Principles of Biology (BST 11052)

Year I Semester I
Practical No 1:
Laboratory safety, biological and chemical waste disposal and handling of laboratory
equipment
Course coordinator: Dr. Sarath Bandara

Background
This practical aims to educate students on how to work safely in a laboratory avoiding health
risks and accidents, how to dispose biological and chemical waste materials avoiding or
minimizing the environmental pollution and risks, and how to handle laboratory equipment
properly avoiding damages to both user and equipment itself.

Students should be able to:

• Demonstrate proper laboratory safety practices

• Explain laboratory safety signs and guide lines
• Demonstrate correct handling of laboratory equipment
• Demonstrate safe disposal of biological and chemical waste
• Demonstrate precautions and safety activities to avoid laboratory hazards
• Demonstrate ability to act appropriately in case of emergencies

General safety guidelines

• Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses
are prohibited within the laboratory.
• Food and drink should not be stored in laboratory refrigerators, freezers, cabinets, or on
shelves, countertops, or bench tops.
• Running or jumping in the laboratory is not permitted
• Stored items or equipment shall not block access to the fire extinguisher(s), safety
equipment, or other emergency items
• No student should work alone in the laboratory
• Lab jacket or coat should be worn in the laboratory to protect from splashes and spills of
chemicals and contact with other harmful materials (Lab jackets or coats should have snap
fasteners rather than buttons so that they can be readily removed)
• Long hair, ties, scarves and earrings should be secured.
• Mouth pipetting is never allowed
• Wear shoes that adequately cover the whole foot
• Keep pens and pencils out of your mouth
• Multi-outlet plugs should not be used unless they have a built-in circuit breaker as this
may causes overheating damage and possible electric fire.

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12. Use equipment (glassware, Bunsen burner, etc.) in the correct way, as indicated by the
supervisor.
13. Never leave experiments while in progress.
14. Never place the container directly under your nose and inhale the vapors.
15. Never mix or use chemicals not called for in the laboratory exercise.
16. Never touch, taste, or smell any reagents.
17. Never point the open end of a test tube containing a substance at yourself or others.
18. Make sure no flammable solvents are in the surrounding area when lighting a flame.
19. Do not remove any equipment or chemicals from the laboratory.
Eye Safety
Eye goggles should be worn:

• When working with certain caustic reagents and/or solvents, or concentrated acids and
bases.
• When working with reagents under pressure.
• When working in close proximity to ultra-violet radiation (light).
Safe handling of glassware
1. Glass breakage is a common cause of injuries in laboratories. Only glass in good
condition should be used
2. Protect hands with leather gloves when inserting glass tubing. Hold elbows close to the
body to limit movement when handling tubing
3. Clean all glassware properly after use
4. Use glassware of the proper size for experiments. There should be at least 20% free space
5. Conventional laboratory glassware must never be pressurized or used with vacuum
6. Never evacuate scratched, cracked, or etched glassware. Always check for stars or cracks
before use

Safe handling of chemicals

Working with potentially harmful chemicals is an often occurrence in the laboratory. Students
are expected to adhere to the safety precautions while working with hazardous chemicals and
they are expected to get familiarized with the following pictograms and hazard codes widely
used to mark risks.

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Many laboratory accidents occur by carrying chemicals from one place to another or
transferring them from one container to another. The chemicals used in a laboratory are often
corrosive, toxic or flammable and any accident involving these has the potential for personal
injury. Therefore, it is good practice to assume that all chemicals are potentially hazardous.

When large bottles of acids, solvents, or other liquids are transported within the laboratory
without a cart, only one bottle should be carried at a time. The bottle should be carried with
both hands, one on the neck of the bottle and the other underneath. Do not hook a finger through
the glass ring on top of the bottle, allowing it to dangle while being transported. Never carry
or attempt to pick up a bottle by the cap.

Check the label to verify it is the correct substance before using it. Weigh out or remove only
the amount of chemical you will need. Do not return the excess to its original container, but
properly dispose of it in the appropriate waste container. Hold containers away from the body
when transferring a chemical or solution from one container to another. To avoid dangerous
splatter, ALWAYS ADD ACID TO WATER when you need diluted solutions. Dispose of all
chemical waste properly. Never mix chemicals in sink drains. Sinks are to be used only for
water and those solutions designated by the instructor.

In case of hazardous chemical spill and it poses an immediate danger, leave the spill site and
warn others to stop entering to the spill site and report to any authorized person.

Remove contaminated clothing. Flush skin/eyes with water at least 15 to 30 minutes; use soap
for intermediate and final cleaning of skin areas. Protect yourself, then remove injured
person(s) to fresh air, if safe to do so. If flammable vapors are involved, do not operate
electrical switches. Turn off electric at the mains. Try to turn off or remove heat sources where
safe to do so. Do not touch the spill without protection gloves. Exterior doors and windows
should be opened to ventilate non-toxic vapors. Use absorbents to collect substances. Reduce
vapor concentrations by covering the surface of a liquid spill with absorbent. For small
quantities of inorganic acids or bases, use a neutralizing agent.

In case of solid chemical spill, sweep spilled solids of low toxicity into a dust pan and places
them into a container suitable for that chemical. Additional precautions such as the use of a
vacuum cleaner equipped with a HEPA filter may be necessary when cleaning up spills of more
highly toxic solids.

1. All chemicals in the laboratory are to be considered dangerous. Do not touch, taste, or
smell any chemicals unless specifically instructed to do so.

2. Check the label on chemical bottles twice before removing any of the contents. Take only
as much chemical as you need.

3. Never return unused chemicals to their original containers.

4. Never use mouth suction to fill a pipette. Use a rubber bulb or pipet pump.

5. When transferring reagents from one container to another, hold the containers away from
your body.

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6. Acids must be handled with extreme care. You will be shown the proper method for diluting
strong acids. Always add acid to water, swirl or stir the solution and be careful of the heat
produced, particularly with sulfuric acid.

7. Handle flammable hazardous liquids over a pan to contain spills. Never dispense
flammable liquids anywhere near an open flame or source of heat.

8. Never remove chemicals or other materials from the laboratory area.

9. Take great care when transferring acids and other chemicals from one part of the
laboratory to another. Hold them securely and walk carefully.

Ethidium bromide waste management & disposal

Ethidium bromide (3,8 diamino-5-ethyl-6-phenyl phenanthridinium bromide) is mutagenic and

moderately toxic and must be handled with care. You should always wear lab coat and gloves
when working with ethidium bromide. If ethidium bromide powder form has to be used to
prepare solutions, perform this process in a fume hood. Protective gloves, a lab coat, and eye
protection must be worn at all times. Do Not Use sodium hypochlorite (bleach) to treat
ethidium bromide. Bleach treatment can produce mutagenic products and leave behind up to
20% of the original ethidium bromide.

Ethidium bromide waste disposal

Ethidium bromide waste should NOT be poured down the drain or thrown in the trash, unless
the waste has been deactivated or filtered. All the contaminated gloves, electrophoresis gel
should be disposed to a dedicated bin for ethidium bromide waste.

Bis-Acrylamide handling, waste management & disposal

When handling Bis-Acrylamide you should always wear gloves, eye protection and Lab coat.
Dispose remaining acrylamide in original chemical containers by polymerization. For that add
100μl TEMED and 100μl of 10% Ammonium Persulfate (APS) and shake it thoroughly and
allow to react over night. Polymerized waste should be disposed into a dedicated bin for poly
acrylamide wastes.

Phenol/Chloroform handling, waste management & disposal

All the activities involved phenol/ chloroform should be conducted in a fume hoods. Handle
Phenol or Phenol/Chloroform formulation only with gloves, eye protection and Lab coats.
These liquids have to be disposed in dedicated organic solvent containers.

Organic solvent handling, waste management & disposal

Most organic solvents are flammable liquids and also toxic and environmentally hazardous.
Chlorinated organic solvents (solvents containing chlorine, e.g. dichloromethane, chloroform,
epichlorohydrin, carbon tetrachloride) should be disposed separately from non-chlorinated
(e.g. acetone, acetonitrile, ethanol, isopropanol, methanol, xylene) organic solvents into
separate waste containers.

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Safe handling of Biological waste
Bacteria, viruses, fungi or other infectious agents are studied because they may cause disease,
they can help us understand the natural world, and for many other reasons including the
possibility of industrial applications. Biohazards are infectious agents or hazardous biological
materials that present a risk or potential risk to the health of humans or animals. The risk can
be direct through infection or indirect through damage to the environment. Unlike chemical
hazards, infectious agents have the ability to replicate and to give rise to large numbers of
organisms when small numbers are released from a controlled situation. The biological and
physical nature of human pathogens can be categorized into risk groups (RG) based on the
transmissibility, invasiveness, virulence (i.e., ability to cause disease), and the lethality of the
specific pathogen. Risk groupings of infectious agents (RG1 through RG4) generally
correspond to biosafety levels (BL1 through BL4), which describe containment practices,
safety equipment, and facility design features recommended for safe handling of these
microorganisms.

Biosafety Level 1
Agents are not associated with disease in healthy human adults which requires standard
laboratory facilities and standard microbiological practices.

Biosafety Level 2
Agents are associated with human disease which is rarely serious. Treatment is usually
available. This requires more sophisticated personal protection and engineering controls (e.g.,
facilities and equipment) than are available in standard laboratories, as well as special handling
and decontamination procedures.

Biosafety Level 3
Agents are associated with serious or lethal human disease; treatment may be available; low
community risk. This which requires very sophisticated personal protection, specialized
facilities and engineering controls; as well as special handling and decontamination procedures.

Biosafety Level 4
Agents are associated with serious or lethal human disease; treatment is not usually available,
high community risk.

Biohazardous waste must be decontaminated before the end of each working day and before
final treatment and disposal. Decontamination is the process of reducing the number of disease
producing microorganisms and rendering an object safe for handling. Decontamination is best
accomplished by steam sterilization in a properly functioning autoclave.

As chemical surface decontamination, ethanol and isopropanol are effective against some
vegetative forms of bacteria, fungi, and hydrophobic (enveloped) viruses, but will not destroy
spores or hydrophilic viruses. The recommended strength is 70–90%. Alcohol can also be used
for disinfection of instruments or surfaces that have low organic burden. Alcohol-based hand-
rubs are recommended for the decontamination of lightly soiled hands in situations where
proper hand-washing is inconvenient or impossible.

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Safe handling of laboratory equipment

The types of equipment and instrumentation used in the laboratory are highly diverse. Although
each will have its own specific safety requirements, there are some general guidelines to follow
whenever operating any lab equipment and instrumentation.

• Always refer the manufacturer’s operating manual of the instrument

• Never remove hazard-warning labels from an instrument.
• Disconnect equipment from the power-source after use
• Use protective equipment recommended by the manufacturer when using the instrument
(hearing protection, face shield, etc.)
• Clean properly the equipment after use
• If you do not know how to operate a machine or equipment, newer try to use it by your
own

Laboratory staff or demonstrators will train you how to use the following laboratory
equipment and instrumentation properly.

• Laboratory refrigerator
• Laboratory freezer
• Autoclave
• Centrifuge
• Lamina flow
• Fume hood
• PCR machine
• Micro pipettes
• pH meter
• Hot plate
• Magnetic stir
• Gel documentation system
• Electric balancer
• Electrophoresis system
• Water bath
• Laboratory oven

Laboratory housekeeping
• Keep work area neat and free of any unnecessary objects.
• Thoroughly clean your laboratory work space, glass ware and equipment at the end of the
laboratory session.
• Do not block the sink drains with debris.
• Never block access to exits or emergency equipment.
• Inspect all equipment for damage (cracks, defects, etc.) prior to use; do not use damaged
equipment.
• Never pour chemical waste into the sink drains or wastebaskets.
• Place chemical waste in appropriately labeled waste containers.
• Properly dispose of broken glassware and other sharp objects
• Properly dispose of gloves, filter paper, and paper towels in the laboratory.

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Before you leave the lab
Switch off
• The ventilation in hoods that are not used
• All apparatus (heaters, stirrers)
• All lights

Close
• The solvent cabinets
• All windows
• The covers of the used solvent containers

Put away and store

• chemicals
• solvent cans

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 2:
Basics of Microscopy, care and safe handling of microscope
Course coordinator: Dr. Sarath Bandara
Background
This practical aims to provide students with basic information on microscopy, handling and care
of microscope. The purpose of using a microscope is to enlarge small details of specimen, thus
making them visible to the human eye. The enlarged image is visually observed through the
eyepieces which are typically of 10 x magnification. The image formation within the compound
microscope takes place in two major steps: First, the objective forms the slightly magnified, real
intermediate image. Second, this image is further enlarged by the eyepiece, which acts like a simple
magnifying loupe. This virtual image is viewed with the human eye lens apparatus and projected
onto the retina. Microscope is expensive laboratory equipment. Safety handling and caring of
microscope is of paramount importance. Proper care and maintenance of your microscope can
extend its life by many years.

Students should be able to:

• Identify the parts of light microscope

• Describe the functions of different parts of the microscope
• Demonstrate care and safety handling of the microscope
• Calculate the total magnification of a specimen viewed through the microscope
• Demonstrate focusing steps of a microscope
• Identify the different objective lenses

Parts of a compound light microscope

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Stand (arm) - All other parts of the microscope are attached to the arm
Stage - The platform on which the microscope slides rests and the clamping device that secures
the slide. A mechanical stage is used when working at higher magnifications where delicate
movements of the specimen slide are required.
Stage Clips - are used when there is no mechanical stage. The viewer is required to move the
slide manually to view different sections of the specimen.
Mechanical stage control knobs - These knobs, under the stage, move the stage front to back
and the slide from side to side.
Light source (Lamp or illuminator) - The lamp on/off switch is located on the base. The light
intensity can be adjusted with this on/off switch. The lamp should be adjusted to a medium
level at the start of viewing. The lamp or illuminator usually has a blue filter that rests on the
light housing or under the condenser.
Condenser (Aperture or diaphragm) - The condenser is located directly beneath the stage.
Used to adjust the amount of light reaching the specimen. It is adjusted with a thin, black lever
under the stage. It has a dramatic effect on the contrast observed in the specimen and may need
to be adjusted frequently.
Objective lenses - There are three lens systems: the eyepieces (ocular), the objectives (four),
Coarse and Fine Focus knobs - located on both sides of the microscope and used for focusing
image. The larger, inner coarse adjustment knob moves the stage up and down much faster and
farther than the smaller, outer fine adjustment knob. The coarse adjustment knob is used

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ONLY with the low power (4X, 10X) objectives. When focusing under the 40X or 100X
objective, ONLY use the fine adjustment, never the coarse adjustment.
Eyepieces (ocular) - is what you look through at the top of the microscope. Typically, standard
eyepieces have a magnifying power of 10x. Optional eyepieces of varying powers are available,
typically from 5x-30x. The eyepieces are held rather loosely in the eyepiece tubes. Never
remove the eyepieces from the eyepiece tubes. A rubber eye shield should be on the top of each
eyepiece. The width between the eyepieces should be moved until a full circle (the viewing
field) is visible with both eyes simultaneously.

Eyepiece Tube - holds the eyepieces in place above the objective lens. Binocular microscope
heads typically incorporate a diopter adjustment ring that allows for the possible
inconsistencies of our eyesight in one or both eyes. The monocular (single eye usage)
microscope does not need a diopter. Binocular microscopes also swivel (Interpupillary
Adjustment) to allow for different distances between the eyes of different individuals.

Objectives - There are four objectives: Scanning 4X (red), Low power 10X (yellow), high
power 40X (blue), and Oil immersion 100X (white, oil immersion objective). The objectives
are attached to a rotating nosepiece (nose turret).
Nosepiece - Holds the objective lenses

Coarse and Fine Focus knobs - are used to focus the microscope. Increasingly, they are
coaxial knobs - that is to say they are built on the same axis with the fine focus knob on the
outside. Coaxial focus knobs are more convenient since the viewer does not have to grope for
a different knob.

Focusing of the Microscope

First turn on the microscope lamp.

Start with Clean Lenses:

It is important that microscope lenses be very clean. Before viewing through a microscope, use
lens paper to gently clean the lenses.

Begin at Low Power Magnification:

Always begin by viewing the object through a low power lens. Depending on how small the
object is, start with the scanning or low-power objective. Using low-power objective lens, get
the target object centered in the field-of-view and focus as much as possible, first by using the
coarse focus and then fine-tuning the clarity of the image with the fine focus. Once the object
is in focus, switch to the next higher objective power. Do not change the focus or manipulate
the focus knobs in any way while changing objectives.

Adjustments for oil immersion objective lens:

Oil Immersion Objective (100X): This lens must be used with a specially formulated oil that
creates a bridge between the tip of the objective and the cover slip. Since the refractive indices
of air and this lens are different, the lens will not work without this special oil!

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To use the lens, first make sure the specimen is in focus under the high power (40X) objective.
Next, move the high-power objective out of position, place a small drop of oil on top of the
cover slip above the specimen to be viewed and move the oil immersion lens into place. Use
the fine adjustment knob to bring the specimen into focus. When you are finished, make sure
to clean the lens and slide with lens paper. Also, it is extremely important that this oil does not
contact any of the other objectives. If this should happen, clean the lenses immediately. Never
manipulate the coarse focus at oil immersion. Manipulating the coarse focus at high power can
smash the lens into the slide, potentially damaging the scope and the specimen.

Magnification:

Magnification is defined as the degree of enlargement of an object provided by the microscope.

Magnification of a microscope is the product of individual magnifying ability of ocular lens
and objective lens.

Magnification of ocular lens Magnification of objective Total magnification

lens
10X 4X 40X
10X 10X 100X
10X 40X 400X
10X 100X 1000X

Resolving power:
It is defined as an ability to distinguish between two particles situated very close.

Microscope care and handling

1. Handle with care
Improper handling is a common cause of many problems that occur with microscopes.
When carrying a microscope, hold it by the base and the metal support arm. The stage
on a microscope is the flat plate where the slides are placed for observation. Avoid
picking your microscope up by the stage or the eyepiece holder, as this can cause
misalignment.

2. Always place the microscope on a flat, level, firm bench, free from vibration.

3. Look after lenses

When using your microscope, the objective lens is lowered to adjust the focus.
However, be careful not to let the lens touch the slide you’re looking at, as this can
damage the lens. Furthermore, dirty lenses are notoriously difficult to clean.

4. Keep covered
Microscopes should always be covered with dust covers when not in use. Whether
transporting or storing your instrument, make the most of the microscope bag and
remember to keep your microscope covered when not in use. The microscope’s eye
tubes also need to be kept dust free. If the eyepieces need to be removed, cover the
tubes with caps and store them with the microscope.

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5. Store safely
Ensure you store your microscope in a clean, dry space with good ventilation. Salt air
or damp, for example, can cause damage to equipment over time. Expensive, precision
equipment should not be stored next to solutions that may leak. Similarly, keep your
microscope away from areas with potentially corrosive chemical fumes. Such fumes
can destroy lenses or corrode metal parts.

6. When storing the microscope, place the objective lenses on the lowest setting. Use the
coarse adjustment to put the nosepiece on its lowest setting. Make sure the light is
shut off.

7. Keep clean
Oil immersion is a technique used to increase the resolving power of a microscope.
Both the objective lens and sample are immersed in a transparent oil of high refractive
index so that high magnifications can be achieved while still maintaining good
resolution. It is essential to ensure careful cleaning takes place immediately after using
immersion oil and do not use damaging solvents.

8. Take care of bulbs

After using your microscope, turn off the illuminator and wait for it to cool down before
putting it away. Allowing the bulb to cool will extend its life and avoid the unnecessary
cost of expensive replacements. Similarly, if used constantly on full power, the bulb will
overheat and blow. Remember too, to turn the illuminator off when not in use.

9. Clean carefully
Microscope lenses are delicate. Treat them carefully to avoid any scratches. Use an
aspirator to remove dust. Moisten special lens paper with distilled water or appropriate
cleaning solution. Rubbing gently in a circular motion will remove any sticky residue.
Never use anything abrasive on microscope lenses.

10. Refer to the user’s manual

Your microscope should be sold with a user’s manual and specialist spanners as
required. Always refer to the manual when making any adjustments to the microscope
and use the supplied spanners. Never use force, inappropriate tools or over-tighten when
making adjustments to your microscope, as this will only result in equipment damage.

11. Maintain your microscope

An annual maintenance check of microscopes is always a good idea. Moving parts
should be cleaned and lubricated. Similarly, inspect the power cords and plugs for
safety.

Principles of Biology (BST 11052)

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Year I Semester I
Practical No 3: Preparation of temporary slides for observing bacteria, fungi, plant and
animal cells using light microscope
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Prepare temporary slides for observing bacteria, fungi, plant and animal cells under the
light microscope
• Demonstrate proper handling of a microscope to observe specimens
• Observe plant cells, animal cells, bacteria and fungus under different magnifications

Temporary preparation of specimens for light microscopy can be made for quick preliminary
investigation. This may involve sectioning, staining and mounting. Fresh material may be
hand-sectioned with a razor blade. There are four common ways to mount a microscope slide
as described below

Dry Mount
In a dry mount, the specimen is placed directly on the slide. A cover slip may be used to keep
the specimen in place and to help protect the objective lens. Dry mounts are suitable for
specimens such as samples of pollen, hair, feathers or plant materials.

Wet Mount
In a wet mount, a drop of water is used to suspend the specimen between the slide and cover
slip. Place a sample on the slide. Using a pipette, place a drop of water on the specimen. Then
place on edge of the cover slip over the sample and carefully lower the cover slip into place
using a toothpick or equivalent. This method will help prevent air bubbles from being trapped
under the cover slip. Your objective is to have sufficient water to fill the space between cover
slip and slide. If there is too much water, the cover slip will slide around. Take a piece of paper
towel and hold it close to one edge of the cover slip. This will draw out some water. If too dry,
add a drop of water beside the cover slip. Practice this until you get used to it. Wet mounts are
suitable for studying water-bound organisms such as bacteria, alga, paramecium, or bodily
fluids such as saliva, blood and urine.

Section Mount
In a section mount, an extremely thin cross-section of a specimen is used. Using a microtome, cut
a thin slice of your selected specimen such as an onion, and carefully set it on your slide. Then
follow the instructions for a dry or wet mount. A stain can often be applied directly to the specimen
before covering with a cover slip. Section mounts are suitable for a wide variety of samples such
as fruit, vegetables and other solids that can be cut into small slices.

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Smear
A smear is made by carefully smearing a thin layer of the specimen across a slide and then
applying a cover slip. Typically, a smear should be allowed to air dry before applying a stain.
Smears are suitable for samples such as bacterial species.

Staining
Stains are used to help identify different types of cells using light microscopes. They give the
image more contrast and allow cells to be classified according to their shape (morphology). By
using a variety of different stains, you can selectively stain different areas such as a cell wall,
nucleus, or the entire cell. Stains can also help differentiate between living or dead cells. Stains
tend to be grouped as neutral, acidic or basic, depending upon their chemical makeup and will
attract or repel different organisms accordingly. For example, scientists and health
professionals use Methylene Blue, a slightly alkaline stain, to reveal the presence of
deoxyribonucleic acid, more commonly known as DNA.

Stain Types
Iodine
Is one of the more commonly available stains and is used to identify starch in a variety of
samples. It will stain carbohydrates in plants and animal specimens brown or blue-black.
Glycogen will show as red.

Methylene Blue
Is an alkaline stain useful in identifying acidic cell nuclei and DNA in animal, bacteria or blood
samples. It’s also useful in aquariums to prevent the spread of fungal infections in fish.

Gram's Stain
Is one of the most frequently used processes in identifying bacteria – used daily in hospitals. It
is a primary test that quickly and cost effectively divides bacteria into one of two types: Gram
positive or Gram negative.

Staining steps
1. Prepare a wet mount slide.
2. Collect a drop of stain with an eye dropper or pipette.
3. Put a drop of stain on an outer edge of your cover slide.
4. Place a piece of paper towel against the opposite side of your cover slip, right up against
the edge. This will help draw the stain under the cover and across the specimen.
5. You may need to add another drop to ensure complete coverage.
6. The slide is now ready for viewing.

Apparatus and Materials:

Microscope Forceps
Slides Razor Blade
Coverslips Plant Material
Iodine solution Bread mold
Methylene Blue (0.5% to 1%) Pond water
Scissors Yogurt
Tissues Sterile cotton swabs / tooth pick

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Procedure:
Preparation of temporary plant tissue slide

1. Get a transparent layer of onion epidermis using tweezers.

2. Place the piece flat in the center of the slide. Be careful that the piece does not fold over.
3. Cover the onion skin with one or two drops of water. Gently lower the cover slip over the
slide.
4. Observe the slide under the microscope. Sketch what you see under each magnification
on the Observing Cells page.
5. Switch to low power and remove the slide.
6. Put on rubber gloves and safety goggles. Place a drop of iodine or methylene blue stain at
one edge of the cover slip.
7. Hold a piece of paper towel at the other end of the cover slip. The paper towel will draw
the iodine stain under the cover slip and across the onion skin.
8. Observe the slide under the microscope again. Sketch what you see under each
magnification.

Preparation of temporary animal cell slide

• Take a clean cotton swab (or tooth pick) and gently scrape the inside of your mouth.
• Smear the cotton swab on the centre of the microscope slide for 2 to 3 seconds.
• Add a drop of methylene blue solution and place a coverslip on top. Concentrated
methylene blue is toxic if ingested.
• Remove any excess solution by allowing a paper towel to touch one side of the coverslip.
• Place the slide on the microscope and observe.

Preparation of temporary fungal cell slide

Mold formation
1. Leave the food in open air for 30 minutes to an hour
2. Place the food (fruit, bread etc) in a moist bag (plastic paper bag)
3. Place the bag in a dark and warm place
4. Check after about 5 days - If the mold has formed, it is ready for microscopy

Slide Preparation
1. Using a dropper, place a drop of water at the center of a glass slide
2. Using a toothpick, scrap a little mold off the bread and introduce it in to the drop of
water in the glass slide
3. Gently place a cover slip on the glass slide (at an angle to remove air and lower it
gently)
4. Place the slide on the microscope for viewing starting with low power to high
power
5. A drop of methylene blue can be added to increase visibility.

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Preparation of bacterial cell slide

1. Take a very small drop of yogurt with the toothpick and smear it for 2 to 3 seconds on
the slide.
2. Place a small drop of methylene blue solution on a microscope slide (optional).
3. Place a coverslip on top. Remove excess solution around the coverslip with a paper
towel or tissue.
4. View in the compound microscope at 4 x or 10 x initially, before moving to higher
magnification. Bacteria will appear small even at the highest magnification.

Preparation of pond water slide

1. Using a dropper, place two or three drops of pond water at the center of a clean,
sterile microscopic slide.
2. Place a clean, sterile cover on top of the water drop (This should be done carefully,
placing the slide on one edge at a 45-degree angle and gently laying it on top of the water
to allow for even spreading of the water sample and remove bubbles)
3. Touch a piece of blotting paper on one side of the slide to absorb any excess water.
4. Place the slide on the microscope stage for observation.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 4: Preparation of Chemical Solutions
Course coordinator: Dr. Sarath Bandara

Background:

The ability to successfully make solutions is a basic laboratory skill performed in virtually all
biological experiments. A solution is a homogenous mixture of solute dissolved in the solvent.
Solutions can be described by their solute concentration (mol L-1 or M), a measure of how
many moles of solute are present per unit volume of solution, by their weight (w/v), a measure
of how much weight of the solute is in the given volume of solution, and by volume (v/v), a
measure of how much volume of solute is in the given volume of solution.

Solute - The substance which dissolves in a solution (NaCl, KCl, Glucose, etc.)

Solvent - The substance which dissolves another to form a solution. (Water, Methanol, Ethanol
etc.). For example, in a sugar and water solution, water is the solvent; sugar is the solute.

Solution - A mixture of two or more pure substances. In a solution one pure substance is
dissolved in another pure substance homogenously. For example, in a sugar and water solution,
the solution has the same concentration throughout, ie. it is homogenous.

Mole - A fundamental unit of mass used by scientists. This term refers to a large number of
elementary particles (atoms, molecules, ions, electrons, etc.) of any substance. 1 mole is 6.02
x 1023 molecules of that substance (Avogadro's number).

Students should be able to:

• Demonstrate chemical solution preparation ability
• Calculate the amount of solute required for preparation of a particular chemical solution
• Demonstrate correct handling of laboratory equipment being used in chemical solutions
preparation

To prepare a solution, two or more substances are mixed together in known quantities. This may
involve weighing a precise amount of dry material or measuring a precise amount of liquid.
Preparing solutions accurately will improve the precision and success of the experiment

Solution 1: Percentage by weight to volume (w/v)

Formula-
% w/v = [Mass of solute (g) / Volume of solution (ml)] x 100

Example- preparation of a 100 ml of 10% (w/v) NaCl solution

A 10% (w/v) NaCl solution has ten grams of NaCl dissolved in 100 ml of solution.

The solute occupies space in the solution, the volume of the solvent needed is almost always less
than the desired volume of solution. For example, if you are going to prepare a 10% (w/v)

Page | 17
NaCl solution, it would be incorrect to add 100 ml of water to 10 g of NaCl because that would
produce more than 100 ml of solution.

According to the above formula, if you dissolve 5 g of NaCl, to make up a total volume of 50
ml of solution then you have made a 10% w/v solution of NaCl.

Procedure: Preparation of a 50 ml of 10% (w/v) NaCl solution

First, express the percent of solute as a decimal: 10% = 0.1

Multiply this decimal by the total volume: 0.1 x 50 = 5 g (NaCl needed).

Weigh 5g of NaCl. Pour it into a graduated cylinder or volumetric flask containing about 30ml of
water. Once the sodium chloride has dissolved completely (swirl the flask gently if necessary), add
water to bring the volume up to the final 50 ml. Caution: Do not simply measure 100ml of water
and add 10g of sodium chloride. This will introduce error because adding the solid will change the
final volume of the solution and throw off the final percentage.

1. Weighing of 5 g of NaCl
2. Pouring into volumetric flask
3. Addition of about 30 ml water in the beginning, dissolving completely by shaking and
finally add more water to bring the volume up to the 50 ml mark.

Solution 2: Percentage by volume to volume (v/v)

When the solute is a liquid, it is sometimes convenient to express the solution concentration as
a volume percent.

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Formula:
% v/v = [Volume of solute (ml) / Volume of solution (ml)] x 100

Example 1: preparation of a 100 ml of 10% (v/v) Ethanol solution

A 10% (v/v) ethanol solution has 10 ml of Ethanol dissolved in 100 ml of solution.

Procedure: Preparation of a 100 ml of 10% (v/v) ethanol solution

In a measuring cylinder, add 10ml of ethanol in a little less than 90 ml of water and mix well.
Now bring the final volume of solution up to 100 ml with the addition of more water carefully.

Example 2
Preparation of 1000ml of a 70 % (v/v) ethanol solution.

Procedure:
First, express the percent of solute as a decimal: 70% = 0.7

Multiply this decimal by the total volume: 0.7 x 1000 = 700ml (ethanol is needed).

Subtract the volume of solute (ethanol) from the total solution volume:

1000ml (total solution volume) - 700ml (ethanol volume) = 300 ml (water needed)

Add 700 ml ethanol in a little less than 300 ml of water and mix well. Now bring final volume
of solution up to 1000ml with the addition of more water.

Solution 3: Molar Solutions

Molar solutions are the most useful in scientific experiments because they directly relate the
moles of solute to the volume of solution.

Formula:
Mole/L or (M) = moles of solute / 1 L of solution
Mole/L or (M) = [weight of solute (g) / molecular mass (g mol-1)] / 1 L of solution

Example 1:
-1
Preparation of 1 L of 1 mole L NaCl solution
The molar mass of NaCl is 58.44 g. (The molar mass of Na is 22.99 g, the molar mass of Cl is
35.45, so 22.99 g + 35.45 g = 58.44 g).

If you dissolve 58.44 g of NaCl in a final volume of 1 liter solution, you have made a 1 mole L-
1
NaCl solution, or 1 M NaCl solution.

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Example 2:

Preparation of 1 L of 0.5 mole L-1 NaCl solution

0.5 M NaCl solution requires 0.5 x 58.44 g of NaCl = 29.22 g (NaCl is needed)
If you dissolve 29.22 g of NaCl in a final volume of 1 liter solution, you have made a 0.5 mole
L-1 NaCl solution, or 0.5 M NaCl solution.

Example 3:

Preparation of 500 ml of 0.5 mole L-1 solution

1 L of 0.5 M NaCl solution requires 0.5 x 58.44 g of NaCl = 29.22 g
500 ml of 0.5 M NaCl solution requires 0.5 x 29.22 g of NaCl = 14.61 g
If you dissolve 14.61 g of NaCl in a final volume of 500 ml solution, you have made a 0.5 mole
L-1 NaCl solution, or 0.5 M NaCl solution.

Procedure:
Weigh 14.61 g of NaCl. Pour it into a graduated cylinder or volumetric flask (500 ml) containing
about 450 ml of water. Once the sodium chloride has dissolved completely, add water to bring
the volume up to the final 500 ml. Label the solution (Concentration, solution name and Date).
Caution: Do not simply measure 500 ml of water and add 14.61 g of sodium chloride. This will
introduce error because adding the solid will change the final volume of the solution and throw
off the final concentration.

Solution 4: Acid and base dilution

When a solution of acid or base is mixed with water some precautions must be taken. Protective
gloves should be worn. It is better to add the acid solution in water and not the opposite because
the water in contact with a large amount of acid can cause temperature rises more or less violent
which projects some acid droplets. This precaution especially concerns highly acidic solutions.
Dilution is the addition of more solvent to produce a solution of reduced concentration.

Formula:

C1V1=C2V2

C1 = the initial concentration of solute (the concentration of acid or base you have)
V1 = the initial volume (the volume of acid or base you need to dilute)
C2 = the final concentration (the concentration you want)
V2 = the final volume (the volume you need)

Example 1:

Preparation of 100 ml of 0.2 M HCl solution from 1M HCl solution.

Procedure:
• Use the equation C1V1 = C2V2 to calculate the amount of 1M HCl solution required (V1)
to make 100 mL of 0.2M HCl solution.
• 1 M x V1 = 0.2 M x 100 ml
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 • Measure the 1 M HCl amount required in to a graduated cylinder.
• Pour around 70 ml of water in to a 100 ml volumetric flask.
• Carefully add 1 M HCl amount measured gradually by gentle mixing
• Fill distilled water in to the volumetric flask until the meniscus of the liquid reaches the
calibration mark on the neck of the vol flask.
• Label the solution.

Assignment:

The following solutions are commonly used in plant DNA extraction. Please explain how to
prepare those solutions.

1. 500 ml of 2% (w/v) hexadecyltrimethylammonium bromide (CTAB) solution.

2. 500 ml of 1.4 M NaCl solution.
3. 500 ml of 20 mM Ethylenediaminetetraacetic acid (EDTA) solution (molar mass of EDTA
= 292.24 g).
4. 500 ml of 0.2 M sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) solution.
5. 100 ml of 70% (v/v) ethanol.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 5: Diffusion and Osmosis
Course coordinator: Dr. Sarath Bandara

Background:

Diffusion is the movement of molecules form a high concentration to a low concentration, is

the process by which nutrients and wastes move toward and away from cells. This happens
because of random molecular motion. Molecules move around randomly until there is an even
mixture throughout the container in which they are enclosed. The overall effect is that molecules
move "down" a concentration gradient from a region of high concentration to a region of low
concentration.

Figure 1: Diffusion

Osmosis is the movement (diffusion) of solvent molecules across a differentially permeable

membrane along the concentration gradient. Cell membranes do not allow all molecules to cross
them. They are said to be "selectively" or "differentially" permeable. Only certain molecules can
cross the membrane into or out of a cell. (If there is a salt concentration gradient across the
membrane, water will move across the membrane down the concentration gradient while the salt
cannot. If there is more salt and less water inside a cell than outside, water will flow into the cell
from the surrounding environment. This process is called osmosis).

When the environment outside a cell has a lower concentration of dissolved solute molecules
than inside the cell, the solution is said to be hypotonic, and water will move from the solution
into the cell. If the surrounding solution has a higher concentration of dissolved solute molecules
than the cell, the solution is hypertonic. In that case, water will move from the cell out into the
surrounding solution. An isotonic solution is one in which the concentration of dissolved solute
molecules is the same inside and outside the cell, and there is no net movement of water across
the membrane. When cells are placed in a hypertonic solution, water flows out of them and they
shrink or shrivel up. When cells are placed in a hypotonic solution, water flows into them. If the
cell does not have a cell wall or some other means of protecting the membrane, it will burst in
a hypotonic solution.

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 Figure 2.

Figure 2. A. Plant cell placed in pure water. This cell will become inflated because the water
outside the cell is at a higher concentration than the water inside the cell. As water moves in
by osmosis the vacuole fills up and presses out against the cell wall. B. A cell placed in a salt
solution. This cell will lose water as the water moves by diffusion from higher to lower
concentration. The cytoplasm of this cell has shrunken in a process called plasmolysis. C. A
cell in water equal to the concentration inside the cell. This cell has no overall gain or loss of
water because whatever moves out will be replaced by water moving in.

Students should be able to:

• Explain diffusion and Osmosis

• Explain hypertonic, hypotonic and isotonic solution
• Explain the role of cell membrane in osmosis

Apparatus and Materials:

Beakers Potato tuber

Water Clock
Dye or food coloring Tissues
NaCl solution knife
Test tubes

Procedure:

Diffusion
1. Obtain beakers filled with clean water.
2. Add a drop of dye to the center of the beaker.
3. Do not disturb the beaker and Observe what happens.

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Osmosis
1. Cut the potato tuber in to approximately equal size of strips.
2. Record the initial length and the weight of potato slices.
3. Prepare a series of NaCl solution (0%, 1%, 3%, 5%, and 10% w/v) in test tubes or beakers.
4. Place a slice of potato in each test tube.
5. Allow the potato to stay in the test tubes for 30 min (be sure to record your start time!).
6. Remove the potato slices from the test tubes and dry out excess water using a tissue.
7. Record the features (Length, Weight) of potato slices after the experiment.
8. Observe and record differences between texture and the features of the strips.
9. Discuss with your lab group: “what happened?”

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 6: Use of Micropipettes
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Explain the basic parts of micropipette.

• Demonstrate the measuring of different volumes with P10, P20, P200 and P1000
micropipettes.
• Demonstrate the ability to adjust and read the volume indicated on P 10, P20, P200 and
P1000.
• Demonstrate safety handling of micropipettes.

Background:
Micropipettes are the standard laboratory equipment used to measure and transfer small volumes
of liquids which are generally less than 1 ml of liquid (1000 µl = 1 ml). The scales on micro pipettes
are in microliters (µl). It is essential that you master their use if you are to be successful in your
experiments. Each micropipette has a specific volume range. These pipettes are expensive (Around
29000.00 Srilankan rupees per pipette), precision instruments and must be used with extreme care
to insure their accuracy, reliability and the safety of the instrument.

Please NEVER DO the following four thins when you are using a micropipette.
1. Don't rotate the volume adjustment knob beyond the upper or lower range of the pipette.
2. Don't use a pipette without a tip in place. Doing so could ruin the precision piston that
measures the volume of fluid.
3. Don't lay down or invert a pipette that has a filled tip. Fluid could run back into the piston.
4. Don't let the plunger snap back after withdrawing or ejecting fluid. Doing so could
damage the piston.

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Always adhere to the following steps when micropipette is used

1. Always select the SMALLEST size pipet that will handle the volume you wish to move
to achieve the greatest accuracy.
2. Set the desired volume by turning the volume adjustment knob clockwise to increase
volume or counterclockwise to decrease volume.
3. Firmly press a new tip onto the pipette. Do not touch the tip with your hands. Note that tips
also come in different sizes—be sure to use one that has the proper capacity for your sample.
4. To draw the set volume of liquid, Push the plunger down slowly to the point of first
resistance: this is the load volume. The plunger will stop at two different positions when
it is depressed.
5. While holding the plunger at the load volume position, put the tip into the solution so that
it is immersed just enough to cover the end (3-4 mm), not as deep as possible.
6. Slowly release the plunger to draw up the liquid making sure to keep the tip immersed.
7. The second stopping point can be found when the plunger is depressed beyond the initial
resistance position until it is in contact with the body of the pipette. This second stopping
position is used for the complete discharging of solutions from the plastic tip.
8. To remove the tip, press the tip ejector on the back of the grip while holding the tip over
an appropriate waste container.
9. Whenever pipettes are stored after the laboratory activities, adjust the pipettes to their
maximum volume to maintain the accuracy of the pipettes.

The plunger will stop at two different positions when it is depressed

Page | 26
Setting the desirable volumes on micropipettes

A simple check for proper calibration of the micropipette

Check the calibration of your micropipette by using the fact that 1 ml of deionized (or distilled)
water has a mass of 1 g. Pipet a range of volumes spanning the pipette's useable range and
weigh them on a top loading balance having at least 3 decimal place accuracy. Pipets having
greater than 5 % error should be recalibrated.

Page | 27
Principles of Biology (BST 11052)
Year I Semester I
Practical No 7: Use of basic laboratory equipment
Course coordinator: Dr. Sarath Bandara

Background:
Working safely with laboratory equipment are essential parts of the research activities. Many
of the accidents and equipment damages that occur in the laboratory can be attributed to
improper use or maintenance of laboratory equipment. This practical discusses prudent
practices for handling equipment used frequently in laboratories.
Students should be able to:

• Demonstrate proper handling of commonly used laboratory equipment (Refrigerator,

Freezer, Microwave oven, Centrifuge, Autoclave and Refractometer).

Refrigerators and Freezers

• Make sure the doors are properly closed. Do not keep opening the doors while not using.
Careful labeling of samples placed in refrigerators and freezers with both the contents and
the owner's name is essential. Do not use water-soluble ink; labels should be a waterproof
or covered with transparent tape.
• All materials must be properly capped and sealed. Avoid using of foil or parafilm as a
primary method for sealing the container.
• Do not overload your refrigerator.
• Do not store food in refrigerators located in technical areas.
• DNA and RNA samples should be stored separately in a dedicated area of freezer

Microwave Ovens
• Never operate ovens with the doors open, to avoid exposure to microwave.
• Never attempt to heat flammable liquids or solids, hazardous substances or
radioactive materials in any type of microwave oven
• Do not place metal items inside the microwave, including aluminum foil and plastic-
coated magnetic stirrer bars.
• Do not heat sealed containers in a microwave.
• Set the power and timings correctly. Do not overheat.

Centrifuge
• A centrifuge is a device that separates particles from a solution
through use of a rotor. In biology, the particles are usually cells,
subcellular organelles, or large molecules (DNA, Proteins, RNA
etc.), all of which are referred to here as particles.
• BALANCE THE LOAD IN ROTOR each time the centrifuge is
used. The disconnect switch should automatically shut off the
equipment when the top is opened.
• Do not overfill the centrifuge tubes. Ensure that they are hung
properly

Page | 28
 • Ensure that the lid is closed before starting the centrifuge.
• Do not overload a rotor beyond the rotor's maximum mass without reducing the rated
rotor speed.
• Follow the manufacturer's instructions for safe operating speeds. Do not run a rotor
beyond its maximum rated speed.

Autoclave
• Autoclaves provide a physical method for disinfection and
sterilization. They work with a combination of steam,
pressure and time. Autoclaves operate at high temperature
and pressure in order to kill microorganisms and spores

• The sterilization conditions: The sterilization time will vary

according to the contents reaches 121°C and 15 pounds per
square inch (PSI). It can be variable with a minimum of 12-
15 minutes. Several trays with large bags or containers
loaded in the autoclave will require a longer time to reach
121°C and should be set accordingly. Unless specifically
instructed, the chamber temperature is set to 121°C.

• Always use personal protective equipment (PPE)

including a lab coat, heat resistant gloves, and eye
protection, especially when unloading the autoclave when
using an autoclave.
• Do not autoclave flammable, reactive, corrosive, toxic, or radioactive materials.
• Check that plastics are compatible with the autoclave. Not all plastics can be
autoclaved.
• Inspect glassware for cracks. Do not autoclave cracked or compromised glassware.
Never sealing containers; under pressure they pose an explosion risk.
• For liquids, leave caps loose or cover with foil to allow steam penetration and prevent
explosion.
• For bagged items, loosely tape or tie closed. Leave an opening for steam to penetrate
the bag load
• Inspect for spills or debris inside the autoclave; check door gasket for cracks or
bulges.
• Ensure that the jacket has reached sufficient pressure to start a cycle.
• Place items in an autoclave tub on rack. Never place items directly on the autoclave
bottom or floor.
• Do not overload the autoclave. Allow sufficient space between items for steam.
• Add water if needed (100mL of water can be added to the autoclave pan to ensure
even heating of liquids).
• Close and lock the door. Ensure the door is secure before starting a cycle.
• Select appropriate cycle (e.g. dry heat, sterilize media, sterilize biohazardous waste
• about 20 minutes into the cycle to verify the autoclave has reached sterilization
temperature (121°C).
• Waiting for the pressure to reach zero and the temperature is at or below 121°C before
opening the door at the end of a cycle to avoid steam burns and shattered glassware.
Do not stand directly in front of the door.

Page | 29
• Do not open the autoclave door during a cycle! If necessary, abort the cycle and wait
until the chamber depressurizes.
• If cycle fails, notify the person responsible for the autoclave. Items may not be
sufficiently decontaminated if the cycle did not complete UNLOAD
• When the cycle is complete, verify that chamber temperature has dropped and pressure is
zero.
• Slowly open the door to allow steam to escape gradually. Keep your face away from the
door.
• Allow items to stand in the autoclave for 10 minutes.
• Cautiously remove items, and place in a safe area to cool. Do not agitate containers as
boiling or superheated liquids can explode if moved too quickly

Refractometer

• There are two types of handheld refractometers: analog and digital. They work on the
principle that light entering a prism has a unique characteristic. That characteristic is
represented by a value on a scale in units known as ºBrix.
• Operation consists of placing 1 or 2 drops of the water sample on the prism, closing a
glass plate over the sample, then looking through the eyepiece for the reading.
• The water sample is sandwiched between the measuring prism and the cover plate.
Light traveling through the sample is either passed through or totally internally
reflected.
• The net effect is that a shadow line is formed between the illuminated area and the dark
area. It is at the point that this shadow line crosses the scale that a reading is taken.

The field of view in an analog
refractometer remains blue
when only light passes through
the prism.
When water is placed on the solution containing sugar will display
prism, a contrast line develops at the percent sucrose in ºBrix units.
the “0” mark on the scale. (The sample placed on this prism is
displaying 17 ºBrix.)

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 8: Fermentation (Anaerobic respiration of yeast)
Course coordinator: Dr. Sarath Bandara

Background

Yeast (Eukaryotic single-celled microorganisms, a member of the kingdom fungi) needs

energy (as ATP) to be at their optimum in order for it to survive and reproduce. This energy is
produced by a biochemical reaction known as cellular respiration. Yeast can undergo both
aerobic and anaerobic respiration. When sugar is available along with oxygen, yeast is in the
phase of cellular growth, it grows and multiplies. When oxygen gets depleted and still sugar is
available yeast will continue to use it to make energy by anaerobic respiration. During this
process, glucose is not completely broken down and much less energy is released than during
aerobic respiration.
Anaerobic respiration:
In the absence of oxygen to accept the electrons in NADH generated through glycolysis, some
organisms can still respire anaerobically, using inorganic molecules to accept the electrons. For
example, many bacteria use sulfur, nitrate, or other inorganic compounds as the electron
acceptor in place of oxygen.

Fate of Pyruvate with and without oxygen

Page | 31
Fermentation:
The fate of the pyruvate that is produced by glycolysis depends upon which of these two
processes takes place. The aerobic respiration path starts with the oxidation of pyruvate to a
molecule called acetyl-CoA, which is then further oxidized in a series of reactions called the
Krebs cycle. The fermentation path, by contrast, involves the reduction of all or part of
pyruvate. In the absence of oxygen, aerobic metabolism cannot occur, and cells must rely
exclusively on glycolysis to produce ATP. Under these conditions, the 2 electrons (a hydrogen
atom) of NADH generated by glycolysis are donated to organic molecules in a process called
fermentation.

Ethanol Fermentation

Glucose → Ethanol + Carbon dioxide

(C6H12O6 → 2C2H5OH + 2CO2)

Eukaryotic cells are capable of only a few types of fermentation. In one type, which occurs in
single-celled fungi called yeast, the molecule that accepts hydrogen (2 electrons) from NADH
is pyruvate, the end product of glycolysis itself. Yeast enzymes remove a terminal CO2 group
from pyruvate through decarboxylation, producing a two-carbon molecule called acetaldehyde.
The CO2 released causes bread made with yeast to rise, while bread made without yeast
(unleavened bread) does not. The acetaldehyde accepts two electrons (a hydrogen atom) from
NADH, producing NAD+ and ethanol (ethyl alcohol). This particular type of fermentation is
of great interest to humans, since it is the source of the ethanol in wine and beer. Ethanol is a
byproduct of fermentation that is actually toxic to yeast; as it approaches a concentration of
about 12%, it begins to kill the yeast. That explains why naturally fermented wine contains
only about 12% ethanol.

Page | 32
Lactic Acid Fermentation

Most animal cells regenerate NAD+ without decarboxylation. Muscle cells, for example, use
an enzyme called lactate dehydrogenase to transfer a hydrogen atom from NADH back to the
pyruvate that is produced by glycolysis. This reaction converts pyruvate into lactic acid and
regenerates NAD+ from NADH. It therefore closes the metabolic circle, allowing glycolysis to
continue as long as glucose is available. Circulating blood removes excess lactate (the ionized
form of lactic acid) from muscles, but when removal cannot keep pace with production, the
accumulating lactic
acid interferes with muscle function and contributes to muscle fatigue.

A comparison between aerobic and anaerobic respiration

Aerobic Anaerobic

Oxygen Needed Not needed

Glucose
breakdown Complete Incomplete

End Animal cells: lactic acid

Carbon dioxide and water
product(s) Plant cells and yeast: carbon dioxide and ethanol

Energy
Relatively large amount Relatively small amount
released

Page | 33
Students should be able to:

• Explain anaerobic respiration

• Demonstrate ethanol fermentation by yeast

Activity:

• Prepare two glucose solutions (250 ml of 10 % (w/v).

• Add 5 g of yeast into one solution, mix well and keep for about 1 hour. The
other solution is the control experiment.
• Observe any differences between 2 solutions.
• Measure the brix values of both solutions.
• Measure the pH values of both solutions.
• Among yourselves, discuss and explain the reasons for any differences between 2
solutions.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 9: Spectroscopic analysis of Chlorophyll
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Demonstrate the isolation of plant pigments.

• Demonstrate the correct handling of spectrophotometer.
• Explain the absorbance spectrum of chlorophyll.
• Explain the importance of pigments in photosynthesis.

Background:
Molecules that absorb light in the visible range are called pigments. Pigments capture energy
from sunlight. In photosynthesis, the wavelength of light absorbed depends upon the specific
pigment. Organisms have evolved a variety of different pigments, but there are only two
general types used in green plant photosynthesis: carotenoids and chlorophylls. Chlorophylls
absorb light (photons) within narrow energy ranges. Two kinds of chlorophyll in plants,
chlorophylls a and b, preferentially absorb violet-blue and red light. Neither of these pigments
absorbs light (photons) with wavelengths between about 500 and 600 nanometers, and light of
these wavelengths is, therefore, reflected by plants. When these photons are subsequently
absorbed by the pigment in our eyes, we perceive them as green. Chlorophyll a is the main
photosynthetic pigment and is the only pigment that can act directly to convert light energy to
chemical energy. However, chlorophyll b, acting as an accessory or secondary light-absorbing
pigment, complements and adds to the light absorption of chlorophyll a. Chlorophyll b has an
absorption spectrum shifted toward the green wavelengths. Therefore, chlorophyll b can absorb
photons chlorophyll a cannot (Figure 1). Chlorophyll b therefore greatly increases the
proportion of the photons in sunlight that plants can harvest. An important group of accessory
pigments, the carotenoids, assist in photosynthesis by capturing energy from light of
wavelengths that are not efficiently absorbed by either chlorophyll.

Figure 1. The absorption spectrum of chlorophyll and Carotenoi

Page | 35
Activity

Plant leaf pigment extraction and analysis of the absorption spectrum

• Weigh about 0.5 g of any type of plant leaves and wash well with distilled water.

• Cut leaves into tiny pieces (removing leaf veins) and then, in the fume hood, use a mortar and
pestle to grind the spinach leaves with about 10 mL of 80 % (v/v) acetone (~2 minutes).
(Acetone is a volatile organic liquid so you may need add more because of evaporation.)

• Centrifuge the homogenized sample at 10,000 rpm for 15min at 4 0 C.

• Collect 0.5 ml supernatant and mixed with 4.5 ml of 80 % (v/v) acetone (This is done for
dilution. Check absorbance spectrum of pigments with several dilution for an optimum
graph).

• Analyze the absorption spectrum of the mixture (range of wavelength from 300 nm to 800
nm) using the spectrophotometer.

• Discuss your results.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 10: Lab discussion on Photosynthesis and respiration
Course coordinator: Dr. Sarath Bandara

Lab discussion on Photosynthesis and Respiration

Activity
• Group discussion and presentation on photosynthesis
• Group discussion and presentation on Respiration
• Discuss among yourselves and get help from demonstrators to prepare your
presentations.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 11: Mitosis (Eukaryotic Cell Division)
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Prepare specimens for microscopic observation of cell division.
• Explain mitosis in eukaryotic cell division.
• Visualize different phases of mitosis

Background:
Mitosis is the nuclear division of a eukaryotic cell which produces two daughter cells identical to
the dividing parent cell. In eukaryotes, mitosis is important for growth, replacing old cells and
tissue repair. For single-celled, and some multi-cellular eukaryotes, mitosis is part of asexual
reproduction. Mitosis is a one portion of the cell cycle. In eukaryotic cells, one complete cell cycle
is broken into interphase, mitosis and cytoplasmic division (cytokinesis) (Figure 1). The cell spends
most of its time in interphase (G1, S and G2), during which the cell synthesizes cytoplasmic and
nuclear components (e.g. proteins, carbohydrates). DNA replication also occurs in S phase of the
interphase. This process involves the precise duplication of all the DNA in the nucleus, in
preparation for mitosis. Mitosis is the physical division of the nucleus (During M phase), creating
two identical nuclei, one for each daughter cell. Cytoplasmic division (During C phase) results in
the partitioning of the cytoplasmic components and the physical separation of the two new daughter
cells.

Figure 1. The phases of cell cycle.

The M phase where nuclear content is divided into two, has been divided into four sub phases
as below (Figure 2). Mitosis (separation of the two genomes or replicated nuclear content or

Page | 38
chromosomes) occurs in four stages -prophase, metaphase, anaphase, and telophase - and is
followed by cytokinesis (division into two separate cells).

Figure 2. Mitosis and cytokinesis.

The structure of eukaryotic chromosomes at the beginning of prophase
During the S phase, each chromosome replicates to produce two sister chromatids, which
remain attached to each other at the centromere. The centromere is a point of constriction on
the chromosome, containing a specific DNA sequence to which is bound a disk of protein
called a kinetochore. This disk functions as an attachment site for fibers that assist in cell

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division. Each chromosome’s centromere is located at a characteristic site (Figure 3). The cell’s
DNA replicates only during the S phase of the cell cycle. After the chromosomes have
replicated in S phase, they remain fully extended and uncoiled. This makes them invisible
under the light microscope. In G2 phase, they begin the long process of condensation, coiling
ever more tightly. When the chromosome condensation initiated in G2 phase reaches the point
at which individual condensed chromosomes first become visible with the light microscope,
the first stage of mitosis, prophase, has begun. The condensation process continues throughout
prophase.

Figure 3. Condensed eukaryotic chromosomes structure (Before and after the replication).

Activity: Squash preparation of plant (onion) root tips for observing mitotic stages
The genetic information of all organisms resides in the individual DNA molecules or
chromosomes. An onion cell possesses eight chromosomes whereas human cells possess 46
(23 pairs) chromosomes. Cell division occurs rapidly in growing root tips of sprouting seeds or
bulbs. An onion root tip is a rapidly growing part of the onion and thus many cells will be in
different stages of mitosis. The onion root tips can be prepared and squashed in a way that
allows them to be flattened on a microscopic slide, so that the chromosomes of individual cells
can be observed easily. The super coiled chromosomes during different stages of mitosis
present in the onion root tip cells can be visualized by treating with DNA specific stains, like
Feulgen stain and Acetocarmine stain.

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Material required

Onion or other plant with root tips Acetocarmine stain 1 M HCl

Scissors
Forceps
Razor blade
Microscopic slides and cover slips
Water bath
Light Microscope

Procedure

• Cut the root tips 5 to 8 mm from the tip of the freshly sprouted root and put them into an Eppendorf tube w
• Wash and clean them with distilled water.
• Fill half the tube with 1M HCL and incubate in a water bath for 12 mins at 60 Co.
• Remove the HCl from the tube and wash the root tips 3 times with distilled water.
• Put acetocarmine stain to the tube in such a way that all the root tips are immersed in the stain and keep fo
• Remove the stain and wash the root tips 3 times with distilled water.
• Carefully remove the root tips with forceps, cut into small pieces with a razor blade and transfer onto mic
• Carefully place a coverslip and blot the excess water with a blotting paper.
• Squash the slide with your thumb using a firm and even pressure. (Avoid squashing with such force that
slides).
• Observe it under a compound microscope in 10x objective. Scan and narrow down to a region containing d
to 40x for a better view.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 12: Simple DNA extraction from plant tissues
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Demonstrate simple and quick (miniprep) DNA isolation procedure from plant tissues.
• Prepare chemical solutions required for simple DNA isolation method.
• Explain the role of different chemicals in the extraction solution.

Background:
The isolation of good quality DNA from plants is complicated due to the presence of phenolic
compounds, highly viscous polysaccharides, glycosides, flavonoids and DNA degrading
enzymes (Endonuclease / DNase). Sodium dodecyl sulphate (SDS) is an anionic detergent used
in DNA extraction from plants animals and bacteria. The main purpose of adding SDS into
DNA isolation solution is for disruption of lipid bilayers of the cell membrane and cell
organelles including nuclear membrane. SDS also denature proteins and inactivates many DNA
degrading enzyme (DNase). This ensures that all the lipid bilayers are broken down and DNA
are release to the extraction solution as well as the DNA is safe from the released degradative
enzymes due to the denaturing ability of SDS.
DNA is polar and negatively charged molecules. This property accounts for water solubility,
as water is also polar. The positive charges of water interact with the negative charges of DNA
and make a solution. To recover DNA from an aqueous solution, the DNA must be precipitated
out of a solution. Since water has a relatively weak positive charge, this precipitation is done
by providing a stronger positively charged ion in the solution. Sodium is the perfect candidate
for this. Once DNA has been removed from the nucleus of a cell and allowed to mix with water,
the introduction of sodium ions creates a temporary attraction between sodium and the DNA.
The DNA is temporarily neutralized and then easily disassociated from the water. At this stage
the introduction of an alcohol forces the DNA and sodium ions to become even more tightly
bonded, since alcohol is very nonpolar. Ethanol or isopropyl alcohol can be used. Once DNA
is disassociated from the water and tightly bound to the sodium, it will precipitate out of the
solution after addition of alcohol. DNA is less soluble in isopropanol or ethanol leading to get
precipitated. NaCl also helps to remove proteins that are bound to the DNA. It also helps to
keep the proteins dissolved in the aqueous solution so they don't precipitate in the alcohol along
with the DNA.

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Activity:
Preparation of DNA extraction solution:

• Prepare 50 mL of 1 % SDS solution having 0.5 M NaCl.

DNA isolation procedure:

1. Measure about 200 mg of healthy immature leaf sample and put it into a mortar.
2. Add 0.5 mL of extraction buffer and grind well with a pestle and add more 1.2 mL of
extraction buffer into the mixture and mix.
3. Transfer the content into an Eppendorf tube (micro centrifuge tube).
4. Centrifuge at 13000 rpm for 4 min at room temperature.
5. Transfer the supernatant into new microcentrifuge tube and add equal volume
of isopropanol.
6. Mix gently and place on ice for 5 min.
7. Centrifuge at 13000 rpm for 5 min at room temperature.
8. Discard the supernatant.
9. Wash the DNA pellet with 0.5 ml of 70 % ethanol.
10. Centrifuge at 13000 rpm for 2 min.
11. Discard the supernatant carefully without damaging to the pellet.
12. Air dry the pellet for 15 min and dissolve in 100 µL of distilled water.
13. Store the DNA sample at -21oC.

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Principles of Biology (BST 11052)
Year I Semester I
Practical No 13: How to make an agarose gel for electrophoresis
Course coordinator: Dr. Sarath Bandara

Students should be able to:

• Demonstrate the preparation of agarose gel for electrophoresis.
• Explain the role of agarose gel in biomolecule (DNA, RNA and proteins) size separation.
• Demonstrate the safety handling of ethedium bromide during gel preparation.

Background:
Agarose gel electrophoresis is a routinely used method for separating biomolecules such as
proteins, DNA and RNA. After melting, agarose forms three dimensional matrix (gel) with
channels and pores through which biomolecules can pass (DNA, RNA and proteins). Gels are
described in terms of (w/v) percentages. 0.7%, 0.8%, 1%, and 1.2% are pretty common gel
percentages. For example, a 1% gel would be 1g agarose in 100 mL TAE. (Tris-Acetate-EDTA
which is a buffer used for agarose gel preparation and used for electrophoresis). The pore size
of a 1% (W/V) gel has been estimated from 100 nm to 500 nm.
Nucleic acid molecules (DNA is negatively charged) are size separated by the aid of an electric
field where negatively charged molecules migrate toward anode (positive) pole. The migration
flow is determined solely by the gel percentage (The higher the percentage the smaller the pore
size) (Table 1), the molecular weight where small weight molecules migrate faster than larger
ones and the voltage applied. In order to visualize nucleic acid molecules in agarose gels,
ethidium bromide (ethidium bromide is a MUTAGENIC compound!!!) or SYBR Green are
commonly used dyes. Illumination of the agarose gels with ~500 nm UV light is subsequently
used for visualizing the stained nucleic acids. In addition to size separation, nucleic acid
fractionation using agarose gel electrophoresis can be used as an initial step for further
purification of a DNA band of interest. This includes excising the desired “band” from a stained
gel viewed with a UV transilluminator. There are different gel tanks and casting trays that vary
in size. The volume of gel you will need to make will depend on the size of the casting tray.
The wells (the holes into which DNA is loaded) of the gel are made by inserting a comb into
the slots in the tray, and as the agarose hardens around the comb, wells are formed. The thicker
you pour your gel, the deeper the wells will be.
Table 1. The suggested agarose concentrations for separation of different ranges of Linear
DNA molecules

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Activity:
To make an agarose gel, first figure out what volume you want. You can pour water into the tray
with the comb held on that and when the wells look deep enough, you can record the volume using
measuring cylinder and make your gel using that volume. Then figure out what mass of agarose to
be used for the percentage gel you want. Measure out the agarose amount required and put the
agarose in an Erlenmeyer flask. Measure out the correct volume of 1X TAE buffer using a
graduated cylinder. Microwave your gel solution to melt completely. During the process of
microwave heating, do not let the solution to get boiled for long time as it may degrade the quality
of the gel matrix. Let it cool on the bench top for a while and then add ethidium bromide (this is a
chemical that intercalates DNA and makes it visible under UV light). EtBr is a potent mutagen,
so make sure you don’t get any on your fingers or on yourself! Don’t spread it around the lab.
You might want to wear gloves and lab coat. The amount of EtBr to add is as follows: of a 0.5
mg/mL stock solution (which is the most common stock solution in the lab is), add 1/1000 to your
gel. For example, 30 µl of EtBr should be added into 30 mL of gel. The casting tray should be
sealed with rubber stripes around the edges that makes a tight seal before pouring the melted gel
solution. It also has notches where you can insert a comb. The size of the comb to be used depends
on the width of the wells you want to have on the gel. Swirl the flask immediately before pouring
the gel into the tray to make sure it’s mostly all at the same temperature; otherwise your gel will
harden in a weird manner and will be ruined. It will take about 20 minutes for a small gel to harden
enough to be used, longer for bigger gels. When you are finished pouring your gel, rinse well your
Erlenmeyer flask with water before you put it in the wash tub as hardened agarose is hard to clean
off of glassware.

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