INDEX

PAGE
S.NO DATE TITLE SIGNATURE
NO
Safety measures while working in
1.
Laboratory

2. Handling of chemical substances

3. Handling and uses of glasswares

Cleaning, drying and sterilization of

4.
glasswares
Weighing and preparation of solutions of
5.
different strengths and their dilution

6. Neutralization of acid and bases

Preparation of buffer of different strength

7.
of pH value
Estimation of fertilizer doses for field and
8.
pot applications

9. Preparation Of Culture Media – Nutrient

Broth, Nutrient Agar And Potato
Dextrose Agar
Sterilization and Disinfection
10.

11. Agarose Gel Electrophoresis

Separation of Protein Molecules Using

12.
SDS- Page Gel

13. Microscopy

14. Microscopic Examination Of Stained

Cell Preparation
15.
Micrometry
 Ex.No :1
SAFETY MEASURES WHILE WORKING IN LABORATORY
Date:

SAFETY MEASURES WHILE WORKING IN LABORATORY

Laboratory can be a place of discovery and learning. However, by the very nature
of laboratory work, it can be a place of danger if proper common-sense precautions
aren't taken. While every effort has been made to eliminate the use of explosive, highly
toxic, and carcinogenic substances from the experiments which you will perform, there is a
certain unavoidable hazard associated with the use of a variety of chemicals and glassware.
You are expected to learn and adhere to the following general safety guidelines to ensure a
safe laboratory environment for both yourself and the people you may be working near.

General Safety Rules

Safe laboratory practice is based on understanding and respect, not fear. The
regulations below are intended to help you work safely with chemical reagents. These
guidelines cover ordinary hazards and apply to any laboratory experiments you will
encounter. Before beginning an experiment, be sure you have this information at hand and that
you understand it. Do not hesitate to consult with your Course Teacher and Lab. Attendant, if
you have questions about any experiment.
Personal and General Laboratory Safety
1. Working alone in the laboratory is strictly forbidden. Students may work in instructional
laboratories only during regularly scheduled times and then only when supervised by an
authorized Lab. attendant or a member of the faculty.
2. Worn safety glasses, aprons and hand gloves as and when required. Splash hazards are most
significant danger present in lab and eyes are extremely sensitive. Aprons are chemical and
flame resistant, and could save you!
3. Contact lenses are not permitted in lab. Contact lenses can absorb chemical from air (fumes),
concentrated and hold them against eye.
4. In laboratory bare foot, long hair and billowy clothing must be restricted. To protect you
from splashes and spills. Long hair must be tied back.
5. Do not work in Laboratory empty stomach, as it may lead to faintness and panic situation.
6. Careful consideration should be given before wearing any jewelry into the lab. Some
chemicals evaporate very quickly. If they get beneath a ring , watch or some form of jewelry,
they can be prevented from evaporating, held against the skin longer and greatly increased the
risk of injury.
7. Always keep exhaust fan on while working in Laboratory.
8. Do not use any equipment unless you are trained and approved as a user by your supervisor.
9. Use every precaution to keep all chemicals off your skin and clothing, out of your nose,
mouth and eyes, and away from flames.
10. Do not eat or drink anything (including water) during working in laboratory.
11. Never use chemicals (salt, sugar, alcohol, bicarbonate, etc.) from the laboratory or
stockroom on food. The source containers may be contaminated or mislabeled.
12. Disposal - Students are responsible for the proper disposal of used material if any in
appropriate containers.
13. Before start working in laboratory you must know the location and proper use of all
laboratory safety equipment, including eyewash, safety shower, fire extinguisher etc.
14. Pipette must be fitted with suction bulbs to transfer chemicals. Do not use mouth
suction.
15. Wash your hands before handling anything (drinks, chewing gum, food etc.) which goes into
your mouth. Wash your hands when you leave the laboratory.
16. You should know how to exit the lab via two different exits, in the event of an
emergency.
17.Never use laboratory glassware as a food or drink container.
18. Never store food or drink in a laboratory refrigerator and never consume ice from a
laboratory refrigerator.
19. All accidents, including contact with chemicals, cuts, burns, or inhalation of fumes must be
reported to Course Teacher immediately. Any treatment beyond emergency first aid will be
referred to the student hospital.
20. Laboratory equipment and work area must be cleaned after finishing work.
21. If leaving a lab unattended, turn off all ignition sources and close the doors
22. Failure to observe laboratory safety rules and procedures may result in injury to you or to
fellow students.
It is in your own best interest to stay alert and to be aware of possible hazards in the
laboratory. Do not hesitate to call unsafe practices by your colleagues to the attention of
the course teacher and laboratory attendant.
Safety Hazards
Accidents in the laboratory are often the result of carelessness or ignorance either by
you or by your neighbors. Stay alert and pay constant attention to your own and to your
neighbors' actions. The safety precautions outlined below will be worthless unless you plan,
understand, and think through the consequences of every operation before you perform it. The
common accidents, which often occur simultaneously, are fire, explosion, chemical and
thermal burns, cuts from broken glass tubing and thermometers, absorption of toxic, but
non-corrosive chemicals through the skin, and inhalation of toxic fumes. Less common, but
obviously dangerous, is the ingestion of a toxic chemical. Each of these types is discussed in
a general way below, and more specific reference to certain hazards will be found in the
individual experiments.
1.Fire: There should never be open flames in the laboratory. Make it a working rule that water is
the only nonflammable liquid you are likely to encounter. Treat all others in the vicinity of a
flame as you would gasoline. Specifically, never heat any organic solvent in an open
vessel, such as a test tube, Erlenmeyer flask, or beaker, with a flame. Such solvents should
be heated in a hood with a steam bath, not a hot plate. Never keep volatile solvents, such as
ether, acetone, or benzene in an open beaker or Erlenmeyer flask. The vapors can and will creep
along the bench, ignite, and flash back if they reach a flame or spark.
2.Explosion: Never heat a closed system or conduct a reaction in a closed system (unless
specifically directed to perform the latter process and then only with frequent venting). Before
starting a distillation or a chemical reaction, make sure that the system is vented. The results of
an explosion are flying glass and spattered chemicals, usually both hot and corrosiv
3.Chemical and Thermal Burns: Many inorganic chemicals such as the mineral acids and
alkalis are corrosive to the skin and eyes. Likewise, many organic chemicals, such as acid
halides, phenols, and so forth are corrosive and often toxic. If these are spilled on the desk, in
the hood, or on a shelf, call for Lab. Attendant in cleaning them up. Be careful with hot plates
to avoid burns. Always assume that hot plates are HOT.
4.Cuts:The most common laboratory accident is probably the cut received while attempting to
force a cork or rubber stopper onto a piece of glass tubing, a thermometer, or the
side-arm of a distilling flask. Be sure to make a proper-sized hole, lubricate the cork or stopper
(lubrication is essential with a rubber stopper), and use a gentle pressure with rotation on the
glass part. Severed nerves and tendons are common results of injuries caused by
improper manipulation of glass tubes and thermometers. Always pull rather than push on the
glass when possible.
5.Absorption of Chemicals: Keep chemicals off the skin. Many organic substances
are not corrosive, do not burn the skin, or seem to have any serious effects. They are, however,
absorbed through the skin, sometimes with dire consequences. Others will give a serious
allergic reaction upon repeated exposure, as evidenced by severe dermatitis. Be careful about
touching your face or eyes in the lab; make sure your hands are clean first. Gloves will be
available in the lab. However, 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 a chemical reagent, remove them, wash your
hands, and get a new pair immediately.
6.Inhalation of Chemicals: Keep your nose away from chemicals. Many of the common
solvents are extremely toxic if inhaled in any quantity or over a period of time. Do not
evaporate excess solvents in the laboratory; use the hood or a suitable distillation
apparatus with a condenser. Some compounds, such as acetyl chloride, will severely irritate
membranes in your eyes, nose, throat, and lungs, while others, such as benzyl chloride, are
severe lachrymators, i.e. they induce eye irritation and tears. When in doubt, use the hood or
consult with the laboratory instructor about the use of chemicals required for your work.
7. Ingestion of Chemicals: The common ways of accidentally ingesting harmful chemicals
are: (1) by pipette, (2) from dirty hands, (3) contaminated food or drink and (4) food use of
chemicals taken from the laboratory. Below are ways to avoid accidental ingestion of chemical
reagents.
Safety Measures - If any accident in Laboratory
1.Eyes: GET HELP IMMEDIATELY! Chemicals in the eyes must be removed at once by
flooding with abundant quantities of water. Help the victim. Place the victim on the floor,
by force if necessary. One person should straddle the victim with knees on the floor, pouring
a moderate stream of water from a flask or beaker onto the bridge of the victim's nose so that
both eyes are flooded. Another person should squat at the head of the victim and roll back the
eyelids of both eyes; use the thumb and forefinger to spread the eyelids open. The victim will not
be able to do this voluntarily. Use at least several liters of water. When it is reasonably
certain that the excess chemicals have been washed away, take the victim for immediate
medical attention.
2.Chemicals on the skin: All chemicals which come in contact with the body should be
considered toxic and washed off completely with soap and water even if they do not
appear to be corrosive. Wash off corrosive substances by flooding with tap water, using the
safety shower if necessary, stripping off any clothing and shoes that are soaked with
chemicals. Except as follows, do not try to neutralize acids with alkali or alkalis with
acid; do not apply ointments or salves.
3.Inhalation of chemicals: Get fresh air. Report to the laboratory attendant all incidents
where prolonged exposure to laboratory fumes has induced faintness or a headache.
4.Ingestion of chemicals: Rush to hospital immediately. Vomiting is often dangerous,
especially if vomit gets into the lungs; most serious poisoning problems are best treated in the
hospital emergency room. If necessary, you can induce vomiting by swallowing as much
warm water as rapidly as possible, addition of one or two teaspoons of table salts per
glass of warm water will help. Vomiting should be encouraged by swallowing
additional water until the vomited liquid is clear.
5.Cuts: Serious bleeding should be controlled by direct pressure on the wound,
preferably with a clean gauze or cloth pad. Minor cuts should be washed with tap water
and allowed to bleed briefly and then press with a clean cotton pad or piece of gauze.
More severe cuts, especially with glass or other foreign objects in the wound, require medical
attention.
6.Clothing fires: Call for help and a blanket. When someone's clothing catches fire, the flame
and smoke can rise and be inhaled. Unless a shower is immediately available or can be
reached without inhalation damage, put the victim prone on the floor, forcibly if
necessary, and roll in a blanket; beat the flames out with hands or smother with heavy
garments.
7.Thermal burns: Any burn that is extensive or severe, or which involves the eyes or face,
should be considered serious. A burn involving the respiratory tract could be critical. Call the
ambulance. Minor burns should be treated by washing the burned area with cold tap
water. Wrapping the burned area lightly with a clean wet cloth or holding it in an ice bath will
help reduce the pain. Do not use ointments or salves unless so instructed by a physician.
8.Faintness:If the victim is conscious, have them sit down and place their head between
their knees. Support them to prevent a fall. If they are weak or have fainted, lay them on their
back on the floor, raise their feet and legs a little above the level of their head. Call for
medical assistance if the victim fails to regain consciousness within a half minute. Insist that
they remain quiet, seated or lying down, for a few minutes after recovery unless it is
necessary to take them to the hospital for treatment.
 Ex.No : 2
Date: HANDLING OF CHEMICAL SUBSTANCES

Chemical substance has definite chemical composition and characteristic

properties. It exists as solids, liquids, gases, or plasma and may change between these
phases of matter with changes in temperature or pressure. Chemical reactions convert one
chemical substance into another. A common example of a chemical substance is water; it has
the same properties and the same ratio of hydrogen (H) to oxygen (O), whether it is from a
river or made in a laboratory. Other examples of common chemical substances are diamond
(carbon), table salt (sodium chloride) and sugar (sucrose).
Chemicals have become a part of our life, sustaining many of our activities,
increasing agricultural productivity, preventing and controlling many diseases,. However one
cannot ignore that many of these chemicals may, especially if not properly used, endanger our
health and polluted our environment. It has been estimated that approximately one
thousand new chemicals come onto the market every year, and about 1,00,000 chemical
substances are used on a global scale. These chemicals are usually found as mixtures in
commercial products.
Most chemical accidents have a limited effect. Rarely there is a disaster in
pesticide manufacturing company in Bhopal, India, in 1984 and atomic energy plant in Japan in
2010, with thousands of deaths and many people permanently disabled. It is not just the worker
handling chemicals who is at risk. We may be exposed in our homes and laboratories through
misuse or by accidents. The environment may be affected, chemicals may pollute the air we
breathe, the water we drink, and the food we eat.
Remember: Chemicals have Power, and that is why they have become an important part
of our life. Respect that power and handle them with care.
How Chemicals Enter Our Body
Chemical substance can cause adverse effects after entering into the body or coming
to contact with it. There are three main ways / routes of exposure, for chemical substances to
enter the human body: Inhalation (breathing in) Absorption (through the skin or eye) Ingestion
(eating, swallowing). The chemical substances used in the laboratory may be dispersed into the
air to form dust, fumes, gas or vapour and can then be inhaled. During handling of liquid
chemicals there is a risk of absorbing harmful amounts of chemical through the skin. Dust
may also be absorbed through the skin of it is wetted form. The protective external layer of skin
may be softened (by toluene, dilute washing soda solution, etc), thus permitting other
 chemicals to enter readily to the bloodstream (such as phenol, benzene, etc). Dangerous
chemicals can enter the body through ingestion as gases, dusts, vapors, fumes, liquids or
solids. Inhaled dust may be swallowed, and food or drink may be contaminated by dirty
hands. Hence, Eating and drinking should be avoided during working in laboratory.
Hazards of Some Common Chemicals Used in Laboratory Sodium hydroxide (NaOH)
Sodium hydroxide exists as flakes (concentrate 100%) or as a liquid in various
concentrations, also known as caustic soda.

 Skin exposure to NaOH can cause nasal irritation, temporary loss of hair, intercellular
edema and burns.

 Contact with the eyes can result in ulceration, perforation, opacification, and blindness.

 Inhalation or ingestion can damage the respiratory and gastrointestinal tracts, coughing,
burns, breathing difficulty, coma and even esophageal cancer.

Hydrochloric acid (HCl)

HCl is a yellowish liquid with a strong biting odor is strongly corrosive.

 Exposure to higher levels: Blindness, rapid breathing, blue coloring of the skin, swelling
and spasm of the throat, suffocation and may develop asthma.

 Long-term exposure to low levels: Respiratory problems, eye and skin irritation, and
discoloration of the teeth.

 Direct contact with conc. hydrochloric acid will lead to serious burning of the skin.
Hydrogen Peroxide (H2O2)
Hydrogen peroxide is a clear liquid. It has strong oxidizing properties and is a powerful
bleaching agent. It is combustible.

 Eye Contact: Causes irritation, redness, and pain.

 Inhalation of high concentrations: Causes extreme irritation of the nose, lung and throat.
Typical symptoms include: headache, dizziness, vomiting and diarrhea.

 Eye Contact: Causes stinging and tearing of the eyes and splashes of high concentrations
cause severe corneal damage.

 Skin contact: Causes a temporary whitening or bleaching of the skin; if the skin is not
washed promptly, redness and blisters may develop.
Handling Liquid Chemicals
Use an appropriate container for the reagent. Avoid measuring volumes of strong acids
and alkaline solutions with your graduated cylinder held at eye level. Support your graduated
cylinder on your bench. Add hazardous liquids a little at a time, inspecting after each addition.
1. Mixing: If liquid chemicals are to be mixed with water, always add the
concentrated chemical to water rather than water to chemical. This keeps the new solution
dilute at all times and avoids many accidents. Usually addition should be done slowly, using
small quantities. It is especially important to add acid to water because of the heat generated.
2. Pipetting: Liquids are drawn into the pipette by applying a slight vacuum at the top, using
a small rubber suction bulb but NEVER THE MOUTH.
3. Heating: Liquids in beakers and flasks can be heated by placing them on a ring or tripod
stand on wire gauze with the container preferably supported by a clamp. Liquid should never
be heated in a graduated cylinder or in other volumetric glassware.
4. Disposal: If the liquid chemical can be disposed of in the sink, dispose of it by rinsing it
down the sink with large quantities of water. Avoid unnecessary splashing during this
process by pouring the chemical directly down the drain while the water is running vigorously.
Handling Solid Chemicals
Solid chemicals are most easily poured by tipping the general supply bottle and slowly
rotating it back and forth. Mere tipping of the bottle alone often causes large chunks to come
out very suddenly which leads to spills. If you use spatula, be sure that it is absolutely clean.
1.Mixing: If the solid is to be mixed with a liquid, add the solid to the liquid.
Additions should be made in small quantities except in special circumstances.
2. Disposal: As per the instructions, if you dispose of any solid chemicals in the sink, flush it
down the drain with copious amounts of running water. All other solids should be disposed of in
special containers provided for this purpose.
Chemical Safety

1. Consider all chemicals to be hazardous unless you are instructed otherwise.

2. Handle chemicals always with great care. Know what chemicals you are using.
Carefully read the label twice before taking anything from a bottle.

3. Make sure all chemicals are clearly and currently labeled with the substance name,
concentration, date, and name of the individual responsible.

4. Hold chemical containers away from your body.

5. Never put your spatula in a bottle to remove solid chemicals. If you do so, you will be
contaminating the chemical. Instead, pour solid directly into your container by tilting the
bottle and rotating it to control the amount dispensed.

6. If the solid chemical substance seems to be tightly packed and will not pour off, close
the container and then gently tap the bottle with the palm of your hand to loosen up the
caked solid.

7. Place solids into a beaker or weighing paper before weighing on a balance. Take care in
the use of the balances; they are expensive.

8. Clean up spills immediately.

9. Many common reagents, for example, alcohols and acetone, are highly
flammable. Do not use them anywhere near open flames.

10. Always pour acids into water. If you pour water into acid, the heat of reaction will cause
the water to explode into steam, sometimes violently, and the acid will splatter.

11. Never allow a solvent to come in contact with your skin. Always use gloves and
protective cloths/equipments.

12. While heating chemical in test tube, never points its mouth forward yourself or any
other people.
13. Never "smell" a solvent! Read the label on the solvent bottle to identify its
contents.

14. Do not waste chemicals; do not take more than what is required. Chemicals used in the
laboratory are costly. Wasted chemicals are not only costly but also polluted our
environment.
 Ex.No : 3

Date: HANDLING AND USES OF GLASSWARES

Laboratory glassware refers to a variety of equipment, used for scientific

experiments and work, especially in agricultural research laboratories. The three most common
types of glass used in laboratory glassware are 1) soft soda-lime glass; 2) hard borosilicate glass;
and 3) pure quartz glass. Most laboratory glassware is manufactured with borosilicate glass,
which is made up of silica and boron oxide, a particularly durable glass that can safely be used
for heating of chemicals over a flame. The borosilicate glasswares are resistance to high
temperature and corrosive material and hence are commonly used for reagent bottles. For
some applications quartz glass is used due to its ability to withstand high temperatures or
its transparency in certain parts of the electromagnetic spectrum as it made up of 99 per cent
silica. In other applications, especially some storage bottles, darkened brown or amber
(actinic) glass is used to keep out much of the UV and IR radiation, so that the effect of light
on the contents is minimized.
While working in a laboratory, you would have handled many different kinds of
glassware, each of which has specific features that make it well-suited to certain
applications. Knowing the differences between the kinds of glassware available to you will help
you to carry out experiments more efficiently. Just like borosilicate glasswares does not use for
Boron estimation. Some common glassware and their uses are given below.

1. BEAKER:
Beakers are cylindrical containers of varied sizes, with a small pouring lip.

Uses: Beakers can be used for mixing and transporting solutions, heating fluids over an
open flame, containing chemicals during a reaction and to hold samples for later utilization.

2. ERLENMEYER FLASK
Erlenmeyer flasks have a short cylindrical neck and have flat bottomed conical base, also
known as conical flask.

Uses: Erlenmeyer flasks are similar in function to beakers, but its conical shape help to
swirl solution without spilling and slow evaporation. In laboratory generally used for volumetric
titrations.
3. BUCHNERFLASK
Buchner flask is almost identical to Erlenmeyer flask, with the addition of a side-mounted
pipe on the neck of the flask, and made up of thick glass to withstand vacuum pressure.

Uses: Buchner flask uses for vacuum filtration or the collect the condensed liquid when
system is under negative pressure.

4. MEASURINGCYLINDER
Measuring cylinders are tall, narrow graduated pipe type containers provided with wide base.

Uses: Used for general purpose measuring volume of liquid. They are not use
for measurement of liquid for quantitative analysis as their printed graduations are generally
accurate about 1 per cent.

5. VOLUMETRIC FLASK
Volumetric flasks have a long neck with etched graduated marking to indicate precise
measurement of a given volume.
Uses: Used to prepare solution of concentration.
6. BURETTE
A burette is a graduated (0 to 50 ml) glass tube with a rubber tube tap or stopcock on the
bottom taper / bottom end.
Uses: Burettes are used for titration. It is also used for precise measurement of solution
in quantitative analysis.
7. PIPETTE
A pipette is a long glass tube, either graduated lengthwise or fixed volume provided with
bulb at center of the tube.
Uses: Pipettes are used to draw precisely measured amounts of liquid. The squeezer
bulbs are used to draw the fluid into pipette.
8. FUNNELS
Round wide mouth conical shaped having narrow tube at bottom end help for funneling.
Uses: Funnels are used for pouring / funneling liquids from one container to another or
for filtering when equipped with filter paper.
9. TEST TUBE
A test tube is a relatively slim finger like vessel with a rounded bottom.
Uses: Test tubes are designed to hold relatively small quantities of chemicals and used to
contain small reactions. In microbial / pathological studies used for isolation of microbial
colonies on solid media.
HANDLING OF GLASSWARES
It is very important that glassware is used and stored properly to prevent failure or injury
during working in laboratory.

 All glasswares should be inspecting before use, scratches greatly reduce its
strength and there is possibility of breakage during working hence discarded.

 Do not let glasswares / apparatus in contact with metal, gravel, stone, pebble even
other glassware to avoid damage.

 Plastic stirring rods and scrapers should be used to prevent scratches and to prolong
life of the glasswares.

 Glass should not be scribed or etched, especially important when it is to be used for
vacuum or pressure work.

 All laboratory glassware should be clean and dry glassware immediately after use to
prevent chemical residue from congealing or hardening.

 Glasswares joints and stopcocks will be greased for smoother application, which will also
prevent leakages and breakages during operation.

 However, when not in use, remove and clean all stoppers, adapters and plugs to prevent
“sticking” problems.

 Glassware or quartzware is to be heated, it must be triple rinsed in deionized or distilled

water after cleaning and dried thoroughly. This will ensure contaminates are not burned
onto glassware permanently.
 Ex.No : 4
CLEANING WASHING AND STERILIZATION OF LABORATORY
Date:
GLASSWARES

Cleaning washing and sterilization of laboratory glasswares

Laboratory procedures require exact methods and should include good glasswares
cleaning to insure excellent lab results. In all instances lab-wares should be physically
clean, including both chemical residue free and grease free, and in many cases even be steril. All
class A glassware that is used in precise measuring of liquids should have fully wettable
surfaces. A good test is to use distilled water and see if the water wets all the inner surface
equally.
Oily or other residues will not only contaminate the reaction and test results but also alter
the measurement of the liquids. Good cleaning practices should also be accompanied by good
inspection of the glass surfaces of chips craks or abrasions which will cause mechanical failure.
Cleaning glasswares is not as simple as washing the dishes. It is generally easier to clean
glasswares if you do it right way.
Cleaning

 Always wash glasswares immediately after use. If a thorough cleaning is not

immediately possible, always allow the glasswares to soak. If not cleaned
immediately some residues may be impossible to remove.

 Most new glass is slightly alkaline and should be washed upon receipt and generally
can be soaked in 1 % HCl or HNO3 solution before wash and deionized water rinse.

 Never soak for long periods in strong alkaline solutions as it will damage the glass.

 Always follow up a soap or acid wash with a good deionized water rinse

 Always use soft brushes with a wooden or soft plastic handles to avoid abrasion. Do
not use wire brushes with a wire core as it can abrade the glass.
Glass cleaners
A great cleaner and also removes organic residues. Use gloves and well ventilate the area
when using chromic acid as it is a carcinogen and very corrosive. Make sure metal clamps or
flanges are removed. It is best to fill the vessel or soak the item in the solution for a short time. In
a plastic tub so that you can contain the wash material, then rinse immediately several times
before proceeding to a detergent wash. Make sure the residual chromic acid is diluted after use
and disposed of properly and according to your local or company regulations.

How to wash Common laboratory chemicals

1. Water soluble solutions: Rinse 3 to 4 times with deionized water. (e.g. sodium chloride or
sucrose solutions).
2. Water insoluble solutions such as solutions in hexane or chloroform, rinse 2-3 times with
ethanol or acetone, rinse 3-4 times with deionized water, then put the glass wares away. In
some solutions other solvent needs to be used for the initial rinse.
3. Strong acids: (Concentrated HCl or H2SO4) Under the fume hude carefully rinse the glassware
with copious volume of tap water. Rinse 3 to 4 times with deionized water.
4. Strong bases: 6 M NaOH or concentrated NH4OH) under the fume hood, carefully rinse the
glassware with copious volume of tap water. Rinse 3-4 times with deionized water.
5. Weak acid: (acetic acid solutions or dilutions of strong acids such as 0.1 M or 1 M HCl or
H2SO4) Rinse 3-4 times with with deionized water before putting the glasswares away.
6. Weak bases : ( 0.1 M and 1 M NaOH and NH4OH) Rinse thoroughly with tap water to
remove the bases, then Rinse 3-4 times with with deionized water before putting the
glasswares away.
Washing Special Glasswares
Glasswares used for organic chemistry
Rinse the glasswares with the appropriate solvent. Use deionized water –soluble contents. Use
ethanol-soluble contents, followed by rinses in deionized water. Rinse with other solvents as
needed, followed by ethanol and finally deionized water. If the glasswares requires scrubbing,
scrub with brush using hot spray water, rinse thoroughly with tap water, followed by rinses with
deionized water.
Burettes
Wash with hot soapy water, rinse thoroughly with tap water, then rinse 3-4 times with
deionized water. Be sure the final rinses sheet off the glass. Burettes need to be thoroughly clean
to be used for quantitative lab work.
Pipettes and volumetric flask
In some cases, there may be the need to soak the glasswares in soapy water. Clean pipettes
and volumetric flask using soapy water. The glasswares may require scruibing with brush. Rinse
with tap water followed by 3-4 times with deionized water.
Sterilizing Glassware and Instruments

 Heated bead sterilization using a glass bead sterilizer. These sterilizers heat to
approximately 275-350° C and will destroy bacterial and fungal spores that may be found
on your instruments. The instruments simply need to be inserted into the heated glass
beads for a period of 10 to 60 sec. The instruments should then be placed on a rack under
the hood to cool until needed.

 Flame sterilization. Instruments can be dipped in ethyl alcohol and flamed to burn off
all bacteria and fungi. Safety is a major concern when using ethyl alcohol. Alcohol is
flammable and if spilled near a flame will cause an instant flash fire. This problem
is compounded in laminar flow hoods due to the strong air currents blown towards the
worker. Fires commonly start when a flamed instrument is thrown back into the
alcohol beaker. In case of fire do not panic. Limiting the supply of oxygen can easily put
out fires.

 Hot-dry sterilization. Metal instruments, glassware, aluminum foil, etc., can be sterilized
by exposure to hot dry air (130°-170°C) for 2-4 hr in a hot-air oven. All items should
be sealed before sterilization but not in paper, as it decomposes at 170°C.

 Autoclaving. Autoclaving is a method of sterilizing with water vapor under pressure.

Nearly all microbes are killed by exposure to the super-heated steam of an autoclave for
10-15 minutes. All objects should be sterilized at 121°C and 15 psi for 15-20 min. Cotton
plugs, gauze, labware, plastic caps, glassware, filters, pipettes, water, and nutrient
media can all be sterilized by autoclaving, but autoclaving is not advisable for metal
instruments due to the rust it may cause.
STERILIZATION OF GLASSWARE

The glassware used for microbial studies need to be cleaned and sterilized properly. If not
done so, the glassware will get contaminated which can cause health-problems as well
as contamination of experimental microbial cultures that are used for the purpose of study
 Ex.No : 5
PREPARATION OF STANDARD SOLUTIONS
Date:

Standard solution means a solution which the concentration is known, irrespective of

the manner in which its concentration is expressed. The concentration of solutions may be
expressed in a number of ways, the general methods are:-

1. The percent solutions

2. Molarity or Molar solutions

o Atomic Weight of element is proportional to the Actual Weight of an element,

hence it is customary to expressed in grams.

o The atomic weight of an element expressed in gram, is called Gram Atomic

Weight or simply gram-atom.

o Similarly, Molecular Weight of any given substance is simply the

Gram Molecular Weight or gram-mole. Gram mole = Mole

Molarity (M)

The conc. of solute in a solution expressed as the number of moles of solute per liter of
solution.

Molality (m)

The conc. of solute in a solution expressed as the number of moles of solute per kilogram
of solvent.
Equivalent weight

Equivalent weight of an element may be defined as that quantity of the element in grams
which reacts with or displace l gram atom of hydrogen or l gram atom of oxygen or l
gram atom of chlorine.

Solution

A solution consists of a Solute (one substance) dissolved in Solvent (Another substance)

Concentration

The concentration of a solution is the amount of the solute dissolved per unit volume or
weight of the solvent.

The concentration of solution can be expressed in a number of ways:

a. Gram of solute per l00 gm of the solvent. (w/w) Per cent

b. Gram of solute per l00 ml of the solvent. (w/v) Per cent

c. Gram of solute per l000 ml (Lit.) of the solvent. (w/v) Molar

d. Grams of solute perl000 gm of the solvent. (w/w) Molal

e. Milliliter (ml) per l000 ml (Lit) of the solvent (v/v)

Normal Solution (N)

Normal Solution (N): A normal solution is one which is prepared by dissolving l gram
equivalent of the solute per l000 ml (Lit.) of the solution. The unit symbol N is used to
denote “mol/L”. Sometimes “Eq/L” also used
 Chemical Equivalent 1N 2N 0.5 N 0.1 N
Substance Weight =Eq.wt. X=Eq.wt.X0.5
2 (N/2)=Eq.wt.X 0.1
=Eq.wt./1
(N/10)
=Eq.wt./ 2 =Eq.wt./ 10

HCI 36.45 36.45 72.90 18.23 3.65

H2SO4 49.01 49.01 98.02 24.51 4.90

H3PO4 32.64 32.64 65.29 16.32 3.26 4

NaOH 40.00 40.00 80.00 19.99 4.00

Ca(OH)2 37.03 37.03 74.05 18.51 3.70

Na2CO3 52.97 52.97 105.94 26.49 5.30

Equivalent weight of Acids:-

The equivalent weight of an acid is that weight of acid which contains one replaceable H
atom. Thus the molecular weight itself is the equivalent weight for monobasic acids for
dibasic acids it is mole weight divided by two, for tri basic molecular weight divided by three.
 Acid Mol. Wt. Valency Eq Wt.

HCI 36.45 1 36.45

H2SO4 98.02 2 49.01

H3PO4 97.93 3 32.64

Equivalent weight of bases

The equivalent weight of an base is that weight of acid which contains one replaceableO H
group. l7.008 g Oh are equivalent to l.008 gms of H. Thus equivalent weight of
NaOH, KOH and NH4OH are equal to its molecular weight, whereas eqyuivalent weight of
Ca(Oh)2,Ba(OH)2the molecular weight divided by two.

Base Mol. Wt. Valency Eq Wt.

NaOH 40 1 40.00

Ca(OH)2 74.05 2 37.03

Na2CO3 105.94 2 52.97

Equivalent weight of Acids

The equivalent weight of an acid is that weight of it which contains one gram
equivalent weight of replaceable hydrogen (All acids contains replaceable H atoms).
Example 1: One mole of HCl contains one replaceable hydrogen atom, hence the gram
equivalent weight of HCl (l:l) i.e. (l+35.5=36.5)

Example 2: One mole of H2SO4 contains two replaceable hydrogen atom, hence the
equivalent weight of H2SO4 (2:l:4) i.e. (2+32.6+63.96= 98.56)

Equivalent weight of Bases

The gram equivalent weight of base is that weight of the base, which contains one
gram equivalent weight of the hydroxyl ion (All acids contains replaceable H atoms)
Example l: One mole of NaOH contains one replaceable hydroxyl ion, hence the gram
equivalent weight of NaOH (l:l:l) i.e. (23+l6+l =40)

Example 2: One mole of Ca(OH)2 contains two replaceable hydroxyl ion, hence the
equivalent weight of Ca(OH)2 (l;2:2) i.e. (40.02+3l.98+2=74)

Dilution of acids

Example l: Prepare 500 ml, lN solution of sulphuric acid (H2SO4)from concentrated

acid (Purity, 99%, Sp. gravity, l.84 & Mol wt., 98.08)

Eq. Wt of H2SO4 = 49.04 (98.08/2 i.e. replaceable H)

Eq. wt. of acid X V2 X Normality X l00 Required

Vol of conc. acid (Vl) =
For the solution l000 X Sp. gravity X Purity (%)
 49.04 X 500 X l X l00
Vl =
l000 X l.84 X 99

V1= 13.46 ml

Ans. l3.46 ml conc. H2S04 is to be diluted in 500 ml Distilled Water in a vol. flask to get
lN solution of H2SO4.

Example l: Prepare 750 ml, 0.lN solution of Orthophosphoric acid (H3PO4) from
concentrated acid (Purity, 93%, Sp. gravity, l.75 & Mol wt., 98.00)

Eq. Wt of H3PO4 = 32.66 (98.00/3 i.e. replaceable H)

Vl = l.43 ml
Ans. l.43 ml conc. H3P04 is to be diluted in 750 ml Distilled Water in a vol. flask to get
0.lN (N/l0) solution of H3PO4.

Parts per million (ppm) Concept

ppm solutions are those solutions in which a substance is present in a very small quantity.
It represents Gram of solute per million grams of solution
OR
Gram of solute per million ml of solution

Thus, lppm of NaCl solution represents:

l ppm NaCl = l mg NaCl / Lit of solution
= l mg NaCl / l000 ml solution
= lµg NaCl / ml of solution
Therefore
l00 ppm NaCl = l00 mg NaCl in l000 ml solution
l000 ppm NaCl = l000 mg NaCl in l000 ml solution
= l gm NaCl / Lit.

Example: How would you prepare l00 ppm phosphate solution by using
KH2PO4 (l00 ppm = l00 mg/L or 0.l gm/L PO4)

Answer: Molecular wt. of KH2PO4 = 39.l+(2xl)+30.97+(4xl5.99)

= l36.03
lgm molecular wt of PO4 = 30.97

30.97 gm of P content in l36.03 gm of KH2PO4

Thus, f or l00 mg or 0.l gm

For, l00 ppm P solution = 0.439 gm KH2PO4

(OR)
l000 ppm P solution = 4.39 gm KH2PO4

Example : How would you prepare l00 ppm Potash solution by using KCl
( l00 ppm = l00 mg/L or 0.l gm/L K)

Answer: Molecular wt. of KCl = 39.l + 35.45 = 74.55

l gm molecular wt of K = 39.l
39.l gm of K content in 74.55 gm of KCl
Thus, for l00 mg or 0.l gm

For, l00 ppm K solution = 0.l9l gm KCl

(OR)
l000 ppm K solution = l.9l gm KCl
 Ex.No : 6
NEUTRALIZATION OF ACID AND BASES
Date:

For thousands of years people have known that vinegar, lemon juice and many
other foods taste sour. However, it was not until a few hundred years ago that it was
discovered why these things taste sour - because they are all acids. The term acid, in fact,
comes from the Latin term acere, which means "sour". While there are many slightly
different definitions of acids and bases, in this exercise we will introduce the
fundamentals of acid/base chemistry.

In the seventeenth century, the Irish writer and amateur chemist Robert Boyle first labeled

Acids taste sour, are corrosive to metals, change litmus (a dye extracted from lichens)
red, and become less acidic when mixed with bases.

Bases feel slippery, change litmus blue, and become less basic when mixed with acids.

Acids:

For example, hydrochloric acid (HCl) dissolves in water as follows:

H2O
HCl H+(aq) + Cl-(aq)

Base:

For example, a typical base according to the Arrhenius definition is sodium

hydroxide (NaOH):

H2O
NaOH Na+(aq) + OH-(aq)
WHAT IS AN ACID OR A BASE?

In a broader way

Acid: a substance which when added to water produces hydrogen ions [H+].

Base: a substance which when added to water produces hydroxide ions [OH-].

Acids and Bases as being "strong" or "weak". Strong compounds are compounds
that completely break up in water. In other words, if we're talking about a strong acid, all

of the H+ ions break away from the molecule in water. For strong bases, all of the OH-
ions break away from the molecule in water. There is a difference between a "strong"
acid and a "reactive" one. Strong acids are all reactive, but some "weak" acids can also be
extremely reactive. A good example of a weak, reactive acid is hydrofluoric acid, HF.

 A base is a proton (hydrogen ion) acceptor.

 The HA is an acid because it is donating a proton (hydrogen ion) to the water.
 The water is a base because it is accepting a proton from the HA.
 The reversible reaction contains two acids and two bases. We think of them in pairs,
called conjugate pairs.

 When the acid, HA, loses a proton it forms a base, A-. When the base, A-, accepts a
proton back again, it obviously reforms the acid, HA. These two are a conjugate pair.
 Members of a conjugate pair differ from each other by the presence or absence of the
transferable hydrogen ion.

 If you are thinking about HA as the acid, then A- is its conjugate base.

 If you are thinking about A- as the base, then HA is its conjugate acid.
 The water and the hydroxonium ion are also a conjugate pair. Thinking of the water as
a base, the hydroxonium ion is its conjugate acid because it has the extra hydrogen
ion which it can give away again.
STRONG ACIDS AND STRONG BASES

The common acids that are almost one hundred percent ionized are: HNO3 - nitric acid
HCl - hydrochloric acid H2SO4 - sulfuric acid HClO4 - perchloric acid HBr -
hydrobromic acid HI - hydroiodic acid

The acids on this short list are called strong acids, because the amount of acid
quality of a solution depends upon the concentration of ionized hydrogen’s. You are not
likely to see much HBr or HI in the lab because they are expensive. You are not likely
to see perchloric acid in a school setting because it can explode if not treated carefully.
Other acids are incompletely ionized, existing mostly as the unionized form.
Incompletely ionized acids are called weak acids, because there is a smaller
concentration of ionized hydrogen’s available in the solution. Do not confuse this
terminology with the concentration of acids. The differences in concentration of the
entire acid will be termed dilute or concentrated. Muriatic acid is the name given to an
industrial grade of hydrochloric acid that is often used in the finishing of concrete.
In the list of strong acids, sulfuric acid is the only one that is diprotic, because it
has two ionizable hydrogen’s per formula (or two mols of ionizable hydrogen per mol
of acid). (Sulfuric acid ionizes in two steps. The first time a hydrogen ion splits off of the
sulfuric acid, it acts like a strong acid. The second time hydrogen splits away from the
sulfate ion, it acts like a weak acid.) The other acids in the list are monoprotic, having
only one ionizable proton per formula. Phosphoric acid, H3PO4, is a weak acid.
Phosphoric acid has three hydrogen ions available to ionize and lose as a proton, and so
phosphoric acid is triprotic. We call any acid with two or more ionizable hydrogen’s
polyprotic.
Likewise, there is a short list of strong bases, ones that completely ionize into
hydroxide ions and a conjugate acid. All of the bases of Group I and Group II metals
except for beryllium are strong bases. Lithium, rubidium and cesium hydroxides are not
often used in the lab because they are expensive. The bases of Group II metals,
magnesium, calcium, barium, and strontium are strong, but all of these bases have
somewhat limited solubility. Barium hydroxide has a high enough solubility to really call
it the only dibasic strong base. Magnesium hydroxide has a particularly small solubility.
Potassium and sodium hydroxides both have the common name of lye. Soda lye (NaOH)
and potash lye (KOH) are common names to distinguish the two compounds.
 Sr. No Name Formula
1 Lithium hydroxide LiOH
2 Sodium hydroxide NaOH
3 potassium hydroxide KOH
4 Rubidium hydroxide RbOH
5 Cesium hydroxide CsOH
(Mg(OH)2
6 Magnesium hydroxide
7 Calcium hydroxide Ca(OH)2
8 Strontium hydroxide (Sr(OH)2
9 Barium hydroxide Ba(OH)2

The bases of Group I metals are all monobasic. The bases of Group II metals
are all dibasic. Aluminum hydroxide, Al(OH)3 is tribasic. Any material with two or
more ionizable hydroxyl groups would be called polybasic. Most of the alkaline organic
compounds (and some inorganic materials) have an amino group - (NH2) rather than an
ionizable hydroxyl group. The amino group attracts a proton (hydrogen ion) to become -

(NH3 )+. (The dash before the (NH3)+ or (NH2) indicates a single bonding electron, so
this is attached to something else by a covalent bond.) By the Lowry- Brønsted definition,
an amino group definitely acts as a base, and the effect of removing hydrogen ions from
water molecules is the same as adding hydroxide ions to the solution.

PROPERTIES OF ACIDS

For the properties of acids and bases we will use the Arrhenius definitions.

Acids release a hydrogen ion into water (aqueous) solution. You will usually see the
formula for an acid with the ionizable hydrogen at the beginning, such as HCl,
hydrochloric acid, or H(C2H3O2), acetic acid.

Acids neutralize bases in a neutralization reaction. An acid and a base combine to

make a salt and water. A salt is any ionic compound that could be made with the anion
of an acid and the cation of a base. The hydrogen ion of the acid and the hydroxide ion
of the base unite to form water.

Acids corrode active metals. Even gold, the least active metal, is attacked by an acid,
a mixture of acids called 'aqua regia,' or 'royal liquid.' When an acid reacts with a
metal, it produces a compound with the cation of the metal and the anion of the acid
and hydrogen gas.

Acids turn blue litmus to red. Litmus is one of a large number of organic compounds that
change colors when a solution changes acidity at a particular point. Litmus is the oldest
known pH indicator. It is red in acid and blue in base. The phrase, 'litmus test,' indicates
that litmus has been around a long time in the English language. Litmus does not change
color exactly at the neutral point between acid and base, but very close to it. Litmus is
often impregnated onto paper to make 'litmus paper.
Acids taste sour.

Stomach acid is hydrochloric acid. Although tasting stomach acid is not pleasant,
it has the sour taste of acid. Acetic acid is the acid ingredient in vinegar. Citrus fruits such
as lemons, grapefruit, oranges, and limes have citric acid in the juice. Sour milk, sour
cream, yogurt, and cottage cheese have lactic acid from the fermentation of the sugar
lactose.

PROPERTIES OF BASES

Bases release a hydroxide ion into water solution. (Or, in the Lowry
- Brønsted model, cause a hydroxide ion to be released into water solution by accepting a
hydrogen ion in water.)

Bases neutralize acids in a neutralization reaction.

The word - reaction is: Acid plus base makes water plus a salt. Where
'Y' is the anion of acid 'HY,' and 'X' is the cation of base 'XOH,' and 'XY' is the salt
in the product, the reaction is: HY + XOH HOH + XY

Bases denature protein. This accounts for the "slippery" feeling on hands when exposed
to base. Strong bases that dissolve in water well, such as sodium or potassium lye are
very dangerous because a great amount of the structural material of human beings is
made of protein. Serious damage to flesh can be avoided by careful use of strong bases.

Bases turn red litmus to blue. This is not to say that litmus is the only acid - base
indicator, but that it is likely the oldest one.
Bases taste bitter. There are very few food materials that are alkaline, but those that are
taste bitter. It is even more important that care be taken in tasting bases. Tasting of bases
is more dangerous than tasting acids due to the property of stronger bases to denature
protein.

Neutralization

As you can see from the equations, acids release H+ into solution and bases release OH-.

If we were to mix an acid and base together, the H+ion would combine with the OH- ion
to make the molecule H2O, or plain water:

H+(aq)+OH-(aq) H2O

The neutralization reaction of an acid with a base will always produce water and a
salt, as shown below:

Acid Base Water Salt

HCl + NaOH H2O + NaCl

HBr + KOH H2O + KBr

Acid Base Salt

HCl + NaHCO3 H2CO3 + NaCl

In this example, the carbonic acid formed (H2CO3) undergoes rapid decomposition
to water and gaseous carbon dioxide, and so the solution bubbles as CO2 gas is released.
pH

Under the Brønsted-Lowry definition, both acids and bases are related to the
concentration of hydrogen ions present. Acids increase the concentration of hydrogen
ions, while bases decrease the concentration of hydrogen ions (by accepting them). The
acidity or basicity of something, therefore, can be measured by its hydrogen ion
concentration.
In 1909, the Danish biochemist Sören Sörensen invented the pH scale for
measuring acidity. The pH scale is described by the formula:

For example, a solution with [H+] = 1 x 10-7 moles/liter has a pH equal to 7 (a

simpler way to think about pH is that it equals the exponent on the H+ concentration,
ignoring the minus sign). The pH scale ranges from 0 to 14. Substances with a pH

between 0 and less than 7 are acids (pH and [H+] are inversely related - lower

pH means higher [H+]). Substances with a pH greater than 7 and up to 14 are bases

(higher pH means lower [H+]). Right in the middle, at pH = 7, are neutral substances, for

example, pure water. The relationship between [H+] and pH is shown in the table below
alongside some common examples of acids and bases in everyday life.

[H+] pH Example

1 X 100 0 HCl

1 x 10-1 1 Stomach acid

1 x 10-2 2 Lemon juice

Acids
1 x 10-3 3 Vinegar

1 x 10-4 4 Soda

1 x 10-5 5 Rainwater
 1 x 10-6 6 Milk

Neutral 1 x 10-7 7 Pure water

1 x 10-8 8 Egg whites

1 x 10-9 9 Baking soda

1 x 10-10 10 Tums® antacid

Bases
1 x 10-11 11 Ammonia

1 x 10-12 12 Mineral lime - Ca(OH)2

1 x 10-13 13 Drano®

1 x 10-14 14 NaOH

A base is more dangerous than tasting acids due to the property of stronger bases to
denature protein.

The pH Scale

pH is a measurement of the H+ concentration in a liquid. If there's a high H+

concentration, the pH indicates that you've got a very acidic solution. If the solution is

neutral, there's only a small H+ concentration, and the pH reflects that. If the solution is

basic, there's almost no H+ concentration, and you can tell that by the pH number. pH is

nothing more than a way of telling how concentrated an H+ solution is.

 As you can see, pH values between 0 and 7 are acidic and pH values between 7 and 14
are basic. pH values of exactly seven are called "neutral" solutions - if the pH is 6.99 it's an
acidic solution and if it's 7.01 it's basic. However, people usually refer to solutions with a pH
between 6 and 8 as being "neutral" because they're mostly neutral.
 Ex.No : 7
PREPARATION OF BUFFER OF DIFFERENT STRENGTH OF PH VALUE
Date:

The pH value which is measure of the hydrogen (or hydroxyl) ion activity of the soil water system
indicates whether the soil is acidic, neutral or alkaline in reaction. Since the crop growth suffers much both
under very low (strongly acidic) as well as high (alkali) pH, suitable reclamation measures become
necessary. Presence of neutral soluble salts is not normally reflected in the pH but when in large excess they
suppress the ionic activity.

The instrument for the pH measurement commonly used a glass electrode pH meter with calomel
reference electrode including salt bridge. Most digital pH meters have single (Combined ) electrode
assembly. The instrument being a potentiometer, the pH scale has to be calibrated before use with buffer
solution of known pH values.

Standard buffer solutions :

These may be of pH 4.0, 7.0 or 9.2 in pure water or in other range of expected pH value. In case of
buffer tablets (available commercially) a single piece of table is to be dissolved in freshly doubled distilled
water and made up to 100 ml. It is necessary to prepare a fresh buffer, after every few days as the solutions
do not keep for long. A 0.05 M analytical grade (AR) potassium hydrogen phthalate (KHC 8H4O4) mol. Wt.
204.14) gives a pH of 4.0 (actually 3.97) at 25 0C and this can also be used as a standard buffer. A stock
solution of 0.25 M is prepared by dissolving 51.04 g of pure dry salt in warm water and making up to 1 litre.
Three to four drops of toluence are to added to prevent growth of mould. A solution of 10 ml of this solution
to 50 ml with water gives 0.05 M solution.

(Note :- The buffer tablets and the prepared solutions of different pH such as 4.0,7.0 and 9.2 and
10.0 are commercially available in the market.)
 Ex.No : 8
ESTIMATION OF FERTILIZER DOSES FOR FILED AND POT
APPLICATIONS
Date:

Fertilizers are materials (solid, liquid or gas) containing one or more nutrient
elements in the form of chemical compounds of the organic or inorganic nature. These
occur either as a natural deposit or synthesized in chemical factory. These may be
graded as concentrated plant foods. Upon application to soil, these materials dissolve
fully or sparingly in the soil water system and release the nutrient(s) in the ionic form
which are absorbed by the plants .However, some fertilizers after application to soil,
undergo chemical transformations before releasing the nutrients in plant available form.
Irrespective of the mode of release, a nutrient has to be present in a specific ionic form,
so that plants can absorb it from soil solution through the root system.

The amount of fertilizer needed by a soil depends up on its nutrient supplying

capacity to crop. Hence, it is generally advocated that the fertilizers should be applied on
the soil test base.
The seventeen nutrients recognized as essential for plant growth are
carbon,hydrogen,oxygen,nitrogen,phosphorous,potassium,calcium,magnesium,sulphur,ir
on,maganese,zinc,copper,boron,molybdenum,chlorineand nickle.
Plants need a variable quantity of nutrient within a narrow range and in an
optimum proportion with respect to other nutrients for their vegetative growth and
ultimate yield.

Principles of Fertilizer Application

➢ How much to apply

➢ When to apply

➢ How to apply

➢ Basal dose Kg/ha

➢ Top dressing

➢ As per soil test values

 Approaches of fertilizer application

➢ Recommended dose of fertilizer

➢ As per soil test

➢ Expected Yield

Classification of soil as per available nutrients

Class Available

N P K O.C. RDF for crops

Very low

Low

Medium

Medium high

High

Very high
Example
Quantity of fertilizer required for rainfed cotton

(RDF 60:30:30 NPK/ha)

Sr. Name of Nutrient C.F. Basal 30 DAS

No Fertilizer content
1 Urea + 46 2.17 65 65
SSP + 16 6.25 187 00

MOP 60 1.66 50 00
2 20:20:0 + 5.00 150 0

MOP 1.66 50 0
Urea 2.17 0 65
3 19:19:19 + 5.26

Urea 2.17 0 65
4 DAP + 2.17

Urea + 2.17

MOP 1.66

C.F. = Calculation factor

If available NPK in the soil are 200:30:600 (Kg/ha) and expected yield will be
20q/ha. Calculate NPK for rainfed cotton.

Nutrients Formula Quantity and

Nutrient required
kg/ha
N (13.1 x 20)-(0.75 x 200) =(262-
150) 112

P (6.83 x 20)-(2.84 x 30) =

(137-85) 52

K (8.75 x 20) – (0.17 x 600) =(171-102)

69

RDF 112:52:69 NPK/ha

How to calculate the fertilizer for the experimental plot

If recommended dose of cotton (rainfed is 60 :30 :30 kg NPK ha-1 . The

fertilizers are to be given in the form of Nitrogen – as Urea , Phosphorous - as Single
super phosphate and potassium as murate of potash . Plot size of the layout is is 24 sq
mt.

Then how much for 24 sq mt .?

1 ha = 10,000 sq mt.

N:

Means, 144 gm N = 144 X 2.17 = 312.48 gm urea is required for 24 sq mt area.

SSP:

Means, 72 gm P = 72 X 6.25 = 450 gm SSP is required for 24 sq mt area.

MOP:

Means, 72 gm K = 72 X 1.60 = 115.2 gm MOP is required for 24 sq mt ar

Urea is having 46 % Nitrogen
i.e., 100 kg of urea is containing 46 % Nitrogen
How much urea is required for 1 kg nitrogen application?
100/46 = 2.17 kg Urea

Hence 2.17 is a calculation factor for Nitrogen (if fertilizer is to given in the form of urea.)

SSP contains 16% P

i.e. 100 kg of SSP contains 16 % P.
How much SSP is required for 1 kg of P application?
100/16 = 6.25 Kg SSP

Hence 6.25 is a calculation factor for Phosphorous in the form of SSP.

MOP contains 60% K

i.e. 100 kg of MOP contains 60 % K.
How much MOP is required for 1 kg of K application ?
100/60 = 1.66 Kg MOP
Hence 1.66 is a calculation factor for Potassium in the form of MOP.

DAP
Di-ammonium phosphate contains 18 % N and 46 % P
1 kg of DAP will supply 0.18 % N and 0.46 % P. for 30 kg of P 65 kg of DAP is
required. This quantity of DAP will also supply 11.7 kg nitrogen, along with 30 kg P,
hence,
Quantity of N is to be deducted from the 30 kg N requirement (30-11.7=18.3)
then it will comes to 18.30 kg
N hence, 18.30 X 2.17 = 39.71 kg urea. Therefore 40 kg of urea is to be supplied as
basal dose to fulfill the recommended dose of nitrogen (30 kg N) and MOP – 50 kg
as a basal for the supply of K. Again remaining dose of nitrogen for top dressing is to
be supplied through urea. Hence ,30 X 2.17 = 65 kg Urea is to be supplied top
dressing ( 30 kgN).

Application of micronutrient fertilizer

If micronutrient deficiency is occur as per the soil testing report then apply
the micronutrient as per recommendation given
For spraying of Urea and DAP and Micronutrients, the quantity of
fertilizer use as follows
Urea 1 % ------ 100 g Urea dissolved in a 10 lit water

Urea 2 % ------- 200 g Urea dissolved in a 10 lit water

DAP 1 % -------- 100 g DAP dissolved in a 10 lit water

DAP 2 % ------ 200 g DAP dissolved in a 10 lit water

Zinc sulphate 0.5 % -------- 50 g of zinc sulphate in 10 lt of water

Ferrous ammonium sulphate 0.5 %---50 g of ammonium sulphate in 10 lt of

water

Copper sulphate 0.4 % ----- 40 g

Ammonium molybadade - 0.05 %------- 5 g of Ammonium sulphate in 10 lt. of

water.

Borax -0.2 % ---------20 g Borax in in 10 lt. of water.

Note:- While spraying the micronutrients add filtered solution of 0.5 % lime.

Pot Experiment

22, 40000 kg is a soil weight for one hectare are . From this we can calculate
the
Weight of manure as well as fertilizer per 10 kg of soil weight required for pot
Experiment.

Example : 1
10 t. FYM is required for one ha. How much for 10 kg soil in a pot ?

10 X 10000 /2240000 = 0.044643 kg per pot = 44.64 g FYM

Example :2

If 60 kg Nitrogen is required for one ha. area of rain fed cotton

 means 130 kg urea is required.
How much quantity is for 10 kg soil in a pot?

(10X 130) / 22,40000 = 0.00058 kg = 0.58 g urea per pot

Accordingly , calculate the quantity of various fertilizers for the pot culture
experiments

Note:- If quantity of fertilizer is very less in such case the quantity of

fertilizer should be dissolve in a water and to be applied uniformly in a pot.
Ex. No : 9 PREPARATION OF CULTURE MEDIA – NUTRIENT BROTH,
Date: NUTRIENT AGAR AND POTATO DEXTROSE AGAR

Aim
To prepare the culture media for the growth of microorganisms using Nutrient medium and
Potato Dextrose Agar medium.
Principle
Bacteria are generally cultivated in broth (medium devoid of agar) and agar media. The
requirement of nutrients is met by supplementing beef extract (source of mineral salts, organic
carbon and nitrogen, vitamins) and peptone (semi digested protein, Nitrogen source). The fungi
(Molds) are generally grown in Potato Dextrose Agar (PDA) medium.
To satisfy the diverse nutritional needs of bacteria and fungi, Microbiologists employ two
major categories of media for routine cultivation. They are:
1. Chemically defined media e.g. Inorganic synthetic broth, Glucose salt broth
2. Complex media e.g. Nutrient medium & PDA
Compositions of Media:
Nutrient Broth
Medium, which is used to produce microbial metabolites and for preservation can be prepared by
the combination of following ingredients:
Peptone : 5.0 g
Beef extract : 3.0 g
Sodium Chloride : 8.0 g
Distilled water : 1000 ml
pH : 7.0 ± 0.02
Nutrient Agar
Medium, which is used for cultivation, isolation and characterization, can be prepared by the
combination of following ingredients:
Peptone : 5.0 g
Beef extract : 3.0 g
Sodium Chloride : 8.0 g
Agar :15.0g
Distilled water : 1000 ml
pH : 7.0 ± 0.02
Potato Dextrose Agar
Potato : 200 g
Dextrose : 20 g
Agar : 15 g
Distilled water : 1000 ml
pH : 5.6 ± 0.02

Materials Required
Bunsen burner, test tubes, Petri dishes, autoclave, laminar air hood, weighing Balance,
Nutrient media, Potato Dextrose medium and cotton

Procedure
Nutrient Broth
1. To prepare 200 ml of broth, accurately weigh the necessary ingredients of the medium and
transfer them into a beaker (500 ml) containing 100 ml of distilled water and mix using
magnetic stirrer.
2. Make up the volume to 200 ml using distilled water and set the pH in pH meter using
Hydrochloric acid (1 N) or Sodium hydroxide solution (1N).
3. Dispense 10 ml of broth to each culture tubes and apply cotton plug to the mouth of the
tubes and cover with aluminum foil or paper and tie by rubber band or thread.
4. Sterilize the contents using Autoclave at 121oC for 15 min.
5. After sterilization, remove the contents and allowed to cool until room temperature, mark
the identification details and store for future use.
Nutrient Agar
1. To prepare 200 ml of broth, accurately weigh the necessary ingredients of the medium and
transfer them into a beaker (500 ml) containing 100 ml of distilled water and mix using
magnetic stirrer.
2. Make up the volume to 200 ml using distilled water and set the pH in pH meter using
Hydrochloric acid (1 N) or Sodium hydroxide solution (1N) and transfer the contents to a
conical flask (500 ml).
3. Add Agar into the media and liquefy using water bath until the agar dissolves.
4. Sterilize the media using Autoclave at 121 ºC for 15 min.
5. Remove the contents and allowed to cool until it attains room temperature in the laminar air
hood.
 6. Wisely pour the molten media into and up to half of the shallow bottom dish before
solidification.
7. Leave the set up until proper solidification and mark the identification details on the bottom
of the Petri-plate. The plate is now ready for inoculation. Store at 40C for future use.
Potato Dextrose Agar
1. To prepare 200 ml of medium, weigh 200 g of sliced (washed but unpeeled) potatoes, add
water (150 ml) and boil for 30 minutes.
2. Filter the extract using muslin cloth, add other ingredients of the medium and make up to
200 ml.
3. Sterilize the media using Autoclave at 121 ºC for 15 min.
4. Remove the contents and allowed to cool until it attains room temperature in the laminar air
hood.
5. Wisely pour the molten media into and up to half of the shallow bottom dish before
solidification.
8. Leave the set up until proper solidification and mark the identification details on the bottom
of the Petri-plate. The plate is now ready for inoculation. Store at 40C for future use.

Result and Observation

Ex. No :10
Date: STERILISATION AND DISINFECTION

Introduction
Sterilisation refers to the anti-microbial process during which all microorganisms are killed
or eliminated in or on a substance by applying different processes. Microbes react in their own way
to the antimicrobial effects of various physical treatments or chemical compounds, and the
effectiveness of treatments depends on many other factors as well (e.g. population density,
condition of microorganisms, concentration of the active agent, environmental factors).
Sterilisation procedures involve the use of heat, radiation or chemicals, or “physical removal” of
microbes. The type of sterilisation should always be chosen as required, by taking into
consideration the quality of materials and tools used and the possible adverse effects of sterilisation
on them.
Sterilisation by heat
The use of dry heat is based on the removal of the water content of microbes and
subsequent oxidation. Open flame can be used for sterilisation if the object is not directly exposed
to flame damage. Different laboratory devices (e.g. scalpel, knife, inoculating loop or needle) can
be sterilised quickly and safely by crossing over open flame or by ignition.
Dry heat sterilisation is performed in a hot air steriliser. It is an electric box with adjustable
temperature like an incubator. In order to achieve uniform chamber temperature, hot air is
circulated. Sterilisation with dry heat is limited to devices made of metal, glass or porcelain, and
other thermo-stabile-materials, like glycerol, soft paraffin, oils and fats. In the dry heat sterilisation
system they have to withstand the temperature needed to kill the spore-forming bacteria (at 160°C
for 45 minutes; at 180°C for 25 minutes; at 200°C for 10 minutes).
The heat conductivity of water is several times higher than that of the air, therefore heat
sterilises more quickly and effectively in the presence of hot water or steam than dry heat.
Boiling is the simplest and oldest way of using moist heat. The temperature of boiling
water does not exceed 100°C at normal atmospheric pressure. Heat resistant, endospore-forming
bacteria can survive the 10-30-minute heat treatment of boiling, so no sterilizing effect can be
expected from boiling.
Pasteurisation is a widespread method – named after Louis Pasteur – to reduce the number
of microorganisms found in different heat sensitive liquids. Milk can be pasteurised by heating to
65°C for 30 minutes or to 85°C for 5 minutes. During ultra-pasteurisation milk is heat-treated at
135-150°C for 2 minutes in a heat exchanger. The temperature and time used for pasteurisation are
suitable to control the presence of some pathogenic bacteria, however endospores and cells of heat
resistant bacteria e.g. Mycobacterium species, can survive.
Tyndallisation (intermittent sterilisation) is an old and lengthy method of heat sterilisation
named after John Tyndall. During this method, a medium or solution is heated to a temperature
over 90°C for 30 minutes for four successive days, and the substances are placed in an incubator at
37°C or stored at room temperature in the intermittent periods. Vegetative forms are destroyed
during the heat treatments. Endospores which can germinate during the incubation period are
destroyed during the consecutive heat treatments. This way, after the fourth day of heat treatment,
no living cells remain in the substance.
Sterilisation by radiation
Other forms of energy [e.g. ultraviolet (UV) and ionizing radiation] are also used for
sterilisation especially for heat-sensitive materials. The full spectrum of UV radiation can damage
microbes but only a small part is responsible for the so-called germicidal effect. Very strong
"germicidal" effect can be achieved around 265 nm, because maximum UV absorption of DNA
occurs at this wavelength. The main cause of cell death is the formation of pyrimidine dimers in
nucleic acids. Bacteria are able to repair their nucleic acid after damage using different
mechanisms; however, beyond a certain level of damage, the capacity of the enzyme system is not
enough and the accumulation of mutations causes death. UV (germicidal) lamps are widely used in
hospitals and laboratories (e.g. in biological safety cabinets) for decontamination of air and any
exposed surfaces. The disadvantage of the use of UV radiation is that it does not penetrate through
glass, dirt films, water, and other substances. Among the high-energy ionizing radiation, γ-rays
from radioactive nuclides are generally used for sterilisation of disposable needles, syringes,
bandages, medicines and certain food (e.g. spices). The advantage of gamma radiation is its deep
penetration through the packaging. Its disadvantage is the scattering in all directions, which
requires special circumstances for application.
Filter sterilisation
The most commonly used mechanical method of sterilisation is filtration (Fig. 3). During
filtration, liquids or gases are pressed through a filter, which (depending on its pore size) retains or
adsorbs (e.g. asbestos filter pads) microbes, thereby the filtrate becomes sterile. The pore diameter
of filters should be chosen carefully so that bacteria and other cellular components cannot
penetrate. Earlier Seitz-type asbestos or different glass filters were commonly used for the
filtration of microorganisms. The modern membrane filters are usually composed of high tensile-
strength polymers (cellulose acetate, cellulose nitrate or polysulfone, etc.). Their operation is based
partly on the adsorption of microbes, partly on a mechanical sieve effect. The pure sieve-based
filters can be beneficial because they do not change the composition of the filtered solution. To
remove bacteria, membrane filters with pore size of 0.22 μm are the best choice. Membrane filters
are biologically neutral; do not hamper life activities of microorganisms remaining on the filter and
do not inhibit their enzyme functions. Furthermore, nutrients can diffuse through the membranes,
so bacteria can be cultured on a variety of media also by placing the filters onto their surface.
Sterilisation by chemicals
A wide range of chemicals is suitable to inhibit or kill microbes. Some of the antimicrobial
agents only inhibit the growth of microorganisms (e.g. bacteriostatic, fungistatic, and virostatic
compounds) while others kill them (e.g. bacteriocidal, fungicidal, and virocidal agents). The -static
or -cidal effect of a substance depends on the applied concentration and exposure time in addition
to its quality. Only –cidal effect substances are used for chemical sterilisation. These substances
have the following requirements: they should have a broad-spectrum effect; they should not be
toxic to higher organisms, they should not enter detrimental reactions to the materials being treated
with, they should not be biodegradable, they should be environmentally friendly, easy to apply and
economical.
The materials used in chemical sterilisation are liquids or gases. Liquid agents are used
especially for surface sterilisation. Among sterilising gases, those working at low temperature
function by exposing the materials to be sterilised to high concentrations of very reactive gases
(e.g. ethylene oxide, betapropiolactone or formaldehyde). Due to their alkylating effect, these
compounds cause the death of microbes by damaging their proteins and nucleic acids. The
chemical agents used for sterilisation must be chemically compatible with the substances to be
sterilised, therefore they have a great importance in sterilisation of pharmaceutical and
thermoplastic materials. The chemicals used by the gas sterilisers are harmful to humans as well.
Therefore, the application of gas sterilisers requires compliance with the precautions by the users.
Procedures of disinfection
Any process aimed at destroying or removing the infectious capability of pathogenic
microbes that generally occur on inanimate objects, is called disinfection. The chemicals used for
disinfection can be classified according to their chemical structure and their mode of action.
Among the alcohols, ethanol and isopropanol are widely used as disinfectants. 50-70% aqueous
solution has excellent antiseptic properties. The action mechanism of alcohols depends on the
applied concentration. Due to the solubility of lipids in 50-95% ethanol solutions, biological
membranes are disintegrated. Alcohols pass through the cell membrane with altered permeability,
denature the proteins inside the cell and have a dehydration effect as well. Absolute alcohol (100%
ethanol) provides the best dehydration effect but does not coagulate the intracellular proteins. 70%
dilution of alcohols is the most effective way to kill the vegetative forms of bacteria and fungi, but
less effective against spores and lipid-enveloped viruses.
Phenol called carbolic acid was first used as a disinfectant by Lister. Phenol denatures
proteins, and irreversibly inactivates the membrane-bound oxidases and dehydrogenases. Due to
the unfavourable physical, chemical and toxicological properties, phenol is no longer used.
However, substituted (alkylated, halogenated) derivatives are often used in combination with
surfactants or alcohols (e.g. cresol, hexachlorophene, chlorhexidine).
The halogens (F, Cl, I, Br) and their derivatives are very effective disinfectants and
antiseptic agents; mainly their non-ionic forms have antimicrobial activity. Chlorine gas is used
almost exclusively for the disinfection of drinking water or other waters. In addition, different
compounds (e.g. chloride of lime, chloramine-B, sodium dichloroisocyanurate) are among the
most widely used disinfectant agents. Sodium hypochlorite (“household bleach” is a mixture of 8%
NaClO and 1% NaOH) is one of the oldest high-bleaching and deodorizing disinfectant. The basis
of the effect of chlorine and its derivatives is that during decomposition in aqueous solution, a
strong oxidant, nascent (atomic state) oxygen ('O'),
is released. Nascent oxygen is very reactive and suitable to destroy bacteria, fungi and their spores
as well as viruses.
Iodine is also a widely used disinfectant and antiseptic agent. There are two known
preparations: tincture of iodine (alcoholic potassium iodide solution containing 5% iodine) and
iodophors (aqueous solutions of iodine complexes with different natural detergents). It is applied in
alcoholic solution to disinfect skin or in aquatic solution for washing prior surgery.
Aldehydes, such as formaldehyde and glutaraldehyde, are broad-spectrum disinfectants.
They are used for decontamination of equipment and devices. Formalin is the 34-38% aqueous
solution of formaldehyde gas. Its effect is based on the alkylation of proteins.
Heavy metals such as mercury, arsenic, silver, gold, copper, zinc and lead, and a variety of
their compounds are highly efficient disinfectants but they are too damaging to living tissues to
apply. They can be used as disinfectants at very low concentrations. Inside the cell, they bind to the
sulfhydryl groups of proteins. Primarily, organic and inorganic salts of silver and mercury-
containing products are commercially available, which have bactericidal, fungicidal and virocidal
effect.
Detergents or surfactants are amphiphilic organic molecules which have a hydrophilic
"head" and a long hydrophobic “tail” . Detergents can be non-ionic, anionic or cationic according
to the charge of the carbon chain. Nonionic surfactants have no significant biocidal effect and
anionic detergents are only of limited use because of their poor efficiency. The latter group
includes soaps, which are long chain carboxylic acids (fatty acids) of sodium or potassium salts.
They are not disinfectants on their own, but are efficient cleaning agents due to their lipid-
solubilizing effect. Cationic detergents, such as quaternary ammonium salts, are the best
disinfectants.

Observation and Results

Ex. No : 11
AGAROSE GEL ELECTROPHORESIS
Date:

Aim
To separate the isolated DNA fragments for molecular characterization of DNA molecules
Introduction
Agarose gel electrophoresis is the easiest and most popular way of separating and
analyzing DNA. Here DNA molecules are separated on the basis of charge by applying an electric
field to the electrophoretic apparatus. Shorter molecules migrate more easily and move faster than
longer molecules through the pores of the gel and this process is called sieving. The gel might be
used to look at the DNA in order to quantify it or to isolate a particular band. The DNA can be
visualized in the gel by the addition of ethidium bromide.
Agarose is a polysaccharide obtained from the red algae Porphyra umbilicalis. Its
systematic name is (1 4)-3, 6-anhydro-a-L-galactopyranosyl-(1 3)-β-D-galactopyranan. Agarose
makes an inert matrix. Most agarose gels are made between 0.7% and 2% of agarose. A 0.7% gel
will show good separation for large DNA fragments (5-10kb) and a 2% gel will show good
resolution for small fragments with size range of 0.2-1kb. Low percentage gels are very weak
(Note:- it may break when you lift them) but high percentage gels are usually brittle and do not set
evenly. The volume of agarose required for a minigel preparation is around 30-50ml and for a
larger gel, it is around 250ml.
Material Required
Buffers and Solutions:
Agarose solutions.
Ethidium bromide.
Electrophoresis buffer (TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-
borate, 1 mM EDTA)).
Nucleic Acids and Oligonucleotides:
DNA samples.
DNA Ladders.
Procedure
Preparation of the Gel
1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are
prepared using a w/v percentage solution. The concentration of agarose in a gel will depend
 on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-
2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.
2. Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel
running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate,
1 mM EDTA).
3. Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave,
but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the
contents to mix well. Repeat until the agarose has completely dissolved.
4. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may
also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30
min, followed by destaining in running buffer for an equal length of time.
Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations.
Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the
staining of DNA are available; however EtBr remains the most popular one due to its sensitivity
and cost.
1. Allow the agarose to cool either on the benchtop or by incubation in a 65 °C water bath.
Failure to do so will warp the gel tray.
2. Place the gel tray into the casting apparatus. Alternatively, one may also tape the open
edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create
the wells.
3. Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature.
Remove the comb and place the gel in the gel box. Alternatively, the gel can also be
wrapped in plastic wrap and stored at 4 °C until use.
Setting up of Gel Apparatus and Separation of DNA Fragments
1. Add loading dye to the DNA samples to be separated. Gel loading dye is typically made at
6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading
dye helps to track how far your DNA sample has traveled, and also allows the sample to
sink into the gel.
2. Program the power supply to desired voltage (1-5V/cm between electrodes).
3. Add enough running buffer to cover the surface of the gel. It is important to use the same
running buffer as the one used to prepare the gel.
4. Attach the leads of the gel box to the power supply. Turn on the power supply and verify
that both gel box and power supply are working.
 5. Remove the lid. Slowly and carefully load the DNA sample(s) into the gel. An appropriate
DNA size marker should always be loaded along with experimental samples.
6. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the
anode (red leads). Double check that the electrodes are plugged into the correct slots in the
power supply.
7. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.
Observing Separated DNA fragments
1. When electrophoresis has completed, turn off the power supply and remove the lid of the
gel box.
2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the
gel tray on paper towels to absorb any extra running buffer.
3. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly
done using a gel documentation system. DNA bands should show up as orange fluorescent
bands. Take a picture of the gel.
4. Properly dispose of the gel and running buffer per institution regulations.

Result
Ex.No : 12
SEPARATION OF PROTEIN MOLECULES USING SDS- PAGE GEL
Date:

Aim
To separate the give protein samples using SDS-PAGE gels and estimate the molecular
weight of the protein.
Introduction
SDS-PAGE is widely used to analyze the proteins in complex extracts. The most
commonly used methods are derived from the discontinuous SDS-PAGE system first described by
Laemmli (1970). The system actually consists of two gels - a resolving (aka running) gel in which
proteins are resolved on the basis of their molecular weights (MWs) and a stacking gel in which
proteins are concentrated prior to entering the resolving gel. Differences in the compositions of the
stacking gel, resolving gel and electrophoresis buffer produce a system that is capable of finely
resolving proteins according to their MWs.
The polyacrylamide gels used to separate proteins are formed by the chemical
polymerization of acrylamide and a cross-linking reagent, N,N’methylenebisacrylamide (opposite
page). Investigators are able to control the size of the pores in the gel by adjusting the
concentration of acrylamide, as well as the ratio of acrylamide to bisacrylamide. Raising either the
concentration of acrylamide or bisacrylamide, while holding the other concentration constant, will
decrease the pore size of the gel. Polymerization occurs because of free oxygen radicals that react
with the vinyl groups in acrylamide and bisacrylamide. The oxygen radicals are generated from the
catalyst, ammonium persulfate (APS), when it reacts with a second catalyst, N,N,N’,N’-
tetramethylethylenediamine (TEMED).

Electrophoresis is the study of the movement of charged molecules in an electric field. The
generally used support medium is cellulose or thin gels made up of either polyacrylamide or
agarose. Cellulose is used as support medium for low molecular weight biochemicals such as
amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger
molecules like proteins. The general electrophoresis techniques cannot be used to measure the
molecular weight of the biological molecules because the mobility of a substance in the gel is
influenced by both charge and size. In order to overcome this, if the biological samples are treated
so that they have a uniform charge, electrophoretic mobility then depends primarily on size. The
molecular weight of protein maybe estimated if they are subjected to electrophoresis in the
presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent mercaptoethanol (ME).
SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear
polypeptide chain coated with negatively charged SDS molecules. Mercaptoethanol assists the
protein denaturation by reducing all disulfide bonds.
Acrylamide + N N’ methylene bis acrylamide

Chemical Polymerization Ammonium persulfate (catalyst) +

TEMED (N, N N’ N’ tetramethylethylene diamine)

Polyacrylamide

Material Required
1. Acrylamide solutions (for resolving & stacking gels)
2. Isopropanol / distilled water.
3. Gel loading buffer.
4. Running buffer.
5. Staining, destaining solutions.
6. Protein samples
7. Molecular weight markers.
8. 30% Polyacrylamide solution(29g acrylamide+1g bisacrylamide in 50 mL of water,
dissolve completely using a magnetic stirrer, make the volume upto 100mL). Keep the
solution away from sunlight.
9. 1.5 M Tris, pH 8.8
10. 1 M Tris, pH 6.8
11. 10% SDS (10 g SDS in 100mL distilled water).
12. 10% ammonium persulfate (0.1 g in 1 ml water). It should be freshly prepared.
13. 10x SDS running buffer( pH ~8.3) - Take 60.6 g Tris base, 288g Glycine and 20g SDS in
separate beakers and dissolve them using distilled water. When properly dissolved, mix
three of them and make upto 2L.(working standard is 1X buffer).
The equipment and supplies necessary for conducting SDS-PAGE includes:
1. An electrophoresis chamber and power supply
2. Glass plates (a short and a top plate)
3. Casting frame
4. Casting stand
5. Combs
Note:
1. Gloves should be worn, while performing SDS-PAGE
2. To ensure proper alignment, all the requirements should be clean
3. Special attention should be paid while using acrylamide (since it is a neurotoxin).
Reagents
Preparation of 12% Separating gel monomer composition (10 ml)
DDl.H2O- 3.4 ml
30% Acrylamide/Bis- 4 ml
1.5M Tris-HCl buffer- 2.5 ml
10% w/v SDS- 0.1 ml
Immediately prior to pouring the gel, add
10% APS- 50 µl
TEMED- 5 µl
Preparation of 5% stacking gel monomer composition (10 ml)
DDl.H2O- 5.7 ml
30% Acrylamide/Bis- 1.7 ml
1.5M Tris-HCl buffer- 2.5 ml
10% w/v SDS- 0.1 ml
Immediately prior to pouring the gel, add
10% APS- 50 µl
TEMED- 5 µl
Coomassie R-250 staining solution composition (1000 ml)
Coomassie R-250- 2.5 g
Methanol – 500 ml
Acetic acid – 100 ml
Distilled water- 400 ml
De-staining solution (1000 ml)
Methanol (50%) – 500 ml
Acetic acid (10%) – 100 ml
Water (40%) – 400 ml
Gel loading buffer:
To make 10 mL of 4X stock:
2.0 ml 1M Tris-HCl pH 6.8.
0.8 g SDS.
 4.0 ml 100% glycerol.
0.4 ml 14.7 M β-mercaptoethanol.
1.0 ml 0.5 M EDTA.
8 mg bromophenol Blue.

Procedure
Assembling the glass plates:
1. Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer)
down first and then place the shorter glass plate on top of it.
2. Embed them into the casting frame and clamp them properly make sure that the bottom
ends of the glass plates are properly aligned.
3. Then place it on the casting stand.
Casting the gels:
1. Prepare the separating gel solution by combining all reagents. Do not add Ammonium per
sulfate and TEMED.
2. Add APS and TEMED to the monomer solution (just before pouring) and mix well by
swirling gently. Pour the solution till the mark. (It is ok if you introduce air bubbles, add a
layer of isopropanol or distilled water on top of the gel so as to level the poured gel.)
3. Allow the gel to polymerize for 20-30 minutes.
4. Prepare stacking gel. Mix all reagents except APS and TEMED. Drain the isopropanol with
strips of filter paper.
5. Add APS and TEMED to the monomer solution (just before pouring) and mix well by
swirling gently. (Make sure you keep the comb ready by the side.)
6. Place a comb in the stacking gel sandwich. Allow it to polymerize for 10 minutes.
Preparation of samples:
Mix your protein in the ratio 4:1 with the sample buffer. Heat your sample by either:
Boiling for 5-10 minutes. (Works for most proteins.) or 65°C for 10 minutes or 37°C for 30
minutes.
Running the gel:
Note : Before running the gel make sure that the gel, gel apparatus and samples are ready.
1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus.(
Make sure that the short plate always faces inside and if you have got only one gel to run
use the dummy plate that is available to balance).
2. When the plates are secured, place them in the cassette and then lock it.
 3. Place them in the gel running tank.
4. Fill the inner chamber of the tank with buffer. (Now it is easy to remove the comb, since it
is lubricated).
5. Remove the comb CAREFULLY (without breaking the well).
[Now the gel is ready to load the samples]
6. Rinse the loading tip a few times with distilled water. (Make sure that all the water is
poured out before loading the samples.)
7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the
well. Rinse the syringe with distilled water after loading for a few times.
8. Attach the power supply by putting the lid (Make sure that the connection is in correct way
ie., black - black and red - red). Set the voltage upto 180 V and run for 1 hour. (Don't allow
the dye front to go out of the gel).
Staining the gel:
After running, switch off the power supply and take out the gel plates, remove the gel. Place the
gel in the staining solution for 30 minutes. Destain the gel until the bands are properly seen.
Determine the approximate molecular weight of the visualised protein bands by comparing them
with the molecular weight ladders (markers).

M- Marker; Lane 1-13- Protein Sample

Result
Ex. No: 13

Date: MICROSCOPY

Introduction
Microbiology, the branch of science that has so vastly extended and expanded our knowledge of
the living world, owes its existence to Antoni van Leeuwenhoek. In 1673, with the aid of a crude
microscope consisting of a biconcave lens enclosed in two metal plates, Leeuwenhoek introduced
the world to the existence of microbial forms of life. Over the years, microscopes have evolved
from the simple, single-lens instrument of Leeuwenhoek, with a magnification of 300*, to the
present-day electron microscopes capable of magnifications greater than 250,000*. Microscopes
are designated as either light microscopes or electron microscopes. The former use visible light or
ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and
fluorescent instruments. Fluorescent microscopes use ultraviolet radiations whose wavelengths are
shorter than those of visible light and are not directly perceptible to the human eye. Electron
microscopes use electron beams (instead of light rays) and magnets (instead of lenses) to observe
submicroscopic particles.
Essential Features of Various Microscopes
Brightfield Microscope: This instrument contains two-lens systems for magnifying specimens:
the ocular lens in the eyepiece and the objective lens located in the nosepiece. The specimen is
illuminated by a beam of tungsten light focused on it by a substage lens called a condenser; the
result is a specimen that appears dark against a bright background. A major limitation of this
system is the absence of contrast between the specimen and the surrounding medium, which makes
it difficult to observe living cells. Therefore, most brightfield observations are performed on
nonviable, stained preparations.
Darkfield Microscope: This is similar to the ordinary light microscope; however, the condenser
system is modified so that the specimen is not illuminated directly. The condenser directs the light
obliquely so that the light is deflected or scattered from the specimen, which then appears bright
against a dark background. Living specimens may be observed more readily with darkfield than
with brightfield microscopy.
Phase-Contrast Microscope: Observation of microorganisms in an unstained state is possible
with this microscope. The optics include special objectives and a condenser that make visible
cellular components that differ only slightly in their refractive indexes. As light is transmitted
through a specimen with a refractive index different from that of the surrounding medium, a
portion of the light is refracted (bent) due to slight variations in density and thickness of the
cellular components. The special optics convert the difference between transmitted light and
refracted rays, resulting in a significant variation in the intensity of light and thereby producing a
discernible image of the structure under study. The image appears dark against a light background.
Fluorescent Microscope: This microscope is used most frequently to visualize specimens that are
chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet (UV) light
obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular lens is fitted
with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths
are blocked or eliminated. Ultraviolet radiations are absorbed by the fluorescent label, and the
energy is re-emitted in the form of a different wavelength in the visible light range. The fluorescent
dyes absorb at wavelengths between 230 and 350 nanometers (nm) and emit orange, yellow, or
greenish light. This microscope is used primarily for the detection of antigen-antibody reactions.
Antibodies are conjugated with a fluorescent dye that becomes excited in the presence of
ultraviolet light, and the fluorescent portion of the dye becomes visible against a black background.
Electron Microscope: This instrument provides a revolutionary method of microscopy, with
magnifications up to 1 million *. This permits visualization of submicroscopic cellular particles as
well as viral agents. In the electron microscope, the specimen is illuminated by a beam of electrons
rather than light, and the focusing is carried out by electromagnets instead of a set of optics. These
components are sealed in a tube in which a complete vacuum is established. Transmission electron
microscopes require specimens that are prepared as thin filaments, fixed and dehydrated for the
electron beam to pass freely through them. As the electrons pass through the specimen, images are
formed by directing the electrons onto photographic film, thus making internal cellular structures
visible. Scanning electron microscopes are used for visualizing surface characteristics rather than
intracellular structures. A narrow beam of electrons scans back and forth, producing a three-
dimensional image as the electrons are reflected off the specimen’s surface. While scientists have a
variety of optical instruments with which to perform routine laboratory procedures and
sophisticated research, the compound brightfield microscope is the “workhorse” and is commonly
found in all biological laboratories. Although you should be familiar with the basic principles of
microscopy, you probably have not been exposed to this diverse array of complex and expensive
equipment. Therefore, only the compound brightfield microscope will be discussed in depth and
used to examine specimens.

Observation and Results

Ex. No: 14
MICROSCOPIC EXAMINATION OF STAINED
Date:
CELL PREPARATION

Introduction
Microbiology is a science that studies living organisms that are too small to be seen with
the naked eye. Needless to say, such a study must involve the use of a good compound microscope.
Although there are many types and variations, they all fundamentally consist of a two-lens system,
a variable but controllable light source, and mechanical adjustable parts for determining focal
length between the lenses and specimen
Components of the Microscope
Stage A fixed platform with an opening in the center allows the passage of light from an
illuminating source below to the lens system above the stage. This platform provides a surface for
the placement of a slide with its specimen over the central opening. In addition to the fixed stage,
most microscopes have a mechanical stage that can be moved vertically or horizontally by means
of adjustment controls. Less sophisticated microscopes have clips on the fixed stage, and the slide
must be positioned manually over the central opening.
Illumination The light source is positioned in the base of the instrument. Some microscopes are
equipped with a built-in light source to provide direct illumination. Others are provided with a
reversible mirror that has one side flat and the other concave. An external light source, such as a
lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of
the mirror is used for artificial light, and the concave side for sunlight.
Abbé Condenser This component is found directly under the stage and contains two sets
of lenses that collect and concentrate light as it passes upward from the light source into the lens
systems. The condenser is equipped with an iris diaphragm, a shutter controlled by a lever that is
used to regulate the amount of light entering the lens system.
Body Tube Above the stage and attached to the arm of the microscope is the body tube. This
structure houses the lens system that magnifies the specimen. The upper end of the tube contains
the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the
objective lenses. Rotation of the nosepiece positions objectives above the stage opening. The body
tube may be raised or lowered with the aid of coarse-adjustment and fine-adjustment knobs that
are located above or below the stage, depending on the type and make of the instrument.
Theoretical Principles of Microscopy
To use the microscope efficiently and with minimal frustration, you should understand the
basic principles of microscopy: magnification, resolution, numerical aperture, illumination, and
focusing.
Magnification Enlargement, or magnification, of a specimen is the function of a two-lens
system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving
nosepiece. These lenses are separated by the body tube. The objective lens is nearer the specimen
and magnifies it, producing the real image that is projected up into the focal plane and then
magnified by the ocular lens to produce the final image. The most commonly used microscopes are
equipped with a revolving nosepiece containing four objective lenses, each possessing a different
degree of magnification. When these are combined with the magnification of the ocular lens, the
total or overall linear magnification of the specimen is obtained.
Resolving Power or resolution although magnification is important, you must be aware that
unlimited enlargement is not possible by merely increasing the magnifying power of the lenses or
by using additional lenses, because lenses are limited by a property called resolving power. By
definition, resolving power is how far apart two adjacent objects must be before a given lens shows
them as discrete entities. When a lens cannot discriminate, that is, when the two objects appear as
one, it has lost resolution. Increased magnification will not rectify the loss and will, in fact, blur the
object. The resolving power of a lens is dependent on the wavelength of light used and the
numerical aperture, which is a characteristic of each lens and imprinted on each objective. The
numerical aperture is defined as a function of the diameter of the objective lens in relation to its
focal length. It is doubled by use of the substage condenser, which illuminates the object with rays
of light that pass through the specimen obliquely as well as directly. Thus, resolving power is
expressed mathematically as follows:

Based on this formula, the shorter the wavelength, the greater the resolving power of the lens.
Thus, for the same numerical aperture, short wavelengths of the electromagnetic spectrum are
better suited for higher resolution than are longer wavelengths. However, as with magnification,
resolving power also has limits. Decreasing the wavelength will not automatically increase the
resolving power of a lens, because the visible portion of the electromagnetic spectrum is very
narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.
The relationship between wavelength and numerical aperture is valid only for increased resolving
power when light rays are parallel.
Therefore, the resolving power is also dependent on another factor, the refractive index. This is
the bending power of light passing through air from the glass slide to the objective lens. The
refractive index of air is lower than that of glass; as light rays pass from the glass slide into the air,
they are bent or refracted so that they do not pass into the objective lens. This would cause a loss
of light, which would reduce the numerical aperture and diminish the resolving power of the
objective lens. Loss of refracted light can be compensated for by interposing mineral oil, which has
the same refractive index as glass, between the slide and the objective lens. In this way, decreased
light refraction occurs and more light rays enter directly into the objective lens, producing a vivid
image with high resolution (Figure).

Figure: Refractive index in air and in mineral oil

Illumination Effective illumination is required for efficient magnification and resolving power.
Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is
the most commonly used light source in microscopy. The light is passed through the condenser
located beneath the stage. The condenser contains two lenses that are necessary to produce a
maximum numerical aperture. The height of the condenser can be adjusted with the condenser
knob. Always keep the condenser close to the stage, especially when using the oil-immersion
objective. Between the light source and the condenser is the iris diaphragm, which can be opened
and closed by means of a lever, thereby regulating the amount of light entering the condenser.
Excessive illumination may actually obscure the specimen because of lack of contrast. The amount
of light entering the microscope differs with each objective lens used. A rule of thumb is that as
the magnification of the lens increases, the distance between the objective lens and slide, called
working distance, decreases, whereas the numerical aperture of the objective lens increases
(Figure).
 Figure: Relationship between working distance, objective, and diaphragm opening
Use and Care of the Microscope
You will be responsible for the proper care and use of microscopes. Since microscopes are
expensive, you must observe the following regulations and procedures. The instruments are housed
in special cabinets and must be moved by users to their laboratory benches. The correct and only
acceptable way to do this is to grip the microscope arm firmly with the right hand and the base
with the left hand, and lift the instrument from the cabinet shelf. Carry it close to the body and
gently place it on the laboratory bench. This will prevent collision with furniture or coworkers and
will protect the instrument against damage. Once the microscope is placed on the laboratory bench,
observe the following rules:
1. Remove all unnecessary materials (including books, papers, purses, and hats) from the
laboratory bench.
 2. Uncoil the microscope’s electric cord and plug it into an electrical outlet.
3. Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will decrease the
efficiency of the microscope. The ocular, scanning, low power and high-power lenses may
be cleaned by wiping several times with acceptable lens tissue. Never use paper toweling or
cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper
moistened with xylol is used to wipe it clean. The xylol is immediately removed with a
tissue moistened with 95% alcohol, and the lens is wiped dry with lens paper. Note: This
xylol cleansing procedure should be performed only by the instructor and only if
necessary; consistent use of xylol may loosen the lens.
The following routine procedures must be followed to ensure correct and efficient use of the
microscope.
1. Place the microscope slide with the specimen within the stage clips on the fixed stage.
Move the slide to center the specimen over the opening in the stage directly over the light
source.
2. Raise the microscope stage up as far as it will go. Rotate the scanning lens or low-power
lens into position. Lower the body tube with the coarse-adjustment knob to its lowest
position. Note: Never lower the body tube while looking through the ocular lens.
3. While looking through the ocular lens, use the fine-adjustment knob, rotating it back and
forth slightly, to bring the specimen into sharp focus.
4. Adjust the substage condenser to achieve optimal focus.
5. Routinely adjust the light source by means of the light-source transformer setting, and/ or
the iris diaphragm, for optimum illumination for each new slide and for each change in
magnification.
6. Most microscopes are parfocal, which means that when one lens is in focus, other lenses
will also have the same focal length and can be rotated into position without further major
adjustment. In practice, however, usually a half-turn of the fine-adjustment knob in either
direction is necessary for sharp focus.
7. Once you have brought the specimen into sharp focus with a low-powered lens, preparation
may be made for visualizing the specimen under oil immersion. Place a drop of oil on the
slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion
objective locks into position. Note: Care should be taken not to allow the high-power
objective to touch the drop of oil. The slide is observed from the side as the objective is
rotated slowly into position. This will ensure that the objective will be properly immersed
in the oil. The fine adjustment knob is readjusted to bring the image into sharp focus.
 8. During microscopic examination of microbial organisms, it is always necessary to observe
several areas of the preparation. This is accomplished by scanning the slide without the
application of additional immersion oil. Note: This will require continuous, very fine
adjustments by the slow, back-and-forth rotation of the fine-adjustment knob only.
On completion of the laboratory exercise, return the microscope to its cabinet in its original
condition. The following steps are ecommended:
1. Clean all lenses with dry, clean lens paper. Note: Use xylol to remove oil from the stage
only.
2. Place the low-power objective in position and lower the body tube completely.
3. Center the mechanical stage.
4. Coil the electric cord around the body tube and the stage.
5. Carry the microscope to its position in its cabinet in the manner previously described.

Materials
Slides
Commercially prepared slides of Staphylococcus aureus, Bacillus subtilis, Aquaspirillum itersonii,
and other alternate slides.
Equipment
Compound microscope, lens paper, and immersion oil.
Procedure
1. Review the parts of the microscope, making sure you know the names and understand the
function of each of these components.
2. Review instructions for the use of the microscope, giving special attention to the use of the
oil-immersion objective.
3. Examine the prepared slides, noting the shapes and the relative sizes of the cells under the
high-power (also called high-dry, because it is the highest power that does not use oil) and
oil-immersion objectives.
4. Record your observations in the Lab Report.

Observation and Results

Ex. No : 15
Date: MICROMETRY

Aim
To measure the dimensions of common microorganisms by calibration and standardization
of microscope with the help of stage and ocular micrometers.
Introduction
Micrometry is the science in which we have some measurement of the dimensions of an object
being observed under the microscope. The method employs some special types of measuring
devices which are so oriented that these can well be attached to or put into the microscope and
observed. The object to be measured is calibrated against these scales. Once we are observing an
object under a microscope by the objective 10X and the eye piece 10X we say that the image that
we are able to perceive is 100 times of the object. We get the magnified view no doubt and also
that it is perfect coordination of the dimensions, but to find out the absolute size of the object will
need precision and that is achieved through the application of micrometers.

Types of Micrometers:
Stage Micrometer: As the name indicates it is for the measurement on the stage of themicroscope
where an object is to be kept.This micrometer is of slides size and has amount of very finely
graduated scale. The scale measures only 1 millimeterand has a least count of 0.01 mm, i.e., 1mm
region is divided into 100 divisions. As1 mm has 1000μ, so one division of stagemicrometer will
be equivalent to 10μ.
Ocular Meter:This is a micrometer which is used insidethe eye piece. The upper eye lens
isunscrewed and the ocular meter is put intothe tube of eye piece, and the eye lens isagain
replaced. There are usually 50 or 100divisions in the ocular meter which areengraved on the glass.
Materials Required
Microscope, stage micrometer, ocular meter, slide of microorganisms (e.g.,bacteria or fungus) to
be measured.
Procedure
To work out the measurements per ocular divisions the stage micrometer is keptunder low power
of the microscope and is observed through the eye piece having ocular meter. Suppose, we have
10Xobjective and 5X eye piece in the microscope with a tube of 170 mm length.At this
magnification the number of ocular divisions coinciding the stage micrometerare observed and
thence calculated for microns per ocular divisions, e.g., let us
take that 6 ocular divisions coincide with 8divisions of stage micrometer. Therefore, 6 ocular
divisions = 8 stage micrometer divisions, or 6 ocular divisions= 0.08 mm (since 1 division =
0.01mm).Thus, the microscope is calibrated for different combinations of eye pieces and objective
lenses and is kept for record. It is to note hence that this calibration will bejust only of the tried
lenses on a particular microscope. In this way take three readings, and themean value of these
readings will be the actual value of one pan of ocular meter.

Observation Result