. The cells are slightly
thick walled. Epidermis shows the presence of numerous multicellular projections called
trichomes. Externally, a thin transparent waxy covering called cuticle, which prevents excessive
evaporation of water, surrounds the epidermis. The epidermis also contains numerous minute
opening called stomata, which are mainly involved in transpiration.
T.S. of a Dicot Stem (Sunflower)
Cross-Section of a Dicot Stem
Hypodermis
Hypodermis is a region lying immediately below the epidermis. It is represented by a few layers
of collenchyma cells with angular thickenings. The cells are compactly arranged without any
intercellular spaces. Hypodermis provides mechanical support and additional protection.
Cortex
Cortex is the major part of the stem represented by several layers of loosely arranged
parenchyma cells. Intercellular spaces are prominent. Cortex is the major storage organ in the
stem.
Endodermis
Endodermis is the innermost layer of cortex represented by compactly arranged barrel shaped
cells, without any intercellular spaces. The endodermis is wavy in appearance. The cells are richly
deposited with starch grains and hence, endodermis is commonly described as starch sheath.
Stele
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Stele is the central cylinder of the stem, consisting of pericycle, medullary rays, pith and vascular
bundles.
Pericycle
Pericycle is the outermost covering of the stele, which lies immediately below the endodermis. It
is represented by a few layers of compactly arranged sclerenchyma cells. Above each vascular
bundle, the pericycle forms a distinct cap-like structure known as bundle cap.
Medullary Rays
found in between the vascular bundles. They are meant for the storage of food.
Pith
Pith is the innermost part of the stem formed by a group of loosely arranged parenchyma cells.
Intercellular spaces are prominent. The pith is also meant for storage of food.
Vascular bundles
They are eight in number, arranged in form of a broken ring. The vascular bundles are conjoint,
collateral and open. Xylem is on the inner surface and phloem on the outer surface. Xylem is
described as endarch.
Diagnostic Features of a Young Dicot Stem
Presence of cuticles and trichomes
Presence of stomata
Presence of a hypodermis made up of collenchyma
Presence of a wavy endodermis containing numerous starch grains.
Presence of a bundle cap above each vascular bundle, formed by
sclerenchyma
Presence of eight vascular bundles, arranged in the form of a broken ring.
Presence of conjoint collateral and open vascular bundles with an endarch
xylem.
Anatomy of a Typical Dicot Root
Example: Sunflower
A transverse section passing through the root of Sunflower reveals the following details.
Epiblema is the outermost covering of the root formed by single layer of compactly arranged,
barrel-shaped, parenchyma cells. The cells are characteristically thin-walled since they are
involved in absorption of water. A cuticle and stomata are absent. Some of the epiblema cells are
produced into long unicellular projections called root hairs. Hence, the epiblema is also known as
piliferous layer.
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T. S. section of Dicot Root
Cortex
Cross section of Diocot Root
Cortex is a major component of the ground tissue of root. It is represented by several layers of
loosely arranged parenchyma cells. Intercellular spaces are prominent. The cortex is mainly
meant for storage of water. The cells also allow a free movement of water into the xylem vessels.
Endodermis
It is the innermost layer of cortex formed by compactly arranged barrel-shaped cells. Some of
the cells in the endodermis are thin-walled and are known as passage cells. The passage cells
allow water to pass into the xylem vessels. The remaining cells in the endodermis are
characterised by the presence of thickening on their radial walls. These thickenings are known as
casparian thickenings. They are formed by the deposition of a waxy substance called suberin. The
casperian thickenings play an important role in creating and maintaining a physical force called
root pressure.
Stele
Stele consists of pericycle, conjunctive tissue and vascular bundles.
Pericycle
Pericycle is a region that lies immediately below the endodermis. It is represented by a single
layer of parenchyma cells.
Conjunctive Tissue
Conjunctive tissue is represented by a group of radially arranged parenchyma cells found in
between the vascular bundles. The cells are specialised for storage of water.
Vascular Bundles
Vascular bundles are described as radial and tetrarch. There are four bundles each of xylem and
phloem occurring alternately. Xylem is described as exarch.
Pith
Pith is absent in the older root.
Diagnostic Features of a Dicot Root
Presence of thin walled cells in epiblema
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Absence of cuticle and stomata
Presence of unicellular root hair
Absence of hypodermis
Presence of passage cells and casparian thickening in the endodermis.
Presence of uniseriate pericycle made up of parenchyma
Presence of conjuctive tissue
Absence of pith
Presence of redial vascular bundle exhibiting tetrach condition with exarch
xylem
Anatomy of the Leaf
Leaf represents an important part of the plant body. It is involved in vital physiological activities
such as transpiration, respiration and photosynthesis. Unlike the stem and the root, the leaf is
flat and hence, the anatomy of the leaf differs very much from that of stem or root.
There are two types of leaves in angiosperms based on the manner of orientation, they are:
•
•
Dorsi-ventral leaves which usually orient at an angle to the main axis and held
perpendicular (normal) to the direction of sunlight.
Example: Leaves of dicots
Iso-bilateral leaves which usually orient parallely to the main axis and held parallel to
the direction of the sunlight .
Example: Leaves of monocots.
A characateristic feature of leaves is the presence of two epidermal layers, one on each surface.
The ground tissue that occurs between the two epidermal layers, is known as mesophyll.
Embedded in the mesophyll are the vascular bundles, commonly known as veins.
Anatomy of a typical Dorsi-Ventral Leaf
Example: Sunflower
A transverse section through the midrib region of a typical dorsi-ventral leaf (Sunflower) reveals
the following structure.
• Epidermis is in two layers, one on each surface of the leaf. Both the layers are composed
of compactly arranged, barrel-shaped cells. Intercellular spaces are absent. A cuticle
surrounds both the layers. Multicellular hairs called trichomes are present on both the
layers. Stomata occur only in the lower epidermis. This condition is described as
hypostomatic.
• Mesophyll is the ground tissue that occurs between the two epidermal layers. It is
exclusively composed of chlorenchyma cells. The mesophyll is characteristically
differentiated into two regions namely, an upper palisade parenchyma and a lower spongy
parenchyma.
a) Palisade parenchyma is composed of two or three layers of elongated, compactly
arranged chlorenchyma cells. Intercellular spaces are absent. The cells contain a
very large number of chloroplasts.
b) b) Spongy parenchyma is composed of a few layers of loosely arranged spherical
or oval chlorenchyma cells with prominent intercellular spaces. These cells contain
very few chloroplasts.
Veins represent the vascular bundles. They are found irregularly scattered in the mesophyll due
to reticulate venation. The largest and the oldest vein is found in the centre. It is known as
midrib vein.
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T.S. through the midrib region of a dorsi-ventral leaf
Each vein has a bundle sheath composed of single layer of compactly arranged barrel shaped
parenchyma cells. The bundle sheath encloses both xylem and phloem. Xylem is found towards
upper epidermis and phloem towards lower epidermis. In the xylem many protoxylem and
metaxylem vessels are found. Protoxylem orients towards upper epidermis. Hence, the vascular
bundle are described as conjoint and collateral with endarch xylem. The bundle sheath of the
midrib vein is connected to the upper and the lower epidermal layers by many layers of
collenchyma cells, representing bundle sheath extensions or hypodermal collenchyma.
Diagnostic Features of a dorsiventral leaf
Presence of two epidermal layers
Presence of cuticle and trichomes in both the epidermal layers.
Hypostomatic condition.
Mesophyll differentiated into upper palisade parenchyma and lower spongy
parenchyma.
Veins irregularly scattered in the mesophyll.
Presence of a bundle sheath made up of parenchyma.
Vascular bundles are conjoint, collateral with endarch xylem.
Presence of bundle sheath extensions made up of collenchyma.
Anatomy of a typical iso-bilateral leaf
Example: Maize
A transverse section passing through the midrib region of an iso-bilateral leaf (Maize) reveals the
following structure.
Epidermis is in two layers, one on each surface of the leaf. Both the epidermal layers are
composed of compactly arranged, barrel shaped cells. Cuticle and trichomes are present in both
the layers. Stomata are found in both the epidermal layers. This condition is described as
amphistomatic. A few cells in the upper epidermis are enlarged to form motor cells called
bulliform cells.
Mesophyll is ground tissue that occurs between the two epidermal layers. It is composed of many
layers of loosely arranged, spherical or oval chlorenchyma cells. Intercellular spaces are
prominent.
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T.S. through an isobilateral leaf
Veins are found parallely arranged in the mesophyll (parallel venation). Each vascular bundle is
surrounded by a bundle sheath composed of a single layer of compactly arranged barrel-shaped
cells. The bundle sheath encloses both phloem and xylem. Phloem is found towards lower
epidermis and xylem towards upper epidermis. In the xylem, only two protoxylem and two
metaxylem vessels are present. The vascular bundle is described as conjoint and collateral with
endarch xylem.
The oldest and the largest vascular bundle is found in the centre. It is known as midrib vein. The
bundle sheath of the midrib vein is connected to the upper and lower epidermal layers by
sclerenchyma cells representing bundle sheath extensions or hypodermal sclerenchyma.
Diagnostic Feature of an isobilateral leaf
Presence of two epidermal layers.
Presence of cuticle and trichomes in both the layers.
Amphistomatic condition.
Presence of motor cells in the upper epidermis.
Presence of undifferentiated mesophyll.
Vascular bundles parallely arranged.
Presence of a bundle sheath around each bundle.
Vascular bundles conjoint, collateral with endarch xylem.
Presence of only two protoxylem and two metaxylem vessels in each bundle.
Presence of hypodermal sclerenchyma.
Observation
Observe the slides under microscope and note down the result on the basis of observation
Result
Microscopic examination of the slide shows the presence of
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Experiment No. 14
Aim: Study of permanent slide of Polytene chromosome .
Polytene chromosome
To increase cell volume, some specialized cells undergo repeated rounds of DNA replication
without cell division (endomitosis), forming a giant polytene chromosome. Polytene
chromosomes form when multiple rounds of replication produce many homologous
chromatids that remain synapsed together. In addition to increasing the volume of the cell's
nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as
multiple copies of genes permits a high level of gene expression. In Drosophila
melanogaster, for example, the chromosomes of the larval salivary glands undergo many
rounds of endoreplication, to produce large amounts of glue before pupation.
Polytene chromosomes have characteristic light and dark banding patterns which can be used to
identify chromosomal rearragements and deletions. Dark banding frequently corresponds to
inactive chromatin, while light banding is usually found at areas with higher transcriptional
activity. The banding patterns of the polytene chromosomes of Drosophila melanogaster were
sketched in 1935 by Calvin B. Bridges, in such detail that his maps are still widely used today.
The banding patterns of the chromosomes are especially helpful in research, as they provide an
excellent visualization of transcriptionally active chromatin and general chromatin structure.
Chromosome puffs are diffuse uncoiled regions of the polytene chromosome that are sites of
RNA transcription. A Balbiani ring is a large chromosome puff.
Polytene chromosomes were originally observed in the larval salivary glands of Chironomus
midges by Balbiani in 1881, but the hereditary nature of these structures was not confirmed until
they were studied in Drosophila melanogaster in the early 1930s by Emil Heitz and Hans
Bauer. They are known to occur in secretory tissues of other dipteran insects such as the
Malpighian tubules of Sciara and also in protists, plants, mammals, or in cells from
other insects. Some of the largest polytene chromosomes described thus far occur in larval
salivary gland cells of the Chironomid genus Axarus.
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Polytene chromosome from Axarus larva
Observation :
Students should observe the permanent slide of the polytene chromosome and should write the
difference between normal chromosome and polytene chromosome.
Result : --------------------------------------------------------------------------
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Experiment No. 15
Aim: Determination of monohybrid ratio.
Principle
Monohybrid ratio is also known as law of segregation. Mendel suggested that each character is
represented by unique factor in the gamete known as gene. These gene are in pairs and are
known as alleles. One allele is dominant and other is recessive. During the gamete formation the
allele segregates or separate with different characteristics. On this basis Mendel suggested the
Law of Segregation.
Monohybrid Ratio
The phenotypic ratio of different types of individuals occurring in the F2 generation of the
monohybrid cross is called the monohybrid ratio. In the Mendelian monohybrid experiments,
this ratio was always 3:1( i.e., 75% is dominant and 25% is recessive).
For example, for one of his monohybrid crosses, Mendel selected true breeding homozygous
parents showing contrasting characters for the height of the plant. He performed the experiment
in three stages as described. The result obtained is shown in Figure below.
Figure : Tall x Dwarf Monohybrid cross showing the result obtained by Mendel
The pure tall is crossed with the pure dwarf parent. According to Mendel, when the diploid
individual (having both the alleles/factors) produces gametes, each gamete receives only one of
the two factors/alleles of a character. No gamete receives both the alleles of a character. Thus,
pure tall parent produces only one type of gametes, i.e. all the gametes possess only (T) factor
for tallness. Similarly, all gametes produced by pure dwarf are of one type only and possess (t)
factor. The fusion of (T) and (t) gametes (fertilization) results in the F1 offspring with (Tt)
genotype. It is heterozygous or a hybrid. Its phenotype (external appearance) is tall because the
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factor for tallness (T) is dominant and expresses itself. The factor for dwarfness (t) is present in
F1 hybrid but, being recessive, does not express itself (remains hidden).
Mendel allowed hybrids to self-fertilize or inbreed to raise F2 generation. The F1 hybrid has
dissimilar alleles (Tt). Therefore, it will produce two types of gametes in equal number i.e. 50%
gametes will have (T) factor and remaining 50% will have (t) factor. Since the pea flower is
bisexual, it produces both male and female gametes. Thus, the F1 hybrid will produce two types
of male gametes (T) and (t) in equal numbers. Similarly, there will be two types of female
gametes (T) and (t) in equal numbers. During self fertilization, the fusion between these male
and female gametes occurs at random. For example, each type of male gamete has an equal
chance to fuse with either (T) or (t) female gametes and vice-versa. This chance fusion, between
two types of male and two types of female gametes will produce a maximum of four
combinations (genotypes) in the F2 progeny. This is shown in the checker board or Punnet’s
Square. These four combinations fall into three categories of the genotypes as follows : 1 (TT), 2
(Tt) and 1 (tt) i.e.
1 Pure tall : 2 Hybrid tall : 1 Pure dwarf
(TT)
2(Tt)
(tt)
This is called 1:2:1 genotypic ratio of a monohybrid cross. However, phenotypically, the
progeny shows 3 Tall and 1 Dwarf individuals (75% Dominant and 25% recessive characters) or
3:1 ratio. This is called monohybrid ratio or phenotypic ratio of a monohybrid cross.
Procedure:
We can determine / demonstrate the above monohybrid ratio using beads. Take beads of two
colors one as dominant & other as recessive. Put equal number of both of the beads of two color
in one bag. Now randomly withdraw two beads from bag at a time, observe various combination
and note down it.
Observation:
The ratio was found to be 1:1.5:1 which is near to 1:2:1. From this experiment, it can be said
that gametes pair randomly and also that genes segregate.
Result:
Monohybrid ratio was determined successfully.
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Experiment No. 16
Aim: Dihybrid Ratio {Law of Independent Assortment}
Principle
A cross between two pure, true breeding parents in which the inheritance pattern of
two allelomorphic pairs is considered (studied) simultaneously is called a dihybrid
cross. The phenotypic ratio obtained in the F2 generation of a dihybrid cross is called
the dihybrid ratio.
A dihybrid is an individual which is double heterozygous (i.e. heterozygous for two pairs of
alleles).
Mendel’s dihybrid cross : Mendel considered two characters in the pea plants simultaneously,
e.g. cotyledon color (yellow / green) and seed shape (round / wrinkled). He selected one variety
of pea which was pure (true breeding) for yellow round seeds and crossed it with another variety
pure for green wrinkled seeds.
All the F1 of this cross were yellow round seeds (green and wrinkled characters did not appear in
F1 hybrids). Mendel anticipated this because, from the earlier monohybrid experiments he knew
that yellow was dominant over green and round was dominant over wrinkled.
P
F1
Yellow Round
X
Green Wrinkled
Yellow Round
Similarly, a cross between yellow wrinkled and green round also produced only yellow round
seeds in F1
P
Yellow Wrinkled
F1
X
Green Round
Yellow Round
Moreover, the reciprocal crosses (interchanging male and female parents) also gave the same
results.
Further, when the F1 dihybrids were self-pollinated or inbred, the F2 generation was always the
same, e.g.
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The analysis of F2 progeny showed four different kinds of phenotypes. These were (1) Yellow
round (2) Yellow Wrinkled (3) Green round and (4) Green Wrinkled in the ratio of 9:3:3:1
respectively. It will be seen that out of these four types, two show the same combinations as the
parents whereas the remaining two are new combinations (recombinants).
The phenotypic ratio of 9:3:3:1 in the F2 progeny of a dihybrid cross is called the dihybrid ratio.
Same results were obtained by Mendel using other pairs of alleles in different combinations.
On the basis of these experiments and their results, Mendel formulated the law of independent
assortment of characters and explained it as follows.
Law of Independent Assortment
"When a dihybrid (or a polyhybrid ) forms gametes, (i) each gamete receives one allele
from each allelic pair and (ii) the assortment of the alleles of different traits during the
gamete formation is totally independent of their original combinations in the parents
In other words, each allele of any one pair is free to combine with any allele from each
of the remaining pairs during the formation for the gametes
This is known as the Law of Independent Assortment of characters. It is also referred to as
Mendel’s third law of heredity.
Explanation of the law of independent assortment: The principle of independent assortment
was explained by Mendel with the help of a dihybrid cross involving characters of cotyledon color
(yellow / round) and seed shape (round / wrinkled).
Mendel crossed a true breeding variety of pea having yellow cotyledons (YY) and round seeds
(RR) with another true breeding variety having green cotyledons (yy) and wrinkled seeds (rr).
The complete result of this cross is shown in the Figure
Thus, the yellow round parent has the genotype (YYRR) and the green wrinkled parent (yyrr).
Since each parent is homozygous for both characters (color and shape), each will produce only
one type of gametes. The (YYRR) parent will produce all (YR) type gametes and the (yyrr) will
produce all (yr) type gametes. All F1 dihybrids resulting from the fusion of these gametes would
be double heterozygous with (YyRr) genotype and appear yellow round. This indicated that in the
dihybrid cross also in each pair, the alleles behaved exactly in the same way as in the
monohybrid cross. Both the dominants (Y and R) expressed themselves in F1 while both the
recessive alleles (y and r ) remained hidden.
Types of gametes formed by F1 dihybrid: According to Mendel, during gamete formation by
the F1 dihybrid, the alleles in both pairs Y-y and R-r first segregate from each other (Law of
segregation). Each pair segregates independently of the pair. Then the alleles enter the gametes.
A gamete can receive only one allele from each pair, i.e. Y or y and R or r. Similarly, a gamete
that receives a factor (gene) for color must also receive factor for shape (a factor for every
character must be present in each gamete). Thus, a gamete that receives Y for color may receive
R or r for shape. This would result in (YR) and (Yr) types of gametes. Similarly, a gamete that
receives y for color may receive R or r for shape. This would give (yR) and (yr) types of gametes.
In other words, the F1 dihybrid would produce four types of gametes (YR), (Yr), (yR) and (yr) in
equal proportions. This is the principle of independent assortment of characters. There will be
four types of male gametes and four types of female gametes formed by the F1 dihybrid.
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This shows the result of the dihybrid cross and independent assortment of character
During self-fertilization or inbreeding of the F1 dihybrids to produce an F2 generation, these male
and female gametes can form maximum to dihybrid unions as shown in the Punnet’s Checkerboard (Figure 7.3). These can be grouped into four kinds on the basis of phenotypic appearance.
i.e. yellow round, yellow wrinkled, green round and green wrinkled in the ratio of 9:3:3:1
respectively. This is called the Phenotypic dihybrid ratio.
Procedure
We can demonstrate the dihybrid ratio by taking beads of different colour as individual gametes
YR----------Dark Yellow
yR ---------White
Yr ----------Green
yr ----------Red
On crossing beads for Dhybrid ratio we obtain the ratio 9: 3: 3: 1
Observation
Yellow
Yellow
Green
Green
and white
and Red
and White
and Red
27 – 9
12 – 4
15 – 5
3–1
Result
Dihybrid ratio was successfully determined
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Experiment No. 17
Aim: Karyotype analysis
Principle
The karyotype is an orderly arrangement of chromosomes according to international conventions.
The chromosomes are cut from a photographic print and arranged on a preprinted form using a
suitable adhesive to fix the chromosome permanently to the sheet.
The newer technique of digital imaging and computer rearrangement of chromosomes are
electronic methods of the same task.
The chromosomes are categorized by size and morphology into seven groups ( A to G groups).
Group A contains chromosomes number 1 to 3; group B contains chromosome number 4 and 5;
group C contains chromosome number 6 to 12; group D contains chromosome number 13 to 15;
group E contains chromosomes number 16 to 18; group F contains chromosome number 19 and
20 and group G contains chromosome number 21 and 22; and last group called sex chromosome
contains chromosome X and Y. further with banding techniques, they are identified individually.
There are 22 pairs of autosomes and a pair of sex chromosomes is present in all normal
metaphases of human cells.
The normal female has 22 pair of autosomes and a pair of X chromosomes.
The normal male has 22 pairs of autosomes, one chromosomes each of X and Y.
Chromosome abnormality syndrome
Downs Syndrome
( Trisomy 21): Down syndrome is a genetic disorder caused by extra genetic material. It affects
over 350,000 people in the United State alone and is the most common (1 in 800 live birth)
imbalance in the number of autosomes in the people. The effect of Down syndrome varies greatly
from person to person but can include mental retardation, eyes that slant upward, and heart
defects.
People with Down syndrome have 3 copies of chromosome 21, for this reason Down syndrome is
also called “Trisomy 21”
Where does the extra chromosome come from? In 90% of trisomy 21 cases the additional
chromosome comes from the mother’s egg.
Turner Syndrome
Turner syndrome affects 60,000 girls and women in the United State. This disorder occurs in 1 in
2000 to 2500 live births, with about 800 new cases diagnosed each year. Symptoms include
short stature and lack of ovarian development. Other feature, such as webbed neck, arms that
turn out slightly at the elbow and a low hairline in the back of head are sometimes seen.
Women and girls with Turner syndrome have only x-chromosome. This is an example of
monosomy .
Where dose the single x-chromosome come from? In 75% to 85% cases, the single xchromosome comes from the mother’s egg .
Klinefelter Syndrome
Klinefelter syndrome occurs in 1 in 500 to 1000 live births. People with this disorder develops as
males with subtle characteristics that becomes apparent during puberty .They are often tall and
usually do not develop secondary sex characteristics such as facial hair, or underarm and pubic
hair
Men and boys with Klinefelter syndrome have a Y chromosome 2X chromosomes. This is an
example of trisomy.
Where does the extra chromosome come from? In about half of Klinefelter cases, the extra X
chromosome is from the mother’s egg, while in the other half of cases the extra X chromosome is
from father’s sperm.
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Karyotyping
of
abnormal
individual
suffering
from
Klinefelter syndrome
Male sex organs; unusually small testes, sterile. Breast enlargement and other feminine body
characteristics. Normal intelligence.
CRI DU CHAT
CRI DU CHAT is a rare syndrome ( one in 50,000 live births ) caused by a deletion on the
short arm of chromosome no. 5.The name of this syndrome is French for “cry of the cat “,
referring to the distinctive cry of the children with this disorder .The cry is caused by abnormal
larynx development , which becomes normal within a few weeks of birth. Infants with CRI DU
CHAT have low birth weight and may have respiratory problems . Some people with this
syndrome have a shortened lifespan, but most have a normal lifespan most having a normal life
expectancy.
Where does the abnormal chromosome no. 5 come from? In 80% of the cases, the chromosome
carrying this deletion comes from the father’s sperm.
Reciprocal Translocation: Philadelphia Chromosome
This person has 46 chromosomes with a translocation of material between chromosome 9 and
chromosome 22 (commonly known as the PHILADELPHIA CHROMOSOME). Detail studies of
the Philadelphia chromosome shows that most of the chromosome 22 has been translocated onto
the long arm of the chromosome 9.
In addition, the small distal portion of the short arm of the chromosome 9 is translocated to the
chromosome 22. This translocation, which is found only in tumor cells, indicates that a patient
has CHRONIC MYLEGENOUS LEUKEMIA (CML) In CML, the cells that produce blood cells for
the body (the haematopoietic cells) grow uncontrollably, leading to cancer.
The connection between this chromosomal abnormality and CML was clarified by studying the
genes located on the chromosomes at the sites of the translocation breakpoints. In one of the
translocated chromosomes, part of a gene called abl (pronounced A-ble ) is moved from its
normal location on chromosome 9 to a new location on chromosome 22. This breakage and
reattachment leads to an altered abl gene. The protein produced from the mutant abl gene
functions
improperly.
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Material:
Human chromosome photograph
karyotype forms
Scissors
Pencil
Tape
Glue
Procedure
1)
Take or prepare normal chart of human chromosomes arranged properly in a
sequence in groups.
2)
In this experiment students are supplied with chromosomes made up of paper.
These chromosomes are prepared by taking the photograph of karyotype of normal
individual as well as abnormal individual.
3)
Chromosomes on these photographs are excised properly into individual
chromosomes by avoiding abnormal cutting of any arm of chromosomes.
4)
Chromosomes from normal individual and abnormal individual are kept separate.
5)
These chromosomes from separate individuals are supplied to the students and
asked to arrange in 23 pairs of homologous Chromosomes.
6)
After arranging in 23 pairs these chromosomes are examined for the presence or
absence of extra part of chromosomes i.e addition or deletion of arm. Or if any extra
chromosomes are left after arranging in abnormal set then these chromosomes are
matched with each pair of Chromosomes and placed in the pair where the match is
proper. This matching will result in the identification of the disease whether it is
trisomy or tetrasomy. Absence of a chromosome in the abnormal set will result in
the identification of the disease as monosomy for that particular pair.
For the identification of chromosome following criteria should be taken into
consideration
a)
b)
c)
d)
e)
f)
Position of centromere
Length of chromosome
Arm ratio
Distribution of chromatin
Banding pattern of chromosome
Number of chromosome
In human being 2n = 46
Normal female 2n = 44+XX
Normal male
2n = 44+XY
These 23 pairs of chromosomes are arranged in 7 groups
7)
In this way comparison is made between normal and abnormal individual
chromosomal chart i.e karyotype and disease is identified.
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Picture of finished karyotype
Graphics of finished karyotype
Observation
After comparison of karyotypes from normal and abnormal individuals it has been found that the
abnormal individual suffers from the disease ------------------------Result
Karyotype analysis shows the presence of disease ----------------------------- in the abnormal
individual
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Experiment No. 18
Aim: Human pedigree analysis
Principle
If more than one individual in a family is afflicted with a disease, it is a clue that the disease may
be inherited. A doctor needs to look at the family history to determine whether the disease is
indeed inherited and, if it is, to establish the mode of inheritance. This information can then be
used
to
predict
recurrence
risk
in
future
generations.
A basic method for determining the pattern of inheritance of any trait (which may be a physical
attribute like eye color or a serious disease like Marfan syndrome) is to look at its occurrence in
several individuals within a family, spanning as many generations as possible. For a disease trait,
a doctor has to examine existing family members to determine who is affected and who is not.
The same information may be difficult to obtain about more distant relatives, and is often
incomplete.
Once family history is determined, the doctor will draw up the information in the form of a special
chart or family tree that uses a particular set of standardized symbols. This is referred to as a
pedigree. Pedigree is the family history expressed in the form of tree diagram. A pedigree is a
diagram of family relationships that uses symbols to represent people and lines to represent
genetic relationships. These diagrams make it easier to visualize relationships within families,
particularly large extended families. Pedigrees are often used to determine the mode of
inheritance (dominant, recessive, etc.) of genetic diseases.
and females by circles . An individual who
In a pedigree, males are represented by squares
exhibits the trait in question, for example, someone who suffers from Marfan syndrome, is
or
. A horizontal line between two symbols represents a
represented by a filled symbol
. The offspring are connected to each other by a horizontal line above the symbols
mating
and to the parents by vertical lines. Roman numerals (I, II, III, etc.) symbolize generations.
Arabic numerals (1,2,3, etc.) symbolize birth order within each generation. In this way, any
individual within the pedigree can be identified by the combination of two numbers (i.e.,
individual II3).
1) Autosomal Dominant Inheritance: The allel responsible for a particular trait is dominant
or locus is present on autosome . This mode of inheritance responsible for fifty-fifty chances for
receving the trait allel
2) Autosomal Recessive Inheritance: The allel responsible for particular trait is present on
autosomal locus and the trait is express in homozygous condition thus, the chance is negligible
however in breeding there is chance for a greater degree of expression of autosomal recessive
inheritance.
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3) Sex linked inheritance: The locus present on X or Y chromosome. Chances of males get
affected are more. Females may be affected may not be or may even be carriers in case of
recessive trait.
4) Mitochondrial Inheritance: Inheritance from mother to offspring’s through agencies of
cytoplasm or trait present on mitochondrial inheritance. Father does contribute to mitochondrial
inheritance.
Dominant and recessive traits
Using genetic principles, the information presented in a pedigree can be analyzed to determine
whether a given physical trait is inherited or not and what the pattern of inheritance is. In simple
terms, traits can be either dominant or recessive. A dominant trait is passed on to a son or
daughter from only one parent. Characteristics of a dominant pedigree are: 1) Every affected
individual has at least one affected parent; 2) Affected individuals who mate with unaffected
individuals have a 50% chance of transmitting the trait to each child; and 3) Two affected
individuals may have unaffected children.
Recessive traits are passed on to children from both parents, although the parents may seem
perfectly "normal." Characteristics of recessive pedigrees are: 1) An individual who is affected
may have parents who are not affected; 2) All the children of two affected individuals are
affected; and 3) In pedigrees involving rare traits, the unaffected parents of an affected
individual may be related to each other.
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The reason for the two distinct patterns of inheritance has to do with the genes that predispose
an individual to a given disease. Genes exist in different forms known as alleles, usually
distinguished one from the other by the traits they specify. Individuals carrying identical alleles of
a given gene are said to be homozygous for the gene in question. Similarly, when two different
alleles are present in a gene pair, the individual is said to be heterozygous. Dominant traits are
expressed in the heterozygous condition (in other words, you only need to inherit one diseasecausing allele from one parent to have the disease). Recessive traits are only expressed in the
homozygous condition (in other words, you need to inherit the same disease-causing allele from
both parents to have the disease).
Penetrance and Expressivity
Penetrance is the probability that a disease will appear in an individual when a disease-allele is
present. For example, if all the individuals who have the disease-causing allele for a dominant
disorder have the disease, the allele is said to have 100% penetrance. If only a quarter of
individuals carrying the disease-causing allele show symptoms of the disease, the penetrance is
25%. Expressivity, on the other hand, refers to the range of symptoms that are possible for a
given disease. For example, an inherited disease like Marfan syndrome can have either severe or
mild
symptoms,
making
it
difficult
to
diagnose.
Non-inherited traits
Not all diseases that occur in families are inherited. Other factors that can cause diseases to
cluster within a family are viral infections or exposure to disease-causing agents (for example,
asbestos). The first clue that a disease is not inherited is that it does not show a pattern of
inheritance that is consistent with genetic principles (in other words, it does not look anything
like a dominant or recessive pedigree).
Observation
1. Pedigree tree of the affected person is prepared by collecting the information about his
family.
2. After examining the pedigree tree of the affected person it has been observed that the
person suffers from -------------------------------------------------------------------------------------------------(Autosomal Dominant Inheritance, Autosomal
Recessive
Inheritance, Mitochondrial Inheritance) for the disease ----------------------------Result:
The pedigree analysis of the affected person shows---------------------------------------------------------------------------- Autosomal Dominant Inheritance, Autosomal Recessive Inheritance,
Mitochondrial Inheritance) for the disease ---------------------.
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Experiment No. 19
Aim: Computer aided visualization of amino acid sequence of protein and its 3D
structure using RCSB PDB (Protein Data Bank).
Introduction
The Protein Data Bank (PDB) is a repository for 3-D structural data of proteins and nucleic
acids. These data, typically obtained by X-ray crystallography or NMR spectroscopy and
submitted by biologists and biochemists from around the world, are released into the public
domain, and can be accessed for free.
Founded in 1971 by Drs. Edgar Meyer and Walter Hamilton Brookhaven National
Laboratory, management of the Protein Data Bank was transferred in 1998 to members of the
Research Collaboratory for Structural Bioinformatics (RCSB). Rutgers University is
the lead site and is currently under the direction of Helen M. Berman. [1]
The Worldwide Protein Data Bank (wwPDB) consists of organizations that act as
deposition, data processing and distribution centers for PDB data. The founding members are
RCSB PDB (USA), MSD-EBI (Europe) and PDBj (Japan). The BMRB (USA) group
joined the wwPDB in 2006. The mission of the wwPDB is to maintain a single Protein Data Bank
Archive of macromolecular structural data that is freely and publicly available to the global
community.
The PDB is a key resource in structural biology and is critical to more recent work in
structural genomics. Countless derived databases and projects have been developed to
integrate and classify the PDB in terms of protein structure, protein function and protein
evolution.
As of 26 September 2006, the database contained 39,051 released atomic
coordinate entries (or "structures"), 35,767 of that proteins, the rest being nucleic acids, nucleic
acid-protein complexes, and a few other molecules. About 5,000 new structures are released
each year. Data are stored in the mmCIF format specifically developed for the purpose.
Note that the database stores information about the exact location of all atoms in a
large biomolecule (although, usually without the hydrogen atoms, as their positions are more of
a statistical estimate); if one is only interested in sequence data, i.e. the list of amino acids
making up a particular protein or the list of nucleotides making up a particular nucleic acid,
the much larger databases from Swiss-Prot and the International Nucleotide Sequence
Database Collaboration should be used.
As of 11 September 2007, the "PDB Holdings List" at RCSB reported the following statistics:
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Note that theoretical models are no longer accepted in the PDB.
22461 structures in the PDB have a structure factor file. 3138 structures in the PDB have an
NMR restraint file.
File format
Through the years the PDB file format has undergone many, many changes and revisions. Its
original format was dictated by the width of computer punch cards.
•
PDB Format Guide - Prepared by the PDB Staff at BNL The PDB format
•
•
•
specification can be found here, and it is vital that you read this before looking at the raw
data.
Recently PDB provides a representation of PDB data in XML format, PDBML format.
ftp.rcsb.org The raw data can be downloaded from here.
PDB format files can be downloaded using HTTP with URLs like this:
•
PDBML
•
•
http://www.pdb.org/pdb/files/4hhb.pdb.gz
(XML)
files
can
be
downloaded
using
HTTP
with
URLs
like
this:
http://www.pdb.org/pdb/files/4hhb.xml.gz
ftp.ebi.ac.uk/pub/databases/rcsb/ Alternate download location for the PDB archive.
www.pdb.org Statistics about the PDB can be found here.
This legacy format has caused many problems with the format, and consequently there are
'clean-up' projects;
•
•
The Molecular Modeling DataBase (MMDB) from NCBI
wwPDB
The MMDB uses ASN.1 (and an XML conversion of this format). The wwPDB members RCSB PDB,
MSD-EBI, and PDBj are working together to make the data uniform across the archive. Some
believe this to be desirable; others argue that, without a universal repository of information (i.e.,
a common dictionary), it is not possible to draw comparisons. Each structure published in PDB
receives a four-character alphanumeric identifier, its PDB ID. This should not be used as an
identifier for biomolecules, since often several structures for the same molecule (in different
environments or conformations) are contained in PDB with different PDB IDs.
If a biologist submits structure data for a protein or nucleic acid, wwPDB staff reviews and
annotates the entry. The data are then automatically checked for plausibility. The source code
for this validation software has been released for free. The main data base accepts only
experimentally derived structures, and not theoretically predicted ones (see protein structure
prediction).
Viewing the data
The structural data can be used to visualize the biomolecules with appropriate software, such
as VMD, RasMol , PyMOL, Jmol , MDL Chime, QuteMol , web browser VRML plugin or
any web-based software designed to visualize and analyze the protein structures such as
STING. A recent desktop software addition is Sirius. The RCSB PDB website also contains
resources for education, structural genomics, and related software.
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The PDB offers three options for searching the databases:•
•
•
Enter a four letter PDB identifier directly (eg:-1gii).
Search using searchlite
Search using the search field interfaces.
Result
By using above resource protein structure have been saved in the .pdb format and ready to
visualize off line using specific software.
Experiment No. 20
Aim: Retrieval of metabolic pathway using internet.
Theory
Number of metabolic pathway resources are available online.
1] WIT (What is there)
2] KEGG (Kyoto Encyclopedia of Genes and Genomes)
3] Path DB (Pathway Database)
1] WIT: provides complete metabolic models for organisms whose genomes have been
completely sequenced. It has currently metabolic models for 39 organisms including metabolisms
and bioenergetic pathway available for transcription, translation, transmembrane transport to
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signal transduction. It provides easy search system and every metabolic outcome provides
clickable links that takes closer to the specific levels of details above that subset of metabolism.
2] KEGG: It provides metabolic overview as illustration, rather than text. It is easy to use for
visually oriented reader. It provides listing on EC no. and their corresponding enzymes broken
down by level. It provides many helpful links to sites describing enzyme and liquid nomenclature
in details. Like WIT, KEGG is searchable by sequence homology by keywords and chemical entity.
Liquid ID codes of two small molecules can be put to find the entire possible metabolic pathway
connecting them.
3] Path DB: It is same as that of WIT and KEGG. It handles information in more flexible way
than the other two.
Result
From the above studies user can get better insight of metabolic pathways their links and
enzymes involved in the pathway.
Flow sheet diagram for KEGG.
Google → KEGG → glycolysis → clickable points for more information on enzyme involed in
pathway. →save search result
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Experiment No. 21
Aim: Retrieval and study of nucleic acid sequence
databanks in Gene Bank.
Theory
Gene Bank, NIH (National institute of health), genetic sequence database is an annotated
collection of publicly available nucleotide and protein sequences. The records within gene bank
represent in most cases single and continue stretches of DNA or RNA with annotations. Gene
bank files are grouped into divisions are phylogenetically based whereas others are based on
technical approach that was used to generate the sequence information.
GBFF (gene bank flat file) is one of the most commonly used formats in the representation of
biological sequences. It can be described in 3 parts,
1] THE HEADER: it contains the information that applied to the whole record. The header is the
most database specific part of the record. It has the following attributes.
a) Locus line:
Synonyms locus name accession no. Length of base pair molecule division code
Locus
AF11785
5925bp
mRNA
PRI
Date of publication
1-september-1994
b) Definition line:
Synonyms- Definition genus species product name.
(Gene symbol) mRNA, complete CDS.
Example: definition Homo sapiens myosin heavy chain
mRNA complete CDS.
c) All these followed by several keywords such as source, organisms , references, blocks,
author, title, general, etc. lastly followed by comment line.
2] The feature table: The middle segment of the GBFF record is the most important direct
representation of the biological information in the record. It has the following attributes:
a) Source feature: it contains many legal qualifiers.
Examples:
Source 1- 5925
/organism- “homo sapiens”
/dbx reference = “Taxon : 9609”
/chromosome = “17”
/ Map = “17 p13 – 1”
/ Tissue type = “skeletal muscle”
b) CDS feature: The amino acid sequence is displayed.
c) Gene feature: It represents the segments of the DNA that can be identified with a name or
arbitrary number.
Examples – Accession number.
d) RNA features: It represents the nucleotides sequence of the given organism.
3] All major nucleotide database flat files ends with “//” on the last line of the record.
Result
The format for the nucleic acid database has been better studied, from the above description and
a sample format is attached in the exhibit 1.
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Experiment No. 22
Aim: Homology searching using BLAST.
Introduction
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for
comparing primary biological sequence information, such as the amino-acid sequences of
different proteins or the nucleotides of DNA sequences. A BLAST search enables a
researcher to compare a query sequence with a library or database of sequences, and identify
library sequences that resemble the query sequence above a certain threshold. For example,
following the discovery of a previously unknown gene in the mouse, a scientist will typically
perform a BLAST search of the human genome to see if humans carry a similar gene; BLAST
will identify sequences in the human genome that resemble the mouse gene based on similarity
of sequence. The BLAST program was designed by Eugene Myers, Stephen Altschul,
Warren Gish, David J. Lipman and Webb Miller at the NIH and was published in J. Mol.
Biol. in 1990.
BLAST
Develope Altschul S.F., Gish W., Miller
d by
E.W., Lipman D.J., NCBI
OS
UNIX,
Linux,
Windows
Mac,
MS-
Genre
Bioinformatics tool
License
Public Domain
Website
ftp://ftp.ncbi.nlm.nih.gov/bl
ast/
BLAST is one of the most widely used bioinformatics programs, because it addresses a
fundamental problem and the algorithm emphasizes speed over sensitivity. This emphasis on
speed is vital to making the algorithm practical on the huge genome databases currently
available, although subsequent algorithms can be even faster.
Examples of other questions that researchers use BLAST to answer are
•
•
•
Which bacterial species have a protein that is related in lineage to a certain protein
with known amino-acid sequence?
Where does a certain sequence of DNA originate?
What other genes encode proteins that exhibit structures or motifs such as ones that
have just been determined?
BLAST is also often used as part of other algorithms that require approximate sequence
matching.
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The BLAST algorithm and the computer program that implements it were developed by
Stephen Altschul, Warren Gish, David Lipman at the U.S. National Center for
Biotechnology Information (NCBI), Webb Miller at the Pennsylvania State
University, and Gene Myers at the University of Arizona. It is available on the web on
the NCBI website. Alternative implementations include WU-BLAST and FSA-BLAST.
The original paper by Altschul, et al, was the most highly cited paper published in the 1990s.
Input/Output
Input and output conform to the FASTA format.
Algorithm
To run, BLAST requires a query sequence (also called the target sequence) to search for, and a
sequence to search against (or a sequence database containing multiple such sequences). BLAST
will find subsequences in the database which are similar to subsequences in the query. In typical
usage, the query sequence is much smaller than the database, e.g., the query may be one
thousand nucleotides while the database is several billion nucleotides.
BLAST searches for high scoring sequence alignments between the query sequence and
sequences in the database using a heuristic approach that approximates the Smith-Waterman
algorithm. The exhaustive Smith-Waterman approach is too slow for searching large genomic
databases such as GenBank. Therefore, the BLAST algorithm uses a heuristic approach that is
less accurate than the Smith-Waterman but over 50 times faster. The speed and relatively good
accuracy of BLAST are the key technical innovation of the BLAST programs.
The BLAST algorithm can be conceptually divided into three stages.
1. In the first stage, BLAST searches for exact matches of a small fixed length W between
the query and sequences in the database. For example, given the sequences AGTTAC and
ACTTAG and a word length W = 3, BLAST would identify the matching substring TTA that
is common to both sequences. These exact matches are known as seeds. By default, W =
11 is used for nucleic seeds.
2. In the second stage, BLAST tries to extend the match in both directions, starting at the
seed. The ungapped alignment process extends the initial seed match of length W in each
direction in an attempt to boost the alignment score. Insertions and deletions are not
considered during this stage. For our example, the ungapped alignment between the
sequences AGTTAC and ACTTAG centered around the common word TTA would be:
..AGTTAC..
| |||
..ACTTAG..
If a high-scoring un-gapped alignment is found, the database sequence passes on to the
third stage.
3. In the third stage, BLAST performs a gapped alignment between the query sequence and
the database sequence using a variation of the Smith-Waterman algorithm.
Statistically significant alignments are then displayed to the user.
Program
The BLAST program can either be downloaded and run as a command-line utility "blastall" or
accessed for free over the web. The BLAST web server, hosted by the NCBI, allows anyone with
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a web browser to perform similarity searches against constantly updated databases of proteins
and DNA that include most of the newly sequenced organisms.
BLAST is actually a family of programs (all included in the blastall executable). These include:
Nucleotide-nucleotide BLAST (blastn)
This program, given a DNA query, returns the most similar DNA sequences from the DNA
database that the user specifies.
Protein-protein BLAST (blastp)
This program, given a protein query, returns the most similar protein sequences from the
protein database that the user specifies.
Position-Specific Iterative BLAST (PSI-BLAST)
This program is used to find distant relatives of a protein. First, a list of all closely related
proteins is created. These proteins are combined into a general "profile" sequence, which
summarises significant features present in these sequences. A query against the protein
database is then run using this profile, and a larger group of proteins is found. This larger
group is used to construct another profile, and the process is repeated.
By including related proteins in the search, PSI-BLAST is much more sensitive in picking
up distant evolutionary relationships than a standard protein-protein BLAST.
Nucleotide 6-frame translation-protein (blastx)
This program compares the six-frame conceptual translation products of a nucleotide
query sequence (both strands) against a protein sequence database.
Nucleotide 6-frame translation-nucleotide 6-frame translation (tblastx)
This program is the slowest of the BLAST family. It translates the query nucleotide
sequence in all six possible frames and compares it against the six-frame translations of a
nucleotide sequence database. The purpose of tblastx is to find very distant relationships
between nucleotide sequences.
Protein-nucleotide 6-frame translation (tblastn)
This program compares a protein query against the all six frame translations of a
nucleotide sequence database.
Large numbers of query sequences (megablast)
When comparing large numbers of input sequences via the command-line BLAST,
"megablast" is much faster than running BLAST multiple times. It concatenates many
input sequences together to form a large sequence before searching the BLAST database,
then post-analyze the search results to glean individual alignments and statistical values.
Homology searching can be done to our sequence using either accesion no. or
complete sequence present in the FASTA format.
Steps involed:- google →blast →select databases for comparing → select program → put
sequence → select visualization options → blast → result.
Result
By using BLAST program the sequence have been searched for homology succesfully and result
have stored in file.
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Experiment No. 23
Aim: Computer aided survey of scientific literature
Introduction
Collection of scientific literature is of paramount importance in the context of execution of
research. To execute any scientific idea or to perform any research activity relevant literature is
required as a reference. This literature can be obtained from various sources such as books,
scientific journals, and computer aided facility such as internet. Though it is not difficult to search
a required literature in the journals and books available in the library, but it is cumbersome. Now
a day’s internet has become the most popular tool for extracting relevant information since
almost all the scientific journals are accessible through internet on one click. Computer aided
survey of scientific literature is quite advantageous since it is cheap, less time consuming, easily
accessible, gives recent and updated information and requires small storage space.
In this experiment students are advised to use various search engines like
www.pnas.org
www.jbc.org
www.ncbi.nlm.nih.gov/Entrez.
www.ncbi.nlm.nih.gov
www.ebi.ac.uk/embl/index.html
www.ddbj.nig.ac.jp/
www.nbrf.geogetown,edu/pirwww/
www.expasy.ch/cgi-bin/sprot-search-de
www.nature.com
www.springerlink.com
www.elsvier.com
www.biologydirect.com
www.genome.org
www.IAS.org
www.JSTOR.org
www.oxfordjournals.org
www.harwordunivpress.org
www.google.com,
www.yahoo.com,
www.sciencedirect.com,
www.rediff.com etc.
These search engines can be used for the collection of scientific data by feeding required
information in the search window of these search engines.
In this experiment students are expected to search scientific literature or assignment given by
counselor.
Result
The required scientific literature has been obtained by using search engine--------------------------
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