Biology Lab : GENERAL SAFETY GUIDELINES

Biology lab safety rules are guidelines designed to help keep you safe during laboratory
experiments. Some equipment and chemicals in a biology laboratory can cause serious harm.
It is always wise to read, understand and follow all lab safety rules.

The following biology lab safety rules are a sample of the most basic rules that should be
followed when in a biology lab. If you have any additional queries regarding any procedures
to be followed in the lab, please contact the instructors (or faculty).

Biology Laboratory Safety Rules

1. Never work alone in the laboratory without permission and prior knowledge of the
instructor.
2. Do not engage in unlawful, mischievous, or unprofessional activities in the laboratory.
This includes not being disrespectful with your instructors and classmates.
3. Students should wash hands thoroughly after first entering the lab.
4. Students are only allowed to have a drink-bottle with lid during lectures and computer
labs. Other that this, they are not allowed to eat or drink anything in the laboratory
without explicit permission from the instructor.
5. Wear appropriate clothing at all times in the laboratory. Wear closed-toe shoes that
cover the top of the foot, unless permission otherwise is given by the instructor.
6. Wear examination gloves and safety glasses when dissecting or handling cadavers,
caustic chemicals, bacterial broth cultures, or as otherwise advised by your instructor.
Seemingly harmless biological samples can be dangerous due to chance of infections.
7. Wear gloves when handling any microorganisms. Wear lab aprons or lab coats as
advised by your instructor.
8. Keep hands away from your face, eyes, and mouth when working with cadavers,
chemicals, preserved specimens, microorganisms, or body fluids. This includes not
applying cosmetics, not adjusting contact lenses, and not biting your finger nails.
9. If any chemicals or other agents splash into your eyes, immediately go to the nearest
sink and flush your eyes with water. Immediately alert the instructor.
10. Report ANY and ALL accidents, spills, BREAKAGES, or injuries to the instructor, no
matter how trivial they appear.
11. Scalpels and other sharp objects can be used only if authorized by the instructor and
only after obtaining proper handling instructions. Use small trays to carry all sharp
objects. When handling sharp objects, point their tips down and away from other
people.
12. Wearing examination gloves, students must not leave the laboratory and must not
touch any equipment such as microscopes, door knobs or any personal items such as
cell phones, keys etc. Gloves after work should be appropriately disposed-off as
biohazardous waste.
13. Do not use any lab equipment without instruction and authorization from the
instructor. Report any damaged or broken equipment to your instructor
immediately.
14. Lab benches should be kept free of extraneous items while conducting experiments.
This includes unnecessary books, backpacks, cell phones, and other personal items.

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 Recommended ATTIRE

ü Lab coats or aprons must be worn at all times in the laboratory.

ü The same lab coat should not be worn in other rooms than the lab, e.g. in lecture
rooms, library, hotel or eateries etc., in order to avoid contamination with adherent
chemicals.
ü Safety goggles must be worn strictly when working with hazardous chemicals in
order to prevent damage to eyes from splash, fumes, vapours and irritating mists of
chemicals.
ü Contact lenses are not allowed. Even when worn under safety goggles, various fumes
may accumulate under the lens and cause serious injuries or blindness.
ü Wear gloves when handling hazardous chemicals. Remove them immediately after
use to avoid spreading contamination.
ü Long pants must be worn in the lab. Shorts are not allowed.
ü Long hair must be tied back and loose or baggy clothing must be avoided.
ü Wearing jewellery such as rings and necklaces are usually discouraged. Chemicals
could irritate the skin underneath rings and dangling jewellery could get caught on
equipment or accidentally fall into a running machine.
ü Closed shoes with non-slip soles should be preferably worn in the lab. Shoes must
completely cover the foot to avoid any chance of accidental exposure to harmful
materials.
ü No sandals/chappals are allowed at any time inside the lab.

LAB COATS, SHOES MUST FOR WORKING IN LAB

LOOSE CLOTHING, SANDALS/ CHAPPALS --- STRICTLY PROHIBITED

INFORM PERSON IN CHARGE IN CASE OF ANY ACCIDENT

Good CONDUCT in lab

ü Conduct yourself in a responsible manner at all times in the laboratory.

ü Work areas should be kept clean and tidy at all times.
ü Eating, drinking, and smoking are strictly prohibited in the laboratory.
ü No unauthorized experiments are to be performed. If you are curious about trying a
procedure not covered in the experimental procedure, consult with your laboratory
instructor.
ü Never taste anything. Never directly smell the source of any vapour or gas; instead by
means of your cupped hand, waft a small sample to your nose. Do not inhale these
vapours but take in only enough to detect an odour, if one exists.
ü Coats, bags and other possessions or belongings should be kept at the places assigned
for them and not on the platform where experiments are to be done. Beware that lab
chemicals can contaminate or sometimes destroy personal possessions.
ü Do not wander around the room, distract other students, or interfere with the
laboratory experiments of others.
ü Do not bump into others while they are working with the chemicals.

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 ü Do not sit on the chemical benches or stand against or by leaning on them. The
surfaces may be contaminated with hazardous chemicals that can cause injury to you
or damage your clothes.
ü Notify the instructor or the lab in-charge immediately in case of an accident.
ü Always wash your hands thoroughly with soap before leaving lab.

Disposal Policies

ü Dispose of broken glassware in the marked cardboard box container. Broken glass
containers are ONLY to be used for broken glass. Always use a broom and dust-pan if
asked to clean up broken glassware.
ü Dispose of used slides in the glass, or plastic, container labeled “Used Slides”.
ü Biohazardous wastes must be disposed in a biohazard waste container. Your instructor
will inform you which disposal containers are to be used with which type of
biohazardous waste (metal sharps, glass, and non-sharps).
ü Contaminated gloves must be disposed of in a biohazard waste container.

Issuing and returning of reagents, chemicals and glassware

ü All the Gloves, chemicals and required glasswares should be issued before starting an
experimental work.
ü One should return all the remaining chemicals, glassware etc. to chemical lab in-
charge while leaving the lab.

Equipment handling

ü Follow the student Teaching Assistant and equipment instructions before handling
any equipment. Set up and use the prescribed instrument as directed in the laboratory
instructions provided.
ü In case of any unusual circumstances, inform the lab in- charge/ instrument in-charge.

PROTECTION AGAINST COMMON LAB ACCIDENTS

1) FIRE
Ø There should never be open flames in the lab.
Ø Never keep volatile solvents, such as ether, acetone, or benzene in an open beaker.
Contact the instructors if you are unsure about the nature of a chemical provided to
you.
Ø Always use gloves to pick up hot objects.
Ø Keep flammables out of the lab.
Ø Ensure that no one smokes in the lab.
Ø Ensure that the machines are properly earthed.
Ø Abnormal smell, sound etc. should be checked out and immediately brought to the
notice of the lab staff.
Ø Ensure that there are no broken or loose wire anywhere.
Ø Familiarize yourself with the emergency plan of the lab and emergency equipment
such as the fire blanket and fire extinguisher and you should have been trained in
using them, if a need arises.

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 Ø The fire alarm should be in proper working condition.
Ø If the fire is due to electrical short circuit switch-off the power from mains.

ELECTRIC SHOCK

Ø When using electrical equipment make sure that all electrical cords are in good
condition. Check the cords for wear and tear, loose plugs, and make sure that the
outlets are properly grounded.
Ø When removing an electrical plug from its socket, grasp the plug, not the electrical
cord.
Ø Hands must be completely dry before touching an electrical switch, plug, or outlet.
Ø Do not overload electrical outlets and keep your work area dry. Do not use damaged
electrical equipment. Report damaged electrical equipment immediately to lab staff.

CUTS
Ø The most common laboratory accident is probably the cut received during improper
handling.
Ø Severed nerves and tendons are common results of injuries caused by improper
manipulation of glass tubes and thermometers.
Ø Position your hands in such a way that sharp items or tools are always pointed away
from your hands and other body parts.
Ø When handling sharp objects, keep a safe distance from your co-workers
Ø In case of a cut, wash with tap water and take medical aid.

ABSORPTION OF CHEMICALS THROUGH SKIN

Ø Some chemicals get absorbed through the skin and can cause severe diseases such as
dermatitis.
Ø Be careful about touching your face or eyes in the lab; make sure your hands are clean
first.
Ø Gloves provide only a temporary layer of protection against chemicals on your skin
and may be permeable to some chemical reagents, without visible deterioration. If
your gloves come in contact with such a chemical reagent, remove them, wash your
hands, and get a new pair immediately.

INHALATION OF CHEMICALS

Ø Keep your nose away from chemicals, do not inhale directly from bottle.
Ø If you inhale vapours accidentally due to a spill, leave the lab immediately and go to
open area to breath fresh air and take medical advice as necessary.
Ø In case of chemical spills evacuate lab and switch on emergency exhaust fans and open
windows.
Ø Wash your hands thoroughly with shop and water before handling anything which
goes into your mouth. Wash your hands every time when you leave the laboratory.

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IDENTIFICATION OF THE SAFETY EQUIPMENT
In order to ensure safety of self as well as others, location of the emergency equipments
should be known and also how to use them. The emergency equipments includes:
Ø Emergency exits, fire escape, and escape routes in general
Ø Alarm systems, telephone, and further emergency call equipment
Ø Extinguishers, fire alarm, and fire blankets
Ø Respiratory masks and related filters, safety showers, and eye wash
Ø First aid kits, stretcher, first aid room and contact number of security, IIT hospital,
lab staff, lab in-charge.

Very Important: WHAT TO BE DONE???

IF A FIRE OCCURS
ü In case of a fire, alert others of the situation and evacuate immediately. After everyone
is safely out, close the doors if possible to help contain the fire.
ü Activate a fire alarm and call emergency number for help.
ü Use the extinguisher to put out or hinder the fire, if the fire can be safely approached
switch off the main electrical supply, close main gas valve if it is safe to do.
ü If you or someone else in the lab is on fire, a fire blanket can be used.
ü Immediate medical aid should be provided to the victim.
ü In the event of a major fire, it helps if someone is there who can identify the types and
amounts of chemicals that are stored in the lab. This will assist emergency and fire
crews in effectively fighting the fire.

IN CASE OF AN ELECTRIC SHOCK

ü Immediately switch off the main power supply.
ü Separate the victim from the electric source.
ü Check pulse, breathing and level of consciousness of the victim.
ü Inform the instructor or the lab-in-charge.
ü Call for an ambulance.
ü Take the victim to the hospital as soon as possible.

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 IN CASE OF CHEMICAL SPILL

ü Do not try to clean up any chemical spill with a cloth or anything without knowing the
toxicity level and nature of the chemical. Use appropriate chemical absorbing powders
to contain spill.
ü Immediately alert the area occupants and the lab in charge and evacuate the area, if
necessary. If a volatile, flammable material is spilled, immediately control sources of
ignition.
ü If there is a fire or medical attention needed, contact the lab supervisor and inform
emergency number available in lab.
ü Attend to people who may be contaminated. Contaminated clothing must be removed
immediately and the skin flushed with large amounts of water for several times and
provided immediate medical attention.

IN CASE OF INGESTION OR INHALATION OF CHEMICALS

ü Move the person to fresh air in case of inhalation.

ü Provide first aid as required.
ü Do not induce vomiting unless advised to do so by a reliable medical authority.
ü Provide emergency medical personnel with the MSDS (material safety data sheet) for
the poisonous product.
ü Always ensure that the victim receives medical attention, even if the exposure seems
minor.

IN CASE OF CUTS

ü Wash the wound and surrounding area with mild soap and running water.
ü Remove any dirt around the wound.
ü Cover with an adhesive dressing or gauze square taped on all sides with adhesive tape.
ü Wounds caused by dirty, soiled or grimy objects should be examined by a physician,
who will determine whether a tetanus immunization is needed.
ü If the wound was caused by an object that has contacted human blood or body fluids,
the victim must be seen by a physician immediately, as immunization or post-exposure
prophylaxis may be required.
ü If a wound is bleeding profusely, the first aider should attempt to stop the bleeding as
quickly as possible. Inform the lab-in-charge and call ambulance or take victim to IIT
hospital, as per situation.

In case of Burns:

ü Run cold water over the burn for a minimum of 30 minutes. If the burn is small enough,
keep it completely under water.
ü If the victim's clothing is stuck to the burn, don't try to remove it. Tear or Cut the Cloth
from the near of the burn which is not stuck to the burns.
ü Cover the burn with a clean, cotton material. Don't use any other thing than Cotton.
Do not apply any soap, ointment, or home remedies.
ü Don't offer the burn victim anything to drink or eat, but keep the victim covered with
a blanket to maintain a normal body temperature until medical help arrives.

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 In case of Electric Shock Burns:

ü Don't touch the victim if he/she is still in contact of electricity. Touch only then when
you are sure that victim is not in contact of electricity.
ü Once the victim is stable, begin to run cold water over the burns for a minimum of 30
minutes.
ü Don't move the victim and don't scrub the burns or apply any soap, ointment, or home
remedies.
ü After flushing the burn, apply a clean, cotton cloth to the burn. If cotton is not
available, don't use anything.
ü Keep the victim warm and still and try to maintain a normal body temperature until
medical help arrives.
IN CASE OF EMERGENCY

If any accident happens, then immediately inform the lab-in-charge and SBL100
course coordinator. If the accident is severe and not in controllable limits then
call the concerned

• Security: 011-2659-1000
• Fire Control Room: 011-2659-6101
• Electrical Maintenance: 011-2659-6140
• IIT Delhi Hospital: Ambulance: 011-2659-1500 and 011-2659-6666
• Head of the Department, Prof. James Gomes: 011-2659-1013(O),
9958481338(M)
• Professor-in-charge, Biology lab, Prof Aditya Mittal: 011-2659-1052
• Safety Incharge faculty, Prof V. Perumal: 011-2659-7532, 7827615550
• SBL100 course coordinator, Prof Ashok Patel: 011-2659-7528, 08588871574
• Lab-In-charge Ms. Pushp Lata: 011-2659-7524

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 Biology Laboratory: Practical
Practical 1
Aim1a: Investigating enzyme catalysis and the effect of varying pH on enzyme activity

Introduction:

Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all catalysts,
speed up reactions without being used up themselves. They do this by lowering the activation
energy of a reaction. All biochemical reactions are catalysed by enzymes. Since enzymes are
proteins, they can be denatured in a variety of ways, so they are most active under mild
conditions. Most enzymes have optimum activity at a neutral pH and at body temperature.

Enzymes are also very specific – they only act on one substrate or one class of related
substrate molecules. The reason for this is that the active site of the enzyme is
complementary to the shape and polarity of the substrate. Typically, only one kind of
substrate will “fit” into the active site.

In this experiment, we will work with the enzyme amylase. This enzyme is responsible for
hydrolysing starch. In the presence of amylase, a sample of starch will be hydrolysed to
shorter polysaccharides, dextrins, maltose, and glucose. The extent of the hydrolysis depends
on how long it is allowed to react – if the starch is hydrolysed completely, the resulting
product is glucose.

In this practical, You will test for the presence or absence of starch in the solutions using
iodine (I2). Iodine forms a blue to black complex with starch, but does not react with glucose.
If iodine is added to a glucose solution, the only color seen is the red or yellow color of iodine.
Therefore, the faster the blue color of starch is lost, the faster the enzyme amylase is working.
If the amylase is inactivated, it can no longer hydrolyze starch, so the blue color of the starch-
iodine complex will persist.

In a complementary approach, You will also test for the presence of glucose in the samples
using Benedict’s reagent. When a blue solution of Benedict’s reagent is added to a glucose
solution, the color will change to green (at low glucose concentrations) or reddish-orange (at
higher glucose concentrations). Starch will not react with Benedict’s reagent, so the solution
will remain blue.

Effect of Enzyme Concentration

During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an enzyme-
substrate complex (ES). This is an equilibrium reaction, and will be favored by a high
concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes
place, and then the product is released.
E + S ⇔ ES ® E + P

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Effect of Temperature

All reactions are faster at a higher temperature. However, enzyme-catalyzed reactions

become slower or stop if the temperature becomes too high, because enzymes become
denatured at high temperatures. Therefore, enzymes have an optimum temperature that
corresponds to maximum activity. (At higher or lower temperatures, the activity of the
enzyme is lower.) The optimum temperature for enzymes of human origin is usually around
body temperature (37°C).

Effect of pH

Each enzyme has an optimum pH. Above or below an enzyme’s optimum pH, its activity is
lower. The optimum pH of a particular enzyme corresponds to the pH of its natural
environment. For many enzymes, this corresponds to pH values of around 7. For pepsin,
which is active in the stomach, the optimum pH is 2 (the pH of the stomach). Trypsin, which
is active in the small intestine, has an optimum pH of 8 that matches the pH of the small
intestine.

Procedure

Important: read the entire procedure and make sure you understand it before you start this
experiment. Before you start each part of the experiment, make sure you will have enough
time to complete it. Advance planning is very important for this experiment.

Preparation:

You will need 1% starch solution for each part of the experiment. Shake up the bottle of
starch, and collect about 50 mL of the solution in a small beaker.

You will also need some of the iodine reagent for each part of the experiment. Iodine solution
at 0.01 M concentration is suitable for starch testing. Make this by 10-fold dilution of 0.1 M
solution. Once made, the solution is a low hazard reagent but may stain skin or clothing if
spilled.

You can prepare your own fresh amylase solution by collecting about 1-2 mL of your own
saliva in a small beaker. (You will need to spit politely into the beaker.) After you have
collected 1-2 mL of saliva, add about 10-20 mL of water to the saliva and mix well. For health
reasons, you should work with your own saliva solution (not someone else’s). Alternatively,
commercial amylase solution at 1% may be used instead of saliva.

You will also need a white spot plate/dimple tile and some clean droppers.
• Test tube rack
• Test tubes, 2 for each pH to be tested, total 10
• Stop clock, Marker pen etc

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Reference tests:

Test for starch

Transfer a few drops of starch to one of the wells in the spot plate. Add one drop of the iodine
reagent. Starch and iodine should react to give a deep blue-black complex.

Test for glucose

Place 3 mL of 1% glucose solution in a test tube. Add 2 mL of Benedict’s solution and heat for
3-4 minutes in a boiling water bath. The reaction should produce a red-orange solid.

We will be using visual tests to evaluate the activity of the enzyme in each part of the
experiment. You will take samples of the enzyme-starch mixture at different times and add
this to drop of iodine reagent on white plate. The resulting color will tell you roughly how
much starch has been hydrolyzed. When the enzyme activity is high, it won’t take very much
time to hydrolyze the starch. When the enzyme is slowed down or is inactive, the blue-black
color will be seen for a longer time. You can assess the relative enzyme activity as follows:

Iodine test for starch Amount of starch remaining Enzyme activity level
Dark blue-black All None (0)
Blue
Most Low (1)

Light brown Some

Moderate (2)
None
Gold/orange High (3)

Experiment:
1. Place single drops of iodine solution in rows on the tile.
2. Label a test tube with the pH to be tested.
3. Use the syringe to place 2 ml of amylase into the test tube.
4. Add 1 ml of buffer solution to the test tube using a syringe.
5. Use another syringe to add 2 ml of starch to the amylase/ buffer solution, start the stop
clock and leave it on throughout the test. Mix using a plastic pipette.

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6. At different time points say roughly 5, 10, 15, 20, 25 and 30 minutes, transfer 2 drops of
each reaction mixture (using clean droppers) to a spot plate having 1 drop of iodine
reagent to each.
7. Record your observations and determine the enzyme activity level for each mixture.
8. Rinse off the spot plate with deionized water.
9. The iodine solution should turn blue-black. If the iodine solution remains gold/orange the
reaction is taking place and the starch has been broken down.
10. Repeat the whole procedure with another of the pH buffers to be used.
11. Plot a graph of time taken to break down starch against pH, or calculate the rate of
reaction and plot rate against pH.

Questions
1. What is the substrate of the enzyme α–amylase?
2. What are the products of the hydrolysis of the substrate?
3. At which pH, was starch hydrolyzed the fastest? Slowest?
4. Speculate the effect of enzyme concentration on enzyme activity.
5. How is the activity of amylase affected by a low pH? By a high pH? Explain.
6. What was the optimum pH for amylase?
7. Pepsin hydrolyzes proteins in the stomach. The pH in the stomach is 2, and the
optimum pH for pepsin is 2. What do you think would happen to the activity of pepsin
when it reaches the small intestine, where the pH is 8? Explain why.
8. In which reaction mixtures did hydrolysis of starch occur?

Health safety and precautions:

Amylase solution and iodine solution are low hazard reagents once made up. Wear eye
protection when handling iodine solution.

11
 Practical-1b
Aim1b: To investigate the Oxygen production by catalase enzymes

Introduction:
How do living cells interact with the environment around them? All organisms are limited by
the chemical reactions that make up their metabolism. These reactions break down the
energy from food consumption or photosynthesis into a usable form.

Hydrogen peroxide (H2O2) is a by-product of respiration and is made in all living cells.
Hydrogen peroxide is highly reactive / harmful and must be removed as soon as it is produced
in the cell. It is a by-product of many metabolic reactions, including the oxidation of fatty
acids in the peroxisome of cells. Catalase is an important enzyme that breaks down hydrogen
peroxide (H2O2) into water and gaseous oxygen, both of which are relatively harmless to cells.
Catalase must be present in peroxisomes to rid the cell of any harmful hydrogen peroxide. In
a study done on fibroblast cells, it was found that a lack of catalase in the peroxisome can
lead to neurological disorders stemming from peroxisomal diseases (Faruk G. Sheikh, K. P.
(1998). A deficiency in catalase can also lead to an increased risk of diabetes and a lower life
span (Laszlo Goth, 2000). If the intracellular concentrations of hydrogen peroxide are not
regulated by catalase the excess H2O2 can react with important cell components, degrading
organelles necessary for survival or acting as an intracellular messenger. This intracellular
messaging can create cascades of reactions that are harmful for cell survival.

All living things possess catalysts, or substances within them that speed up chemical reactions
and processes. Enzymes are molecules that enable the chemical reactions that occur in all
living things on earth. Catalase is used in the food industry for removing hydrogen peroxide
from milk prior to cheese production. Another use is in food wrappers where it prevents food
from oxidizing. Catalase is also used in the textile industry, removing hydrogen peroxide from
fabrics to make sure the material is peroxide-free.

2 H2O2 ® 2H2O + O2

Hereby, we would like to demonstrate catalase and hydrogen peroxide experiment to show
how enzymes act as catalysts by causing chemical reactions to occur more quickly within living
things. This investigation looks at the rate of oxygen production by the catalase in pureed
potato as the concentration of hydrogen peroxide varies. Using a potato and hydrogen
peroxide, we can observe how enzymes like catalase work to perform decomposition, or the
breaking down, of other substances. Catalase works to speed up the decomposition of
hydrogen peroxide into oxygen and water. We will also test how this process is affected by
changes in the pH of the potato. Is the process faster or slower when compared to the control
experiment conducted at room temperature?

Catalase and Hydrogen Peroxide Experiment

Materials
1 Potato (should be at room temperature)
• Hydrogen peroxide solution

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 • Small glass beaker or cup
Procedure

1) Divide the potato into five roughly equal sections. Chop and mash a small sample
(about a tablespoon) and place into beaker or cup.

2) Pour 4 ml hydrogen peroxide 1% into five labelled test tubes.

3) Put one drop of Floursecein dye ( for visual color) to each test tube.

4) Label 5 small vials as follows: pH3, pH5, pH7, pH9, pH11 and dilute catalase into the
appropriate vial as directed below:
pH 3: 0.5 mL catalase 1 Unit/ml or one small piece of potato +0.5 mL pH 3 Buffer
pH 5: 0.5 mL catalase 1 Unit/ml or one small piece of potato + 0.5 mL pH 5 Buffer
pH 7: 0.5 mL catalase 1 Unit/ml or one small piece of potato + 0.5 mL pH 7 Buffer
pH 9: 0.5 mL catalase 1 Unit/ml or one small piece of potato + 0.5 mL pH 9 Buffer
pH 11: 0.5 mL catalase 1 Unit/ml or one small piece of potato + 0.5 mL pH 11 Buffer

5) Incubate at room temperature for 30 minutes.

6) Add the potato with buffer to the tube of Hydrogen peroxide.

7) Carefully observe the air bubbles generated by the reaction.

8) Use stopwatch to note the duration of oxygen production/bubbling at different pH.

9) Plot the bar graph amount of oxygen produced (measure as duration of bubbling)
versus pH.

Observations & Results

Watch each of the potato/hydrogen peroxide mixtures and record what happens. The
bubbling reaction you see is the metabolic process of decomposition. This reaction is caused
by catalase, an enzyme within the potato. You are observing catalase breaking hydrogen
peroxide into oxygen and water. Which potato sample decomposed the most hydrogen
peroxide? Which one reacted the least? Why?

You should have noticed that the at different pH, there may be low to no bubbles. This is
because the pH inactivated the catalase enzyme, making it incapable of processing the
hydrogen peroxide.

+ + + extremely active bubbling; foamy

+ + good bubbling
+ moderate bubbling
0 nonreactive; no bubbling

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Conclusions

Catalase acts as the catalyzing enzyme in the decomposition of hydrogen peroxide. Nearly all
living organisms possess catalase, including us! This enzyme, like many others, aids in the
decomposition of one substance into another and the catalytic activity is dependent on the
availability of optimal conditions including favourable pH. Catalase decomposes, or breaks
down, hydrogen peroxide into water and oxygen, best at neutral pH.

Question?

At what pH potato produced the most bubbles?

Why potato has been used?

Why the bubbles are generated?

Why there is yellow colour in liquid?

Faruk G. Sheikh, K. P. (1998). Abnormality in catalase import into peroxisomes leads to server
neurological disorder. Proceedings of the National Academy of Sciences of the United States
of America, 2961 - 2966.

Laszlo Goth PhD, J. W. (2000). Hereditary catalase deficiencies and increased risk of diabetes.
The Lancet, 1820 - 1821.

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 Practical 2:
Aim 2a: Proteins isolation from natural sources

Introduction:

Protein sources: Plants and animals

Animal products, meat, milk, milk products, egg, poultry and fish are rich sources of proteins
containing a balanced level of amino acids. Plant food items like legumes and nuts are also
good sources of the same. Animal proteins and plant (vegetable) proteins are differentiated
as:

• Compared to plants, Animal protein sources are generally associated with high fat content
and, because of this, when consumed in large amounts, it leads to high risks of diseases,
including high blood pressure and heart diseases

• Animal protein has a balanced combination of all amino acids; hence, it is called complete
protein. In contrast, plant (vegetable) protein is incomplete protein; soybean protein is an
exception to this.

Vegetables, legumes and fruits are good sources of plant proteins. Legumes have a higher
content of protein than vegetables and fruits. Different parts of plants are sources of proteins.
In this practical, we will isolate proteins from plants. These procedures of protein extraction
have wide range applications including in the pharmaceutical industry.

Procedure: Isolation of proteins

1. Crush one gram of the desired natural source sample (leaves, vegetable, fruits, grains etc)
using a mortar and pestle.

2. Add 15 ml of buffer (20 mM Tris pH 7.4 and 150 mM NaCl).

3. Take out in a falcon tube or divide in 2 ml tube for centrifugation to remove the debris.
Centrifuge 15000 rpm for 15 minutes.

4. Transfer 10 ml of supernatant into another clean tube and put on ice.

5. Add 10 ml of 4 M ammonium sulphate, slowly on ice with gentle shaking in 10-15

minutes.

6. Centrifuge at 4 degree, 10000 rpm for 10 minutes.

7. Discard the supernatant carefully, add 100 ul of buffer to resuspend the pellet.

8. Take 10 ul of the sample for Bradford estimation of protein concentration. Add 390 ul of
buffer and 100 ul of Bradford solution, measure OD at 595 nm. Compare with the

15
calibration curve done for BSA in laboratory. Quantify the protein concentration in your
sample.

9. Save the sample in step 8 for next day at -20 degree.

10. Next day your instructor will run the 12 % SDS-PAGE gel followed by Coomassie stain for
protein visualization.

11. You can record the approximate molecular weights of proteins visualised in comparison
to standard protein molecular weight ladder.

16
Practical 2b: Experiment for DNA Extraction from natural source

DNA is the hereditary material in all eukaryotic cells. DNA isolation is a technique which is
the basic prerequisite for most of the genetic engineering technologies. Depending on the
type of DNA and the complexity of the organism from which it is isolated, the procedures
can be optimized for better yield, purity and integrity. In this practical course, we will isolate
DNA from plant source and will separate them by electrophoretic techniques.

Materials required

For this experiment you will need:

• Desired natural source of plant DNA

• Buffer 20 mM Tris pH 7.4 and 150 mM NaCl
• Dish soap
• 3 M sodium acetate pH 5.2
• 100 % cold ethanol
• 70 % ethanol
• Centrifuge
• Electrophoresis system

Procedure:

1. Crush one gram of the desire natural source sample (leaves, vegetable, fruits, grains etc)
in a mortal pistol.

2. Add 15 ml of buffer (20 mM Tris pH 7.4 and 150 mM NaCl). Add 2 drop of dish soap.

3. Take out in a falcon tube or divide in 2 ml tube for centrifugation to remove the debris.
Centrifuge 15000 rpm for 15 minutes.

4. Transfer 3 ml of supernatant in another clean tube, put on ice.

5. Add 0.33 ml of 3M sodium Acetate pH 5.2

6. Slowly add 7.5 ml of cold ethanol to the tube.

7. Centrifuge at 4 degree, 4000 rpm for 10 minutes (DNA precipitation).

8. Discard the supernatant carefully without dislodging the pellet and add 1 ml of 70 %
ethanol to the pellet.

9. Centrifuge again at 4 degree, 4000 rpm for 10 minutes (ethanol wash).

10. carefully discard the supernatant. Allow air dry for 15 minutes.
11. Resuspend pellet with 100 ul MilliQ water or TE buffer.
12. Take 1 ul of the sample for DNA estimation using Nanodrop spectrophotometer.,
Measure OD at 260 nm. Quantify the DNA concentration in your sample.
13. Save the sample in step 11 for next day at -20 degree.

17
14. Next day your instructor will run 1 % Agarose gel followed by DNA stain with EtBr for
DNA visualization.
15. You can record the DNA bands visualised and the approximate molecular weight in
comparison to standard DNA ladder.

Conclusion:

DNA from the desired source has been isolated and visualized by agarose gel
electrophoresis. It can be anything for example banana, oranges, watermelon, apples,
blueberries, blackberries, vegetables (potatoes, split green peas, spinach), grains (raw wheat
germ, oats), meat or even cheek cells from inside your mouth.

So amazing to see the DNA bands.

Questions for understanding:

1. Why can’t we see the DNA with eye? Because of size

2.Why do we use detergent? Because Detergent dissolves the lipids in the cell membranes
and nuclear envelope, releasing the DNA into the solution.

3. Why do we use 3M sodium Acetate pH 5.2 buffer ? Because DNA has a negative electrical
charge (due to the phosphate groups on the DNA backbone) and the electrical charge makes
it soluble. When the salt is added to the fruit mixture, the positively charged sodium ions
(Na+) of the buffer are attracted to the negative charges of the DNA, neutralizing the electrical
charge of the DNA. This allows the DNA molecules to come together instead of repelling each
other.

4. Why do we use alcohol? Because When molecules are soluble, they are dispersed in the
solution and are therefore not visible. When molecules are insoluble, they precipitate (clump
together) and become visible. DNA is soluble in water, which is why it is invisible in the filtered
mash solution. DNA is not soluble in the high salt and alcohol solutions, so the addition of the
alcohol makes the DNA precipitate. Molecules are less soluble at lower temperatures, so we
chill the alcohol to get more of the DNA to precipitate.

Questions ?

1. why banana has been crushed and dissolved in buffer?

2. what if other source is used other than banana?

3. do you think raw banana may have same results?

4. why centrifugation is done?

5. what is role of Amso4 salt?

6. what is role of Alcohol in DNA precipitation?

18
 Practical 3:
Microscopy

Aim3a: Visualization of Leishmania under microscope

Leishmaniasis:
Leishmaniasis is one of the most neglected vector-borne parasitic disease caused by
more than 20 species of the genus Leishmania and is transmitted via female sandflies to
mammalian hosts. Approximately 0.7 – 1.2 million new cases of leishmaniasis emerge in
almost 100 endemic countries per year (Georgiadou, Makaritsis et al., 2015). The vectors of
Leishmania parasites are dipterans of the genus Phlebotomus in the old continent and
Lutzomyia in the new world and both belong to subfamily of Phlebotominae. Diverse
Leishmania species cause different clinical manifestations of leishmaniasis which ranges in
severity.

i. Cutaneous leishmaniasis (CL) is one of the most prevailing form of leishmaniasis and is
characterized by the presence of skin ulcers on bare parts of the body, leading to life-long
scars and serious disability. This form of disease is mainly caused by Leishmania major,
Leishmania tropica, Leishmania mexicana and Leishmania peruviana (Burza, Croft et al.,
2018).
ii. Mucocutaneous leishmaniasis (ML) leads to fragmentary or total destruction of mucous
membranes of the mouth, nose, and throat. L. braziliensis is majorly responsible for causing
this disease but less often L. panamensis/ L. guyanensmay also cause the disease (Georgiadou
et al., 2015).

19
iii. Visceral leishmaniasis (VL) also recognized as kala-azar, is most fatal if not treated. The post-
kala-azar dermal leishmaniasis (PKDL) is a consequence of visceral leishmaniasis that emerge
as macular and nodular rashes usually on the face, trunks, upper arms and other parts of the
body. It is caused by Leishmania donovani and Leishmania infantum (Chappuis, Sundar et al.,
2007).
Treatment of leishmaniasis largely relies on the pentavalent antimonials,
amphotericin B and more recently on miltefosine. However, these drugs are very toxic, costly
and frequent resistance arises against these drugs (Haldar, Sen et al., 2011). In addition, there
is no appropriate diagnostic procedure available to detect the disease apart from the
detection of parasites by bone marrow puncture which requires hospitalization. Though,
vaccine is ultimate choice but no licenced vaccine is available. Therefore, major thrust is
provided by WHO to develop newer target against leishmaniasis. Thus, understanding of the
host-pathogen interactions and key intracellular processes can aid in the identification of
newer molecular targets for therapeutic intervention of leishmaniasis.
Life cycle of Leishmania:
Leishmaniasis is a disease caused by different species of protozoan parasite of the
genus Leishmania, which maintains its life cycle through transmission between an insect
(sandfly) and a mammalian host (human and dog). Leishmania exists in two forms:
promastigote (the flagellated, motile form) and amastigote (aflagellated form). During blood
meal of the sandfly the infective, non-dividing metacyclic promastigotes are regurgitated
along with various salivary components into the mammalian host.

Figure 1: Life cycle of Leishmania [Adapted from (Reithinger, Dujardin et al., 2007)]
Then these metacyclic promastigotes are phagocytosed by neutrophils or macrophages. In
macrophages, metacyclic promastigote establishes an intracellular niche and transform into
aflagellated amastigote. Amastigotes undergo replication inside the phagolysosomal
compartment having low pH and rupture the membrane leading to release of multiple
amastigotes allowing reinfection of other local macrophages. Transmission cycle is completed
when another sandfly ingests the amastigotes during the blood meal. These amastigotes
multiply into procyclic promastigote form in the midgut of sand fly. Procyclic promastigotes

20
migrate towards the stomodeal valve in the anterior midgut and reinitiate division into
infective metacyclic promastigote form (Kaye & Scott, 2011, Podinovskaia & Descoteaux,
2015).

Leishmania Giemsa staining protocol

1. Take 1 ml Leishmania culture in a 1.5 ml microfuge/eppendorf tube.

2. Centrifuge the tube at 7200 rpm for 2 min.
3. Dissolve the pellet in a 1 ml 1X PBS and centrifuge the tube at 7200 rpm for 2 min and
discard the supernatant (Twice).
4. Add 100 ul of 1X PBS to resuspend the pellet.
5. Take 5 ul of this resuspended culture onto a glass slide and make a circular smear with
the help of a 200-ul tip.
6. Let it air dry (Generally takes around 5 minutes).
7. Put the glass slide in a methanol containing slide holder chamber for 15 minutes.
8. Remove the slide from methanol solution and let it air dry (5 to 10 minutes)
9. Once it is dry, Add 1 ml of 1/10th or 1/5th dilution of Giemsa stain diluted in Giemsa
staining solution and incubate it for 15 minutes.
10. Wash the excessive stain by putting the slide in a chamber containing distilled water.
11. Once the excessive stain is washed off let it air dry.
12. Observe the slide under the microscope.

Materials required

1. Methanol Catalog number: 34860-1L-R Sigma Aldrich

2. Giemsa Catalog number: 48900-1L-F Sigma Aldrich
3. Slide holder
4. Cover slip
5. Glass slide
6. Buffer (Giemsa staining solution): 8 mM KH2PO4 (1.088 gram) and 6 mM Na2HPO4
(0.85 gram) for 1L.

21
 Practical 3b: Microscopy

Aim 3b: Observation of bacterial motility under microscope

Introduction:

Aim to perform motility test of a bacteria, by hanging drop preparation, to find out whether
it is motile or non-motile.

Purpose:

Motility means ability of movement by own power. Based on motility, bacteria can be
divided into two groups as follows.

(1) Motile Bacteria:

A bacteria, which has the intrinsic ability of movement in the surrounding medium, in which
it remains suspended, is a motile bacteria.

(2) Non-motile Bacteria:

A bacteria, which does not have the intrinsic ability of movement in the surrounding medium,
in which it remains suspended, is a non-motile bacteria. Non-motile bacteria may show
apparent motility, resulting from their brownian movement caused by the bombardment of
the water molecules in the surrounding medium, on the bacteria cells.

In wet mount, though the shape and size of bacteria can be observed, motility may be
hampered, as the suspension is pressed between the slide and the cover slip. That is why;
hanging drop preparation or motility test is performed for clear observation of the motility of
bacteria, besides their shape and size. It is useful in the identification of bacteria.

Principle:

A very small drop of bacteria suspension is hung from the center of a cover slip into the cavity
of a cavity slide. The hanging drop is observed under a microscope using oil-immersion
objective. If the bacteria are motile, its cells can be seen to have erratic movement in the
surrounding medium.

In contrast, if it is non-motile, its cells remain static in the medium without any movement or
may show brownian movement resulting from the bombardment by the water molecules in
the medium, on the bacteria cells.

Materials required:

Cavity slide, cover slip, petroleum jelly or Vaseline, immersion oil, broth culture of bacteria,
loop and microscope (compound, dark-field or phase-contrast).

22
Hanging drop preparation:

Procedure:

1. Take a clean glass slide and apply paraffin ring, adhesive tape ring to make circular
concavity. (This step is not needed if a glass slide with depression is available).
2. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a
toothpick.
3. Place a loopful of the broth culture to be tested in the center of the prepared
coverslip.
4. Turn the prepared glass slide or concavity slide upside down (concavity down) over
the drop on the coverslip so that the vaseline seals the coverslip to the slide around
the concavity.
5. Turn the slide over so the coverslip is on top and the drop can be observed hanging
from the coverslip over the concavity.
6. Place the preparation in the microscope slide holder and align it using the naked eye
so an edge of the drop is under the low power objectives.
7. Turn the objective to its lowest position using the coarse adjustment and CLOSE THE
DIAPHRAGM.
8. Look through the eyepiece and raise the objective slowly using the coarse adjustment
knob until the edge of the drop is observed as an irregular line crossing the field.
9. Move the slide to make that line (the edge of the drop) pass through the center of the
field.
10. Without raising or lowering the tube, swing the high dry objective into position (Be
sure the high dry objective is clean).
11. Observe the slide through the eyepiece and adjust the fine adjustment until the edge
of the drop can be seen as a thick, usually dark line.
12. Focus the edge of the drop carefully and look at each side of that line for very small
objects that are the bacteria. The cells will look either like dark or slightly greenish,
very small rods or spheres. Remember the high dry objective magnifies a little less
than half as much as the oil immersion objective.
13. Adjust the light using the diaphragm lever to maximize the visibility of the cells.
14. Observe the cells noting their morphology and grouping and determine whether true
motility can be observed.
15. Brownian movement should be visible on slides of all the organisms, but there should
also show true motility.
16. Wash the depression slide and after soaking in lysol buckets or discard the prepared
glass slide.

Note: While examining living organism for the property of active locomotion, it is essential
to distinguish true motility, whereby the organism move in different directions and change
their positions in the field, from either

23
 § Passive drifting of the organisms in the same direction in a convectional current in the
fluid or
§ Brownian movement, which is an oscillatory movement about a nearly fixed point
possessed by all small bodies suspended in fluid and due to irregularities in their
bombardments by molecules of water.

Uses: Presumptive diagnosis of Vibrio Cholerae by identifying bacteria with darting motility
from rice watery stools.

Figure: Hanging drop preparation

Source: https://microbeonline.com/

24
Aim3c: Visualization of cells from plants and observe their behaviour in different solutions.

Introduction

Biologists frequently study the onion cell because onions are readily available and their cells
provide a clear view of all the basic characteristics of plant cell structure. The onion's large
cells can be seen easily under a microscope and also used to learn the fundamentals of cell
biology. The skin (or epidermis) between the dormant leaves of an onion are a single cell thick,
and serve as a classic representation of the internal structure of plant cells. In fact, the term
"cell" came from a pioneer of microscopic biology, Robert Hooke, while looking at epidermal
onion cells under a microscope. He thought these structures resembled the cells that monks
would sleep in.

The onion's cell walls, like those of other plants, are rigid. Cellulose in the cell walls forms
clearly defined polygonal structures.

Water within the cell walls gives the walls strength and helps plants resist the force of gravity.
The cell's cytoplasm and vacuole contribute to the onion's solidity and its characteristic crisp
texture.

Below the cell wall is a layer of liquid called the cytosol, primarily composed of water, salts
and organic molecules. The cytosol also contains organelles, organic structures that manage
all the elements of cell metabolism. The cytosol also carries inclusions, which are starches,
proteins and other elements that act as building blocks for a number of functions. The
nucleus, also found in this cytoplasmic layer, contains the plant's basic genetic material.

The onion has a large vacuole, which contains water and ions and produces the onion's
distinctive aroma and taste.

It is likely (though not impossible) that you will not find chloroplasts in an onion cell. Onions
are underground storage organs, which consist of modified leaves emerging from a very short
stem. This morphology allows vegetative organs of onions to persist underground through
cold winters. In spring, the modified leaves undergo rapid cell division and green leaves
emerge to the surface. It may be difficult to detect chloroplasts within your onion cells.
However, if you look closely you will see small, emerald-coloured organelles within some
cells. These are the dormant chloroplasts, which in spring rapidly replicate producing the
green color of the emerging leaves.

25
Protocol and Procedure:

1. Prepare onion wet mount.

2. Quarter an onion.
3. Isolate a single onion leaf.
4. Break leaf in half and pull thin layer of epidermis, being careful to not let it fold on
itself. If it does fold, attempt to unfold it. You may want to use forceps.
5. Place the flattened onion epidermis on a microscope slide.
6. Place a small drop of iodine on the epidermis. Iodine will stain the nucleus of the cell
so it is visible.
7. Place cover slip on top of the specimen.
8. With a paper towel, remove any excess iodine.
9. Place wet mount on microscope.
10. With the scanning objective (4x), focus the specimen and locate a section where the
epidermis is a single cell thick. This is usually found on the edges of the specimen.
11. Switch to low power objective (10x). Focus in on a section of the epidermis where it is
a single cell thick.
12. Switch to high power objective (40x) and focus, only with the fine focus knob.
13. Draw a representative onion epidermal cell identifying the following structures: cell
wall, cell membrane, nucleus, and nuclear membrane.
14. Identify whether your onion cells have chloroplasts or not, and discuss why?

26
Practical 3d:

Aim: Visualization of animal cells by microscopy

Cheek cells are epithelial cells that line the interior surface of our mouths. The base layer of
cells in an epithelial structure are not actually cells, but a sticky layer on which the cells
anchor. The other surface of the epithelial cell touches the outside world (like skin) or an open
space (like the mouth). Because of their high rate of division, epithelial cells are found tightly
packed together. When you stain your cheek cells, you should be able to distinguish between
the nucleus, cytoplasm, cell membrane. If you are very observant (and lucky) you may
visualize the nucleolus and other organelles with in the cell.

Requirements:

microscope slides
Microscope cover slips
Dropper
Blotting paper/Tissue paper
Microscope

Protocol and Procedure:

1. Using a flat toothpick, very gently scrape the inside of your cheek to obtain cheek
cells.
2. Spread the cells on the end of the toothpick onto the microscope slide.
3. Place a cover slip on the sample. Press down on the coverslip and remove excess
methylene blue with a paper towel.
4. Using the scanning objective (4x), focus the specimen and locate cheek cells. Change
the objective to low power, refocus on a few cheek cells.
5. Finally, visualize your cheek cells at high power. At this point you may need to reduce
your light intensity and adjust the condenser aperture.
6. Once the cells have been found, they can then be viewed at higher magnifications.
You can put different solutions and see how mammalian cells respond to hypotonic,
isotonic & hypertonic Solutions.

27
Results:

Draw diagrams for the cells

What you have observed?
What are changes observed due to different solutions?

Large irregularly shaped cells with distinct cells.

A distinct nucleus at the central part of each individual cell (dark blue in color).
A lightly stained cytoplasm in each cell.

Questions:

Why do we have to Stain the Cells?

The cell has different parts, and those that can absorb stains or dyes are referred to as
chromatic. Having absorbed the stain, these parts of the cell become more visible under the
microscope and can therefore be easily distinguished from other parts of the same cell.

Without stains, cells would appear to be almost transparent, making it difficult to

differentiate its parts.

Methylene blue has a string affinity for both DNA and RNA. When it comes in contact with
the two, a darker stain is produced and can be viewed under the microscope.

The nucleus at the central part of the cheek cell contains DNA. When a drop of methylene
blue is introduced, the nucleus is stained, which makes it stand out and be clearly seen under
the microscope.

Although the entire cell appears light blue in color, the nucleus at the central part of the cell
is much darker, which allows it to be identified.

Other similar fun experiments - Onion Cells , Sugar Crystals, Cork Cells, Taking a look at leaves
and Hair Under the Microscope etc.

28
 Practical 4:
Aim4a: Gene amplification by PCR reaction

Introduction:

Polymerase Chain Reaction (PCR) is used in all areas of biological science research, including
the clinical, forensic and diagnostic fields and the widespread adoption of the PCR technique
has revolutionized life science research (Nobel Prize in Chemistry-1993 to Dr. Karry B. Mullis).
PCR is a powerful biochemical technique that enables large-scale amplification of very small
quantities of DNA. PCR is an efficient method for multiplying specific DNA sequences that was
isolated from a single human cell can be screened for mutations associated with various
genetic disorders.

In 1971, Kjell Kleppe and the Nobel laureate, Gobind Khorana, published a description of a
technique that represented the basic principles of a method for nucleic acid replication: “...
The DNA duplex would be denatured to form single strands. This denaturation step would be
carried out in the presence of a sufficiently large excess of the two appropriate primers. Upon
cooling, one would hope to obtain two structures, each containing the full length of the
template strand appropriately complexed with the primer. DNA polymerase will be added to
complete the process of repair replication. Two molecules of the original duplex should result.
The whole cycle could be repeated, there being added every time a fresh dose of the enzyme.
It is however, possible that upon cooling after denaturation of the DNA duplex, renaturation
to form the original duplex would predominate over the template-primer complex formation.
If this tendency could not be circumvented by adjusting the concentrations of the primers,
clearly one would have to resort to the separation of the strands and then carry out repair
replication. After every cycle of repair replication, the process of strand separation would have
to be repeated”.

The PCR approach was used to screen fertilized human embryos prepared from sperm and
ova from a couple that were carriers of the cystic fibrosis. This genetic disease results from
mutation in the CFTR gene located on chromosome seven. The DNA isolated from a single
embryonic cell was subjected to amplification and then screened for mutations in one of the
two copies of chromosome seven. Embryos that had inherited at least one copy of the wild
type gene were selected for implantation into the mother. Therefore, the couple could be
assured that they would have a child that would not be at risk from cystic fibrosis.

PCR can also be used to detect the presence of unwanted genetic material, as in the case of
a bacterial or viral infection. Conventional procedures that involve culturing microorganisms
or antibodies use can take weeks to complete. PCR offers a quick and easy alternative. In the
diagnosis of AIDS, PCR can be used to detect the small percentage of cells infected with HIV-
1. Following amplification and gel electrophoresis, the presence of an appropriate sized PCR
product indicates the presence of HIV-1 sequence and therefore, HIV infection.

The major advantage of PCR is that any target sequences can be amplified from a single copy
of starting material, even when the template is degraded and contaminated with inhibitors.

29
For example, studies have been performed on DNA extracted from ancient Egyptian
mummies to identify the members of King Tutankhamun’s family.

PCR is a very powerful amplification tool so very little DNA is actually required (usually in the
pg range). To copy DNA, all polymerases require a short sequence of nucleotides to provide a
free 3’OH group. Within cells of most organisms enzymes unwind the duplex and then RNA
polymerase adds priming nucleotides. This priming allows the attachment of DNA polymerase
from where extension can occur. In PCR however, there is a different mechanism of
replication. PCR requires that the flanking sequences of the target DNA be known, so that
single stranded synthetic oligonucleotide primers can be generated. Once these have
annealed to the target DNA the polymerase can synthesize the rest of the chain. Primers can
be specific to a particular sequence, or they can be universal to sequences that are very
common within a DNA molecule allowing for a wide variety of DNA templates

Deoxyadenylate (A), deoxythymidylate (T), deoxyguanylate (G) and deoxyctidylate (C) are
components of the reaction mixture that are the basic building blocks of DNA required for the
synthesis of the primers and for their extension. Note that both the primers and the
deoxynucleotides are added to the mixture in excess.

The most commonly used polymerase in PCR is referred to as Taq-polymerase. This enzyme
was originally found in the 1970's from the thermophilic bacterium Thermus aquaticus.
Before its discovery, PCR was a much longer and costlier process since the thermosensitive
E.coli DNA polymerase used lost its activity during the heating process and fresh enzyme had
to be added at every cycle. Owing that T. aquaticus lives in a hot environment (approx. 75°C),
the enzymes that power its internal metabolism have adapted to these conditions resulting
in heat-stability. This polymerase will therefore, retains its activity when the mixture is heated
and can be used for cycle after cycle. There are some other polymerases also like Phusion
polymerases.

Magnesium ions are cofactors used in PCR specifically by the polymerase. They affect the
annealing of the oligonucleotides to the template DNA by stabilizing the interaction, and also
stabilize the replication complex of the polymerase with the template prime.

A typical PCR reagent mixture is added to a microfuge tube as follows:

• Template DNA
• Forward and Reverse Oligonculeotide Primers
• dNTPs ¾Thermostable DNA Polymerase
• Magnesium Chloride or Magnesium Sulfate
• Other Reagents

The microfuge tube (containing your mixture) is placed into a machine called a thermocycler.

30
First Stage of PCR - Denaturation

The two anti-parallel strands of the DNA double helix are held together by hydrogen bonds
between the complementary bases. The heating process provides enough energy to disrupt
this bonding causing the DNA to "unzip" or denature to single-strands. Note that in reverse
(if the DNA is allowed to cool) the bonds reform and the DNA will renature back to the original
double helix conformation. The thermocycler is set to heat at 95°C for 30 to 90 seconds.

Second Stage of PCR - Annealing

This stage involves the annealing of the oligonucleotide primer sequences to the template
DNA. The primers cannot bind to the target DNA at the high temperature used in the first
stage of PCR, so the microfuge tube is cooled to allow double- strands to form again.

The thermocycler is set to 55-60°C for about 30 to 120 seconds.

The target strands remain denatured since they are at too low a concentration to encounter
each other during this stage, but the primers are at a very high concentration and so hybridize
with their complementary sequences readily. Reassociation of the parent DNA is therefore
successfully avoided.
The annealing temperature is a dynamic variable that affects the yield and specificity of PCR.
To increase the yield of PCR the annealing temperature must be decreased (with a reduction
in specificity). Conversely, to increase the specificity of PCR the annealing temperature must
be increased (with a reduction in yield).

Third Stage of PCR - Extension

The third and final stage of PCR involves the extension of the primed sequences of the target
DNA.
The thermocycler is set to raise the temperature of the microfuge tube to 72°C, which is the
optimal functioning temperature of the Taq polymerase.
Taq polymerase then binds to the 3’ end of the primers and begins adding dNTPs one at a
time as dictated by the template. Extension occurs in the 5' to 3' direction on the growing
strand right up to the end (where the polymerase falls off) until there are 2 partial copies of
the original DNA fragment.

31
The incubation period for this stage depends on the size of the target sequence to be
polymerized and the polymerase used. For example Taq requires 60 seconds per kb of
expected product, therefore this stage would take 3 minutes for a 3kb sequence. This
completes one PCR cycle.

Recycling PCR
After the third stage the mixture is heated again to melt the newly formed duplexes and the
whole process begins again. After every completed cycle newly synthesized DNA strands can
serve as templates for the next cycle. The cycles are generally repeated 30 times. After the
last cycle, an incubation period for 10 minutes at 72°C is performed to allow for any
uncompleted polymerization and then the product is cooled down for analysis or storage.
Since the DNA content is amplified in a logarithmic fashion, theoretically 1 billion copies could
be formed in 1 hour. In practice however, (allowing for the time it takes for the thermocycler
to change temperatures and for the last completion cycle) 1 million copies are ready in 3
hours.

Lastly, before you can analyze your PCR products they must be separated. This employs a
biomolecular separation technique called gel electrophoresis.

Reagents and equipment required for this experiment:

• DNA Template
• Forward and Reverse Oligonucleotide Primers
• dNTPs
• Thermostable Taq-polymerase
• Magnesium Chloride
• Milliq water
• Microfuge tubes
• Thermocycler
• Agarose gel electrophoresis apparatus

Procedure:

Today you will be setting up PCR reactions designed to amplify a gene from yeast encoding
an enzyme known as Glutaminyl cyclase (QC).

32
In mammals this enzyme is responsible for the cyclization of N- terminal glutamine residues
to pyroglutamic acid in biologically active peptides such as thyrotropin releasing hormone and
corticotropin releasing hormone.

The source of DNA will be yeast (Saccharomyces cerevisiae) genomic DNA. It will be supplied
to you at a concentration of 25 ng/μL.
The primers will be supplied at 100 ng/μL and the Taq Polymerase will be supplied at 5
Units/μL.
10x ThermoPol® buffer: 20 mM Tris--HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1%
Triton X--100, pH 8.8.

Each group is to set up one reaction as follows:

30 μl sterile water
3 μl 5’ primer (from 100 nM stock)
3 μl 3’ primer (from 100 nM stock)
2 μl template DNA (from 25 ng/ μL stock)
5 μl dNTP mix (from 10mM stock)
1 μl MgSo4 (from 100 mM stock)
5 μl buffer (10 X stock)
1 μl Taq DNA Polymerase ( from 5 Unit/ μL stock)
50 μl total
You can make master mix and divide 10 ul each ,
Or scaledown the reagents composition to make final volume 10 ul.
( you may need to dilute the Taq Polymerase buffer also )
The tubes will be placed in the thermal cycler.
Run program on thermocycler:

Step 1: 95˚C, 1min

Step 2: 95˚C, 30 sec
Step 3: 55˚C, 30 sec
Step 4: 72˚C, 1 min
Step 5: go to step 2, 35 times
Step 6: 72˚C, 10 min

The samples will be stored at –20 ˚C for next day

or immediately run on an 1% agarose gel.

Your instructor will set for DNA gel horizontal electrophoresis apparatus, run at 80 volts for
40 minutes, visualize under Gel doc unit.

Observation:
You will be able to see the gene amplified and relative molecular weight of DNA in compare
to DNA marker.
Questions?
Why does the specific DNA has some fixed length?
Why we are unable to see the DNA?

33
Practical 4 b:

Aim: Gene manipulation in genetic engineering by restriction enzymes

Introduction:

How can we take genes from one organism and connect them with genes from another
organism? Why would we want to do this? How is DNA analyzed and manipulated using
restriction enzymes? How DNA sequences can be mapped and characterized, such as in the
Human Genome Project and how DNA is cut and arranged during genetic engineering?

Using restriction enzymes, we can cut DNA from any organism and then ligate it together (e.g.
the ampicillin resistance gene and the luciferase gene into the plasmid backbone). This is
extremely useful if you want to have an organism make a protein it doesn't normally make
(e.g. bacteria making insulin). If we take the human insulin gene, cut it out of the human
genome using restriction enzymes, ligate it into a plasmid with the ampicillin resistance gene,
and transform bacteria with this plasmid, then all the ampicillin resistant bacteria will be
making insulin that can be used to treat diabetes.

Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA,
found in bacteria. As they cut within the molecule, they are commonly called restriction
endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence
called Restriction sites to generate a set of smaller fragments.

Restriction enzymes form part of the restriction-modification system of bacterial cells that
provides protection against invasion of the cell by foreign DNA– especially bacteriophage
DNA. But the cells own DNA is not cleaved by these Restriction enzymes. This self-protection
is achieved by the help of the specific DNA methyltransferase enzyme which methylates the
specific DNA sequence for its respective restriction enzymes by transferring methyl groups to
adenine or cytosine residues to produce N6-methyladenine or 5-methylcytosine. An
interesting feature of restriction endonuclease is that they commonly recognize recognition
sequences that are mostly palindromes - they shows the same forward (5' to 3' on the top
strand) and backward (5' to 3' on the bottom strand) sequences. In other words, they are
nucleotide sequences or complimentary strands that read the same in opposite direction.

Restriction enzymes generate three types of DNA ends, all possessing 5´-phos-phate and 3´-
hydroxyl groups:

a) Cohesive 5´ ends:- For example, ends generated by EcoR I:

34
b) Cohesive 3´ ends:- For example, ends generated by Pst I:

c) Blunt ends:- For example, ends generated by Hae III

Sticky ends (Blunt ends) are produced by cutting the DNA in a staggered manner within the
recognition site producing single stranded DNA ends. These ends have identical nucleotide
sequence and are sticky because they can bind to complementary tails of other
DNA fragments cut by the same Restriction enzyme.

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Applications:

Restriction enzymes are powerful tools of molecular genetics used to:

o Map DNA molecules
o Analyze population polymorphisms
o Rearrange DNA molecules
o Prepare molecular probes
o Create mutants
o Molecular cloning

Factors affecting Restriction Enzyme Activity:

Temperature: Most digestions are carried out at 37°C. However, there are a few exceptions
e.g., digestion with Sma I is carried out at lower temperatures (~25°C), while with Taq I at
higher temperature i.e., 65°C. As discussed previously, enzymes work at optimal
temperatures in the physiological settings. Owing to this reason, an enzyme isolated from
E.coli which lives in human intestine has 37°C optimal temperature, but Taq I isolated from
the bacterium Thermus aquaticus, which lives in hot springs have a much higher optimal
temperature.

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Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation mixtures,
which is temperature dependent. Most restriction enzymes are active in the pH range 7.0-
8.0.

Ionic Conditions: Mg2+ is an absolute requirement for all restriction endonucleases, but the
requirement of other ions (Na+/K+) varies with different enzymes.

Methylation of DNA: Methylation of specific adenine or cytidine residues within the

recognition sequence of the restriction enzyme affects the digestion of DNA.

Reagents and equipment:

• Circular plasmid DNA

• Restriction enzymes EcoRI and BamHI
• Cutsmart buffer
• MilliQ water
• Microfuge tubes
• Stopwatch
• 37 degree water bath
• Agarose gel electrophoresis system

Procedure:

1. Label your microfuge tubes as : C-control, SD-single digestion, DD-double digestion

2. Take 5 µl of the circular plasmid DNA in all three tubes
3. add 4 µl , 3 µl and 2 µl of water in tubes C, SD and DD respectively.
4. Add 1 µl of cutsmart buffer in all tubes.
5. Add no restriction enzyme in tube C, while 1 µl of BamHI in tube SD, 1 µl of BamHI and 1 µl
of EcoRI in tube DD.
6. Mix well with pipette
7. Incubate at 37 degree water bath for 1 hour.
8. You can store the reactions at -20 degree or run immediately.
9. run 1 % agarose gel and visualise in gel documentation unit

Questions:
1. why there is circular DNA?
2. why one enzyme shifts the band position up?
3. why there are two bands in DD tube?
4. what is the size of smaller and bigger fragments?

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 Practical 5 :
Chromatography

Aim 5 a: Biomolecule separation by size exclusion chromatography at Physiological pH 7.4

Introduction

Proteins are polypeptides that fold into unique structures suited to perform specific tasks.
The special biological properties that each protein possesses are conferred by the three-
dimensional structure and by the distribution of hydrophobic, nonpolar, positive and negative
charged regions on the surface and in active site crevices. Biochemists make use of these
characteristics to separate a given protein from a mixture of cellular components. Whenever
one isolates a protein, there are several characteristics to determine, including the specific
activity, molecular weight, the size, shape and subunit composition.

There are four major types of liquid chromatography - size exclusion, ion exchange,
hydrophobic interaction and affinity chromatography. Each of these methods offers selected
advantages. Size exclusion chromatography is often used to analyze the proteins in a mixture
on size basis, However ion exchange chromatography separates proteins on the basis of
charge (anion or cation exchange chromatography). Proteins that bind to specific ligands can
be isolated by binding to polymer beads containing covalently bound ligands, a technique
termed affinity chromatography.

Size exclusion chromatography (also called gel filtration chromatography or gel permeation
chromatography) is a method of separating molecular mixtures on the basis of their
distribution between a mobile and a stationary phase. The stationary phase is made of
spherical particles, such as cross-linked dextrans ("Sephadex") or dextran-agarose
copolymers ("Superdex"), of a particular size and porosity that are packed in a column. The
mobile phase is the buffer which is passed through the column. The primary separation
process is the diffusional partitioning of solute molecules between the mobile solvent phase
and the interior solvent spaces of the gel particles. Thus, molecules are fractionated on the
basis of their size and shape. “Size” refers to Stokes radius. Small molecules can diffuse into
pores in the gel particles and their movement through the column is retarded; large
molecules, on the other hand, are excluded from the gel particles because their size is larger
than the pore size of the particles and they move rapidly through the space between the gel
particles. In general, the elution position of a molecule is proportional to its apparent size,
with large molecules eluting first and small molecules at greater elution volumes

The total volume (Vt) of a gel filtration column is given approximately by :

Vt = V0 + Vi
where Vo, the void volume, is the volume of solvent outside the gel particles, and Vi, the
internal volume, is the volume inside the gel beads. A molecule that is completely excluded
from the gel beads elutes at a volume equal to V0. Small molecules (such as sugars and buffer
salts) that freely diffuse into the pores of gel beads elute as volume Vt has passed through
the column. Molecules with intermediate sizes elute at elution volumes, Ve, between V0 and
Vt

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Figure 3-1. Schematic diagram of a column containing inert beads with pores of various sizes
that admit or exclude solute molecules on the basis of size for separation by "size exclusion
chromatography". Large molecules like blue dextran are excluded from the beads, large
proteins can enter large pores, small proteins can enter large or small pores and low
molecular weight solutes such as buffer or salt can diffuse freely throughout the beads and
the bulk solvent.

In this experiment, we are using haemoglobin as our model protein and we are characterizing
it by chromatography technique. Scientists have a keen interest in the elucidation of
molecular structure and its correlation with the function of proteins. Structural information
can be derived from cryo-electron microscopy, and the detailed coordinates of amino acid
residues can be obtained by x-ray diffraction of single crystals of proteins. Inside the cell,
proteins in solution interact with one another and react with substrates. Chromatography
offer the capability to separate proteins in solutions, closer to their native environment.
Moreover, proteins can be isolated in sufficient quantities for functional assays such as
enzyme activity.

In our experiment, a mixture of solutes will be separated by size exclusion on Sephadex G-25.
Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin
roughly 50 μm bead . It is supplied in its dry form. The gel swells in aqueous solutions.
Different types of Sephadex differ in their degree of cross-linking and hence in their degree
of swelling and their molecular fractionation range. Sephadex G-25 separates the
components of a sample into two groups, e.g. high molecular weight substances, which are
excluded from the gel and thus elute first, from lower molecular weight substances that enter
the pores and thus elute later. In this experiment we will use important molecule such as
Haemoglobin which is the oxygen carrier to all parts of body while Folic acid is crucial for
proper brain function and plays an important role in mental and emotional health.

In the case of blue dextran , haemoglobin and Folic acid, the molecules are colored blue, red
and yellow respectively and one can see them elute from the column visually. However,
most proteins do not exhibit a visual color. Instead, they absorb at 280 nm with an
extinction coefficient that is related to the number of aromatic amino acids in the protein.
As you collect fractions, you will need to monitor EACH fraction on the spectrophotometer

39
UV-VIS at 280 nm. You should then plot fraction number versus size of protein. Volume to
determine where the proteins elute, based on an increase in color or absorbance at 280 nm.

Materials:

• Biomolecule mixtures (Blue dextran 2000 kDa, Haemoglobin 64 kDa, Vitamin B9 folic acid
0.44 kDa)
• Column with gel filteration matrix superdex 200
• Buffer1: 50 mM Tris pH 7.4, 500 mM NaCl
• Buffer 2: 50 mM sodium Acetate buffer pH 5.0, 500 mM NaCl
• Test tube
• Microfuge tubes,
• stopwatch
• Spectrophotometer

Procedure:
Day 1

1) You will prepare your superdex column before the start of the lab as the gel needs
time to settle. You can clamp it to the stand in a buret clamp and pass buffer 1 about
20 ml in the column by gravity. This allows the beads to swell and get ready to their
full size before pouring.
2) Obtain 50 uL of each of Blue dextran, haemoglobin and folic acid (which were
prepared 20 mg/ml in buffer 1).
3) Mix these three molecules to give a total volume of 150 uL.
4) DO NOT LET THE COLUMN RUN DRY. Make sure that the level of the buffer at the
top of the column is very close to the top level of the gel. If not, drain the buffer
until it reaches the top of the gel.
5) Using a pipette P200, add your 150 uL sample to the top of the gel with the stopcock
closed. Do not disturb the gel at the top of the column.
6) Place about 12 microfuge tubes under the stopcock. Open the stopcock and allow
the sample to move onto the column.
7) Apply slowly 10 mL of buffer to the top of the column through pasture pipette.
8) Continue collecting volume into a different graduated microfuge tube until the first
appearance of blue color (from blue dextran) is seen to elute from the column.
Record this total volume as accurately as possible (Vo).
9) Collect all the blue dextran into some tubes. Remember to keep filling the buffer at
the top of the column gently—do not disturb the level of the column)
10) After all the blue dextran is past, record the total volume at this point. After some
time red haemoglobin protein should start coming. collect continuously stops until
the yellow folic acid elutes. Record this volume. Drain the column until all yellow
color is removed. Vt is the total volume collected (blue dextran plus Folic acid). Close
the stopcock
11) Make record for the elution volume of blue dextran, haemoglobin and Folic acid.

40
Day 2
Repeat all the steps as above but different pH 5.0
1) You will prepare your superdex column before the start of the lab as the gel needs
time to settle. You can clamp it to the stand in a buret clamp and pass buffer 2 about
20 ml in the column by gravity. This allows the beads to swell and get ready to their
full size before pouring.
2) Obtain 50 uL of each of Blue dextran, haemoglobin and folic acid (which were
prepared 20 mg/ml in buffer 2).
3) Mix these three two molecules to give a total volume of 150 uL.
4) DO NOT LET THE COLUMN RUN DRY. Make sure that the level of the buffer at the
top of the column is very close to the top level of the gel. If not, drain the buffer
until it reaches the top of the gel.
5) Using a pipette P200, add your 150 uL sample to the top of the gel with the stopcock
closed. Do not disturb the gel at the top of the column.
6) Place about 12 microfuge tubes under the stopcock. Open the stopcock and allow
the sample to move onto the column.
7) Apply slowly 10 mL of buffer to the top of the column through pasture pipette.
8) Continue collecting volume into a different graduated microfuge tube until the first
appearance of blue color (from blue dextran) is seen to elute from the column.
Record this total volume as accurately as possible (Vo).
9) Collect all the blue dextran into some tubes. Remember to keep filling the buffer at
the top of the column gently—do not disturb the level of the column)
10) After all the blue dextran has passed, record the total volume at this point. After
some time red haemoglobin protein should start coming. collect continuously stops
until the yellow folic acid elutes. Record this volume. Drain the column until all
yellow color is removed. Vt is the total volume collected (blue dextran plus Folic
acid). Close the stopcock
11) Make record for the elution volume of blue dextran, haemoglobin and Folic acid.
12) You can measure absorbance at 280 nm and plot graph for both days Absorbance
versus elution volume. Alternatively, The presence of color (the particular color as
well as the relative color intensity) was evaluated for each fraction either by visual
examination or by Bradford method.

Questions:

What do you observe?

What is the difference in elution volume of haemoglobin?

Why there is difference in elution volume?

41
 at pH 7.4

at pH 5.0

Conclusion:
In summary, we would like to study on the effect of pH on the structure and function of
human hemoglobin, and demonstrated with direct and convincing evidences that when the
environmental pH is away from normal physiological value, the tetramer haemoglobin would
easily dissociate into dimer by having electrostatic free energy advantage. The tetramer →
dimer dissociation is a α2β2→ 2αβ process and it is reversible if the environmental pH returns
to neutral value. When pH becomes more acidic and alkaline, such as in pH 5.0 and pH 9.0,
dimer Hb will further dissociate into monomer. The dissociation is accompanied with series
changes of protein structure, so that the secondary bond is unable to form between the
subunits to maintain a stable state of dimer, thus causing the dissociation of dimer and
inducing the ferrous iron transform to ferrate iron by peroxidization. Since the dissociation
process involves structure changes, even if the environmental pH returns to 7.4, it is not
reversible. The dissociated Hb is not able to adequately carry and release oxygen to the
tissues in circulation. Therefore, pH dependent Hb dissociation should be avoided in patients
and preserved blood.

Reference: PLoS One. 2013; 8(11): e81708. Pathway and Mechanism of pH Dependent
Human Hemoglobin Tetramer-Dimer-Monomer Dissociations

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 Experiment 6:
Practical 6 a: Antigen Antibody detection

Aim: To detect the presence of Salmonella genus which causes enteric or Typhoid Fever by
using qualitative slide agglutination test.

Introduction:

Widal test is a serological method to diagnose enteric fever or typhoid which is caused by the
infection with pathogenic microorganisms like Salmonella typhi,Salmonella paratyphi A, B and
C. This method of diagnostic test is based upon a visible agglutination reaction either in a test
tube or on a slide between antibodies of patient serum and antigens specifically prepared
from Salmonella sp.

Principle:

Enteric fever or typhoid is a life threatening disease which usually occurs due to the infection
of pathogenic microorganisms, e.g. Salmonella typhi, Salmonella paratyphi A, B and C. These
microorganisms are transmitted to human body through food and drinks contaminated with
fecal matter. Early diagnosis and treatment for this fever are essential to avoid serious clinical
complications. During the course of infection antibodies are produced against Salmonella
antigens. Widal test, a serological method for the detection of Salmonella sp., was developed
by F Widal in 1896. During this test a visible agglutination is formed due to the reaction in a
test tube or on a slide between antibodies present in the infected person's blood sample and
specific antigens of S. typhi and S. paratyphi. For the slide agglutination test, stained
Salmonella antigens are used to detect the presence of specific agglutinin in the patient’s
serum. The slide agglutination test is used as a primary screening procedure.
The organisms causing enteric fever possesses two major antigens namely somatic antigen,
O and a flagellar antigen, H along with another surface antigen, Vi. During infection antibodies
are produced in patient’s sera against Salmonella typhi O and H and Salmonella paratyphi AH
and BH antigens. During infection antibodies are produced in patient’s sera against these
antigens. Antigens specifically prepared from this organism are used in the agglutination test
to detect the presence of antibodies in patients’ sera which are elucidated in response to
infection by these bacteria. There are some agglutinins that are produced in the patient’s
serum during the fever period, which react with somatic antigen O of Salmonella typhi, A or
B of Salmonella paratyphi and then with flagellar antigen H which is common in most of the
Salmonella species. In this test four specific antigen suspensions are used e.g. Salmonella
typhi ( H antigen), Salmonella typhi (O antigen), Salmonella paratyphi - A and Salmonella
paratyphi - B. If agglutination occurs with O antigen then it is considered positive for
Salmonella typhi. If agglutination occurs in A or B antigen then it is confirmed as positive for
Salmonella paratyphi. Agglutination will occur in H antigen circle for all the cases of antigens
like O, A, and B. Salmonella species are characterized by three antigens present on the cell,
as shown in this figure.

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 Antigenic structure of salmonella

O Antigen: This is a somatic antigen and is present on the outer membrane of the cell. Its
specificity is determined by the nature of the repeating units in the outer O-polysaccharide
chain. Somatic antigens are heat stable, alcohol resistant and forms compact and granular
clumps when mixed with O antisera.

Vi antigens: This is a virulence antigen which is a capsular polysaccharide that overlays the O
antigen. This capsule is not necessary for infection but it increases the infectivity by making it
less detectable by the body’s immune system. It is heat labile and can be detected using Vi
antisera. Vi antigen can interfere with O antigen testing.

H Antigens: This is a heat labile flagellar antigen which is inactivated both by boiling and
alcohol. H antigens rapidly form fluffy clumps when treated with the corresponding antisera.
H antigen induces rapid formation of corresponding antibodies as it is strongly immunogenic.

Widal Test Teaching Kit (Slide Test) utilizes the principle for rapid slide agglutination to detect
the presence of Salmonellatyphi and paratyphi. The kit gives direct results in form of visible
agglutination.

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45
Diagrammatic representation of Widal slide test

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47
Aim 6b: Antigen Antibody interaction during diagnostic applications

Introduction:

The key reaction of immunology and immune defence is the interaction of antibodies and
antigens. This interaction is responsible for the body’s defence against viral and bacterial
infections and other toxins. The body’s defence mechanism recognizes foreign substances,
or antigens, and raises specific antibodies against them. Antibodies are proteins produced by
cells of the immune system in response to the exposure of an individual to a foreign substance
(an antigen).

The antibodies bind to the antigens and form large macromolecular complexes. Large
macromolecular complexes are formed due to the fact that each antibody can associate and
bind with more than one antigen and each antigen can be bound by more than one antibody
molecule. The formation of the large macromolecules results in their precipitation and the
resulting precipitate is cleared by the body by various mechanisms. The interaction of antigen
and antibody, resulting in precipitation, is also useful in research and diagnostics.

The specificity of antigen-antibody interactions has led to the development of a variety of

immunologic assays, which can be used to detect the presence of either antibody or antigen.
Immunodiffusion in gels encompasses a variety of techniques, which are useful for the
analysis of antigens and antibodies. An antigen reacts with a specific antibody to form an
antigen-antibody complex, the composition of which depends on the nature, concentration
and proportion of the initial reactants.

Enzyme linked immunosorbent assay or ELISA is a sensitive immunological technique to

detect the presence of a specific antigen (Ag) or antibody (Ab) in a biological sample. It utilizes
the dual properties of antibody molecules being specific in reactivity and their ability to be
conjugated to active molecules such as enzymes. An enzyme conjugated with an antibody
reacts with a chormogenic colourless substrate to generate a coloured reaction product.
ELISA is extensively used for diagnostic purpose which utilizes the dual properties. It requires
an immobilized antigen/antibody bound to a solid support (e.g. microtitre plate or
membrane). There are different types of ELISAs for the detection of a protein of interest in a
given sample. One of the most common ELISA is dot ELISA which can visually detect the
presence of an antigen very quickly. The nitrocellulose dot technique was first developed for
screening large number of hybridoma antibodies in 1983.

Principle:
There are various forms of ELISA for the detection of antigen or antibody based on antibody-
antigen interactions. Dot ELISA, a qualitative ELISA test, can be performed very quickly with
the end detection done visually. Because of its relative speed and simplicity, the dot ELISA is
an attractive alternative to standard ELISA. In Dot-ELISA, small volumes of antibodies are
immobilized on a protein binding membrane (Nitrocellulose) and the other antibody is linked
to an enzyme Horse radish peroxidase (HRP). The test antigen at first reacts with the
immobilized antibody and later with the enzyme-linked antibody. The amount of enzyme
linked antibody bound is determined by incubating the strip with an appropriate substrate
(Hydrogen peroxide, H2O2) and a chromogen [Tetramethylbenzidine (TMB)]. HRP acts on
H2O2 to release nascent oxygen, which oxidizes TMB to TMB oxide, which gives, a blue

48
coloured product. The latter precipitates onto the strip in the area of enzyme activity and
appears as a coloured dot, hence the name Dot-ELISA. The results can be visualized in naked
eye. The enzyme activity is indicated by intensity of the dot, which is directly proportional to
the antigen concentration.

In this figure for Dot ELISA, an antibody is immobilized on a membrane and the test antigen
is first allowed to react with immobilized antibody and then to the HRP-labeled antibody. The
amount of HRP-labeled antibody bound is measured by treating the membrane strip with an
appropriate chromogenic substrate which is converted to a coloured precipitate and appears
as a dot on the membrane

We are going the perform the antigen antibody interaction studies using a kit.

This kit can be used to detect the presence of a test antigen by immobilized antibody bound
to the membrane followed by binding of the antigen to the labeled secondary antibody and
its detection by using appropriate substrate.

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Laboratory reports (for wet labs): Instructions and Formats

• Labsare in two week cycles - e.g. you spend 2 weeks doing experiments
doing one experiment, you can move to next experiment.

• If you miss a lab, there is NO make-up. Just continue with the experiments
that are already going on as per regular schedule. With proper focus and
effort, you will learn what has to be learnt!

• Youneed to make a proper lab record notebook. There will be marks on

the lab record notebook.

• Lab reports must be submitted ONLY in the formats given.

Aim, Methods, Results, Discussions

• Lab reports
must be submitted to YOUR TAs AFTER completion of the first
experiments. For example, if you are doing buffer experiments for 2
weeks, then in the 3rd week you come for your lab to start the Bradford
experiment and submit your lab reports to your buffer TAs.

• TAs will NOT accept lab reports before or beyond YOUR two hour lab
window. For example, if your lab group is Mon 1300-1500 hrs and you
finish a 2 week cycle, then TAs will accept your lab report ONLY within
the first 15 minutes of your next lab - not before 1300 hrs and not
beyond 1315 hrs.

54