Good laboratory practices and

Research Methodology
Dr. Dinesh Wanule
 Safety in laboratory
• Report all accidents, injuries, and breakage of glass or equipment to instructor
immediately.
• Keep pathways clear by placing extra items (books, bags, etc.) on the shelves or
under the work tables. If under the tables, make sure that these items can not be
stepped on.
• Long hair (chin-length or longer) must be tied back to avoid catching fire.
• Wear sensible clothing including footwear. Loose clothing should be secured so
they do not get caught in a flame or chemicals.
• Work quietly — know what you are doing by reading the assigned experiment
before you start to work. Pay close attention to any cautions described in the
laboratory exercises
• Do not taste or smell chemicals.
• Wear safety goggles to protect your eyes when heating substances, dissecting, etc.
• Do not attempt to change the position of glass tubing in a stopper.
• Never point a test tube being heated at another student or yourself. Never look
into a test tube while you are heating it.
• Unauthorized experiments or procedures must not be attempted.
• Keep solids out of the sink.
• Leave your work station clean and in good order before leaving the laboratory.
• Do not lean, hang over or sit on the laboratory tables.
• Do not leave your assigned laboratory station without permission of the teacher.
• Learn the location of the fire extinguisher, eye wash station, first aid kit and safety
shower.
• Fooling around or "horse play" in the laboratory is absolutely forbidden. Students
found in violation of this safety rule will be barred from participating in future labs
and could result in suspension.
• Anyone wearing acrylic nails will not be allowed to work with matches, lighted
splints, bunsen burners, etc.
• Do not lift any solutions, glassware or other types of apparatus above eye level.
• Follow all instructions given by your teacher.
• Learn how to transport all materials and equipment safely.
• No eating or drinking in the lab at any time!
• Using light microscope
Use
• Mount the specimen on the stage
• The cover slip must be up if there is one. High magnification objective lenses can't
focus through a thick glass slide; they must be brought close to the specimen,
which is why coverslips are so thin. The stage may be equipped with simple clips
(less expensive microscopes), or with some type of slide holder. The slide may
require manual positioning, or there may be a mechanical stage (preferred) that
allows precise positioning without touching the slide.
• Optimize the lighting
• A light source should have a wide dynamic range, to provide high intensity
illumination at high magnifications, and lower intensities so that the user can view
comfortably at low magnifications. Better microscopes have a built-in illuminator,
and the best microscopes have controls over light intensity and shape of the light
beam. If your microscope requires an external light source, make sure that the
light is aimed toward the middle of the condenser. Adjust illumination so that the
field is bright without hurting the eyes.
• Use dark field mode (if available) to find unstained specimens. If not, start with
high contrast (aperture diaphragm closed down).
• Adjust the condenser
• The condenser is focusable, position it with the lens as close to the opening in the
stage as you can get it. If the condenser has selectable options, set it to bright
field. Start with the aperture diaphragm stopped down (high contrast). You should
see the light that comes up through the specimen change brightness as you move
the aperture diaphragm lever.
• Think about what you are looking for
• It is a lot harder to find something when you have no expectations as to its
appearance. How big is it? Will it be moving? Is it pigmented or stained, and if so
what is its color? Where do you expect to find it on a slide? For example, students
typically have a lot of trouble finding stained bacteria because with the unaided
eye and at low magnifications the stuff looks like dirt. It helps to know that as
smears dry down they usually leave rings so that the edge of a smear usually has
the densest concentration of cells.
• Focus, locate, and center the specimen
• Start with the lowest magnification objective lens, to home in on the specimen
and/or the part of the specimen you wish to examine. It is rather easy to find and
focus on sections of tissues, especially if they are fixed and stained, as with most
prepared slides. However it can be very difficult to locate living, minute specimens
such as bacteria or unpigmented protists. A suspension of yeast cells makes a good
practice specimen for finding difficult objects.
•
• Start with the specimen out of focus so that the stage and objective must be
brought closer together. The first surface to come into focus as you bring stage and
objective together is the top of the cover slip. With smears, a cover slip is
frequently not used, so the first thing you see is the smear itself.
• If you are having trouble, focus on the edge of the cover slip or an air bubble, or
something that you can readily recognize. The top edge of the cover slip comes
into focus first, then the bottom, which should be in the same plane as your
specimen.
• Once you have found the specimen, adjust contrast and intensity of illumination,
and move the slide around until you have a good area for viewing.
• Adjust eyepiece separation, focus
• With a single ocular, there is nothing to do with the eyepiece except to keep it
clean.
• With a binocular microscope (preferred) you need to adjust the eyepiece
separation just like you do a pair of binoculars. Binocular vision is much more
sensitive to light and detail than monocular vision, so if you have a binocular
microscope, take advantage of it.
• One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus
it. Since very few people have eyes that are perfectly matched, most of us need to
focus one eyepiece to match the other image. Look with the appropriate eye into
the fixed eyepiece and focus with the microscope focus knob. Next, look into the
adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not
the microscope.
• Select an objective lens for viewing
• The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding
specimens. We sometimes call it the scanning lens for that reason. The most frequently used
objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens.
For very small protists and for details in prepared slides such as cell organelles or mitotic
figures, you will need a higher magnification. Typical high magnification lenses are 40x and
97x or 100x. The latter two magnifications are used exclusively with oil in order to improve
resolution.
• Move up in magnification by steps. Each time you go to a higher power objective, re-focus
and re-center the specimen. Higher magnification lenses must be physically closer to the
specimen itself, which poses the risk of jamming the objective into the specimen. Be very
cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when
you switch magnifications the specimen remains in focus or close to focused.
• Bigger is not always better. All specimens have three dimensions, and unless a specimen is
extremely thin you will be unable to focus with a high magnification objective. The higher the
magnification, the harder it is to "chase" a moving specimen.
• Adjust illumination for the selected objective lens
• The apparent field of an eyepiece is constant regardless of magnification used. So it follows
that when you raise magnification the area of illuminated specimen you see is smaller. Since
you are looking at a smaller area, less light reaches the eye, and the image darkens. With a
low power objective you may have to cut down on illumination intensity. With a high power
you need all the light you can get, especially with less expensive
• Care of the microscope
• EVERYTHING on a good quality microscope is unbelievably expensive, so be
careful.
• Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder,
for example.
• Hold the plug (not the cable) when unplugging the illuminator.
• Since bulbs are expensive, and have a limited life, turn the illuminator off when
you are done.
• Always make sure the stage and lenses are clean before putting away the
microscope.
• NEVER use a paper towel, a kimwipe, your shirt, or any material other than good
quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an
optical surface. Be gentle! You may use an appropriate lens cleaner or distilled
water to help remove dried material. Organic solvents may separate or damage
the lens elements or coatings.
• Cover the instrument with a dust jacket when not in use.
• Focus smoothly; don't try to speed through the focusing process or force anything.
For example if you encounter increased resistance when focusing then you've
probably reached a limit and you are going in the wrong direction.
 Maintenance
• Proper storage of the microscope will prevent or reduce problems!
• Optics and mechanisms of the microscope must be protected from:
– Dust and dirt
– Fungus
• Store the microscope
– Under a protective cover
– In a low humidity environment
• Cleaning Solutions and Solvents
• Soap solution for cleaning of body and stage
• Ethyl ether-alcohol, alcohol, or lens cleaner solution for cleaning of lenses
• Refer to manufacturer’s guide for appropriate organic solvent
• Microscope Cleaning Process
1. Eyepiece
2. Objectives
3. Microscope Stage
4. Microscope Body
5. Condenser

• Step 1: Cleaning the Eyepieces

• Blow to remove dust before wiping lens
• Clean the eyepieces with a cotton swab moistened with lens cleaning solution
• Clean in a circular motion inside out
• Wipe the eyepieces dry with lens paper
• Repeat cleaning and drying if required
• Step 2: Cleaning the Objectives
• Objectives are cleaned while attached to
microscope
– Moisten the lens paper with the cleaning
solution
– Wipe gently the objective in circular
motion from inside out
– Wipe with dry tissue or lens cleaning
paper
• Objectives should never be removed from the
nosepiece.
• Wipe the microscope stage using the cleaning
solution on a soft cloth
• Thoroughly dry the stage
• Repeat above steps, if required
• Step 3: Cleaning the Microscope Stage
• Wipe the microscope stage using the cleaning
solution on a soft cloth
• Thoroughly dry the stage
• Step 4 Cleaning the Microscope Body

1. Unplug the microscope from power source

2. Moisten the cotton pad with a mild cleaning agent
3. Wipe the microscope body to remove dust, dirt, and oil
4. Repeat steps1–3, if required
• Step 5: Cleaning the Condenser and Auxiliary Lens
• Unplug the microscope from power source
• Clean the condenser lens and auxiliary lens using lint-free cotton swabs moistened
with lens cleaning solution
• Wipe with dry swabs
• ROUTINE USE pH Meter
• Power On the instrument 15 Min Before use
• Place the electrode in KCL solution
• Adjust temperature using Temperature compensation control Most modern
meters and electrode systems have automatic temperature-compensation
correction
• If not
• Calibrate the instrument
• A considerable variety of instruments from several manufacturers is available and
the user is advised to follow the operating instructions supplied with the
instrument. The glass electrode can be used with strong acids; however, it is
attacked by strong alkaline solutions. Therefore, glass electrodes should never be
left in alkaline solutions for longer than is necessary to measure the pH. The glass
electrode responds rapidly to large pH changes in buffered solutions, but the
• response is slower in poorly buffered, or unbuffered, solutions. Equilibrium is
reached slowly and may require several seconds. Poorly buffered solutions should
be stirred vigorously during measurement to prevent stagnation at the electrode.
• Measurements can be made in partly aqueous solutions but the degree of
hydration of the outer surface of the membrane will alter the potential across the
membrane. Hence, values obtained in non-aqueous, or highly ionic, solutions will
be incorrect.
•
INSTRUMENT CALIBRATION
It is essential that pH electrodes are calibrated regularly using the meter and
• reference electrode with which they are to be used in the laboratory. The
procedure is as follows:
• 1 Turn instrument on and allow adequate time to warm up (15–30 min)
• 2 Position electrodes in holder and plug into instrument
• 3 Set function switch to pH
• 4 Prepare pH buffer solutions for integer pH values. These are available in dry form
in foil packets and are prepared using deionised water. Preprepared liquid buffers
• can also be used
• 5 Set temperature compensation control for temperature of the buffers (not room
• temperature). Most modern meters and electrode systems have automatic
temperature-compensation correction
• 6 Insert electrodes in pH 7.0 buffer
• 7 Adjust calibration control Using Asymmetric knob until meter indicates pH 7.0 –
some modern pH meter models working in calibration mode often recognise the
buffer and take necessary action automatically
• 8 Do not readjust calibration control for
• steps 9 to 12
• 9 Remove electrodes, flush with deionised water and blot gently to remove excess
• water
• 10 Start with the highest 9.0 pH buffer and place
• electrodes in the solution Read and record pH value. The pH 9.0 can be adjusted
using slope Knob The highest pH buffer
• represents the lowest hydrogen ion concentration. Calibration in this manner
• minimises test solution carry-over between measurements
• 11 Repeat steps 9 and 10 with each successively lower-value buffer and record
• all results
• Most modern instruments have a built-in automatic buffer recognition facility and
will automatically identify and set to the appropriate temperature-corrected
calibrationvalues.
• ELECTRODE CARE AND
• MAINTENANCE
• Many types of pH electrode are available but the standard glass or epoxy-bodied
combination electrode is ideal for the majority of tests carried out on aqueous
solutions with a reasonable ionic strength at ambient temperatures and with limited
use in strongly acidic or alkaline solutions.
The following general guidelines indicate the care and maintenance required for pH
electrodes:
• To dry the electrode, use clean soft tissues and blot the liquid from the electrode.
• Immerse in pH 4 buffer for short-term storage. F
• or longer-term storage use the same solution as the reference electrolyte of the
electrode. In most cases this is a 3 mol/L KCl solution.
• Most manufacturers supply a plastic protection cap which is filled with this
solution. Close off the filler opening if there is one. If the electrode will be not
used for a long period of time, you may store it dry to prevent ageing (ageing
takes place only when the electrode is
• The gel electrodes, as these must be stored in a concentrated solution of KCl only.
Never store your electrode in water .
• Always rinse thoroughly with deionised water after use. If the response of a glass
electrode has become sluggish, the recommended treatment consists of 1 minute
in 20% ammonium bifluoride solution, followed by 15 seconds in 6 mol/L
hydrochloric acid. The electrode should then be rinsed thoroughly and soaked for
24 hours in water or in an acidic buffer solution.
• Electrodes that have been allowed to dry out (often indicated by a hard, dry
deposit of KCl crystals) should be soaked overnight in warm deionised water. Liquid
junctions with fibres or ceramic pins occasionally can become blocked due to
crystallisation (eg KCl).
• If soaking in KCl solution does not solve the problem, raising the temperature to
the maximum allowable for the reference system will often help.
• Other types of blockage can also occur (eg in the form of a precipitate [black] of
silver chloride or mercury sulphide in the porous pin). Gentle use of abrasive paper
can sometimes remove the precipitate. In other cases, chemical procedures such
as soaking the electrode for a few hours in an acidic solution of thiourea (1 mol/L
thiourea in 0.1 mol/L HCl) can be used. r Ensure that the electrode is used and
stored within its specified temperature
• range. Extreme changes in temperature between samples will affect response
time, and electrodes used above their temperature range will age rapidly. r Ensure
that air bubbles are not trapped at the bottom of the electrode. If present,
bubbles should be removed by holding the electrode vertically and gently tapping
the electrode body. If the air bubbles are trapped by KCl crystals, heating the
• electrode gently to 60°C (maximum) in a water bath may also prove beneficial.
• Handled carefully – the normal lifetime of glass electrodes is approximately two
years. Occasionally, functional failure occurs before mechanical failure. This is
recognised by a gradually increasing electrode response time, with increasingly
erratic readings. This is a different effect from electrode shock, which also
produces increased response time. Electrode shock is produced by dipping the
• electrode into a high-concentration solution and then immediately afterwards into
a low concentration solution, or vice versa. Thus, if one tries to measure pH 2 and
then pH 11, an increased response time should be expected.
• ELECTRODE CLEANING
• The solution used to clean pH electrodes depends on the presence of possible
• contaminants. Mechanically intact electrodes may show slow response due to
clogging or coating. Table 1 shows some recommended cleaning solutions for glass
electrodes. More detailed information about electrode care and maintenance can
be obtained from the manufacturer.
• ELECTRODE STORAGE
• As a general rule, store the pH electrode in the same solution as the reference
electrolyte of the electrode. In most cases this is a 3 mol/L KCl solution. Most
manufacturers supply a plastic protection cap which is filled with this solution.
Close off the filler opening if there is one. Never store your electrode in
• water as this will cause ions to leach out of the glass membrane, leading to a
sluggish response.
• RECONDITIONING ELECTRODES
• Older electrodes, or electrodes that have been stored dry, may need to be
‘reconditioned’. Recondition an electrode by soaking in pH 4.01 buffer or electrode
 Colorimeter vs
Spectrophotometer
• Like colorimeters, spectrophotometers are used to measure the color absorbing
properties of a substance.
• The key difference between the two is that the spectrophotometer measures the
transmittance and reflectance as a function of wavelength, whereas the
colorimeter measures the absorbance of specific colors.
• Spectrophotometers measure the transmittance and reflectance for all colors of
light and show how they vary as the color is changed. Colorimeters operate only in
the visible portion of the electromagnetic spectrum whereas spectrophotometers
work with infrared as well as visible light. Spectrophotometers will produce valid
results for Beer’s law and can effectively be used as colorimeters but are much
higher in cost and complexity.
 Colorimeter
• Allow the Colorimeter to warm up for about five minutes before calibrating.
• Select the appropriate filter
• Calibrate the Colorimeter. Slide the lid of the Colorimeter open to reveal the
cuvette slot.
• Insert a cuvette, filled with distilled water or other solvent used to prepare your
solutions, for your calibration blank (100% transmittance or 0 absorbance).
Important: Line up one of the clear sides of the cuvette with the arrow at the right
side of the cuvette slot. Slide the Colorimeter lid closed.
• Press the CAL button on the Colorimeter to begin the calibration process. Release
the CAL button when the red LED begins to flash.
• When the red LED stops flashing, the calibration is complete. The absorbance
reading should be very close to 0.000 (100%T).
• Fill a cuvette two-thirds to three-fourths full with liquid, including the calibration
blank, so that the light travels through the liquid reliably. After filling a cuvette
with liquid, seal the cuvette with a cap to prevent spills.
• Remove the blank cuvette from the Colorimeter.
• Continue with data collection.
• Care of Colorimeter
• Proper use and maintenance of all laboratory equipment is necessary for
efficiency and safety in labs. Spectrophotometers are expensive devices. Some
important tips of the safe installation and use of this equipment:
• Use an electrical supply source that conforms with industry standards
• Place the spectrophotometer in a clean environment and away from other devices
that cause vibration (such as centrifuges)
• Ensure routine maintenance by a trained and certified technician. Annual
inspection would typically include the inspection of the area where the device is
installed as well as electrical installation to ensure user safety
• Test the general structure of the device – check buttons, control switches
• Confirm that the mechanical components are in good condition
• Make sure accessories, cable devices and terminals are clean and intact
• Check the electrical components to avoid overheating
• Take care to clean spills carefully and using the right procedures. Cuvettes should
be rinsed in distilled water and with special cleaning material, if recommended by
the manufacturer.
 Maintenance
• One of the most important steps you can take to ensure the accuracy and
longevity of your colorimeter is to maintain it properly. All colorimeters will
eventually suffer from the filter deterioration over time, although some
colorimeters suffer from the effects of filter deterioration at a much slower rate as
a result of design.
• The most important step you can take to increase the longevity of your meter is to
prevent moisture. This can be achieved by storing your meter in a container that
prevents moisture getting in. Professional grade carrying cases provide excellent
waterproof seals and pressure release valves to get the best possible moisture
protection. Combined with preventing moisture from getting to your meter you
will also want to eliminate the moisture already in your meter’s case. The easiest
way of doing this is to throw some desiccant into the case. The most standard
desiccant is silica gel, however, it does lose effectiveness over time. For a long term
solution a ‘rechargeable’ desiccant option provides the most long-term solution.
• Light can also negatively affect the colorimeter. It is best to keep the meter in a
dark location. Anything that blocks light radiation from getting to your meter (and
most importantly the filters) will help prevent damage from radiation.
• The other cause of increased filter deterioration is heat. Your meter’s longevity can
be significantly increased by ensuring that you store the meter in a cool location.
This doesn’t mean the meter has to be kept in the refrigerator, but take care to
avoid storing the meter in a location that will allow it to overheat.
•
Analytical
Preparing the balance for use
balance
• Before weighing anything on the analytical balance you must make sure that it is
leveled and zeroed.
• To check the leveling on the balance, look at the leveling bubble on the floor of the
weighing chamber. If it is not centered, center it by turning the leveling screws on
the bottom toward the back of the balance.
• Once the balance is leveled, close all the chamber doors and press the control bar
on the front of the balance. After a few seconds, a row of zeros will appear. This
indicates that the balance is zeroed and ready for use.
• Weighing a liquid, powder, or granular substance
• These substances must always be weighed using an appropriate weighing
container.
• Place the weighing container on the balance pan and close the doors.
Tare the container by briefly pressing the control bar. The readout will read zero
with the container sitting on the pan. This allows the mass of your sample to be
read directly.
Add the substance to be weighed. Be careful not to spill chemicals on the balance.
If need be, you can remove the container from the weighing chamber while you
add the sample provided that no one presses the control bar before you weigh
your sample.
• With the sample and its container sitting on the pan, close the chamber doors and
read the display to find the mass of your sample.
• Weighing a solid object directly on the balance
• If the object you need to weigh is a solid object take it in watchglass / non sticky
paper (tare the weight of empty watch glass before), you can weigh it directly on
the pan in . Be sure the balance is zeroed. Open the chamber doors, carefully place
the object on the balance pan, close the doors, and read the mass of your object

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• Centrifuge Maintenance and Care
• The centrifugal field which accelerates the separation process also exerts large
forces on the rotor material. If a rotor fails, a tube breaks, or any other incident
occurs, the centrifuge can be severely damaged, as well as possibly endanger
those working in the lab. For this reason, some simple precautions should be
observed to improve safety during centrifugation.
• Sample Retrieval, Cleaning, and Maintenance
• Precautions During Centrifugation
• Avoiding Rotor Failures
• The centrifugal field which accelerates the separation process also exerts large
forces on the rotor material. If a rotor fails, the centrifuge is severely damaged as
well. For this reason, some simple precautions should be observed
• Rotors are designed to be run up to their maximum speed with a load of a specific
weight. One should never attempt to run a rotor at a speed higher than the one
designated by its manufacturer. Also, if high density solutions (greater than 1.2
g/mL, for instance) are used, the run speed must be reduced to prevent undue
stress on the rotor. Consult your instruction manual for exact directions.
• Tube Breakage
• Glass tubes can break during centrifugation, due either to improper loading or
inherent defects. Any glass fragments must be removed from the buckets,
adapters, rubber liners, and rotor chamber before the next run is made. If you find
gray dust, which results from sandblasting of the rotor chamber by glass particles,
it must be cleaned up too. You should make several dry runs without samples, and
clean the chamber between each run to be sure this dust is eliminated from the
centrifuge.
• Chemical Resistance
• If you plan to centrifuge any uncommon solvents or solutions, consult your manual
to be sure they are compatible with the various plastics and metals comprising the
centrifuge, the rotor, the tubes, and other accessories. These same precautions
must be observed with any solvents used for sterilization purposes. A table of 19
chemical resistances for common centrifuge materials is available from Beckman
Coulter.
• Aerosol Generation
• If any liquid is spilled on a rotor, it will be dispersed as a particulate mist when the
centrifuge is run. Part of this mist will be fine enough to form a relatively stable
aerosol which will tend to be dispersed throughout the laboratory. Such spills
should be thoroughly cleaned up before running the centrifuge.
• Handling Human Samples
• Human blood or blood components can transmit an infectious disease or virus if
the patient or donor carries these. Blood should be handled with respect for this
possibility during all laboratory manipulations, including centrifugation.
• When in doubt, refer to your instruction manual
• From time to time, you’ll have questions about the actual operation and
maintenance of your centrifuge. The instruction manual provided with each
instrument is designed to answer these questions. It should be read before making
your first run, and kept handy for future reference.
• Care
• Electrophoresis involves the use of high voltages and carries the risk of electric
shock.
• Many chemicals commonly used in electrophoresis are highly toxic.
• Make sure you have been trained by an experienced worker in the safe use of
electrophoresis equipment.
• Familiarise yourself with the COSHH forms for the chemicals you intend to use.
• Check equipment and wiring before use. Look for signs of damage. Do not use
worn or frayed leads.
• Use only electrophoresis tanks which have a secure design preventing contact with
buffer when connected to a power supply.
• Always disconnect from the power supply before opening.
• Switch off power before moving a tank.
• Clean up spills of electrophoresis buffer or gel mixes immediately – these may
contain toxic chemicals e.g. ethidium bromide or acrylamide.
• Latex gloves often contain small holes – use nitrile (or other suitable) gloves when
immersing hands in electrophoresis buffers or handling gels.
• When using vertical electrophoresis equipment, take care that leakage from the
upper buffer chamber does not cause arcing.
• Emergency action in case of electric shock
• Switch off power at once. Do not attempt to touch the victim until they have
been isolated from the source of electricity. If you are unable to get to the power
supply you will have to insulate yourself so that you do not become a casualty. A
piece of DRY wood or rolled up paper could normally be used.
• Check the casualty’s airway and breathing if unconscious be ready to resuscitate,
call for help immediately
• (From a University phone dial 222 or from a brown phone dial R-999)
• Treat any burns when away from the electricity by flooding the affected part with
water for a minimum of 10 minutes. Remove and constrictions if swelling is likely,
keep injury elevated.
• Do not burst blisters, apply any creams or sprays or apply any plasters. Seek
help from a first aider.
•