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Good Laboratory Practices and

Research Methodology‐ I
Dr. Dinesh Wanule
magnifying power (M) of a lens with focal length (f in millimeters) when viewed by the
naked human eye:
Mnaginfication = 250 / f
Light microscopy
• The light microscope, so called because it employs visible
light to detect small objects, is probably the most well-
known and well-used research tool in biology.
• Types of light microscopes
• The bright field microscope is best known to students
and is most likely to be found in a classroom. Better
equipped classrooms and labs may have dark field and/or
phase contrast optics. Fluorescence and confocal
microscopes are specialized instruments, used for
research, clinical, and industrial applications.
• Other than the compound microscope, a simpler
instrument for low magnification use may also be found
in the laboratory. The stereo microscope, or dissecting
microscope usually has a binocular eyepiece tube, a long
working distance, and a range of magnifications typically
from 5x to 35 or 40x. Some instruments supply lenses for
higher magnifications, but there is no improvement in
resolution. Such "false magnification" is rarely worth the
• A compound microscope is an indispensable instrument in any biological laboratory. It is used for passive observation of structural details of a cell, tissue or organ in sections.
• A modern compound microscope has following structural components.
• Non-Optical Components:
• 1. Base (foot):
• It is U or horseshoe-shaped metallic structure that supports the whole microscope.
• 2. Pillar:
• It is a short upright part that connects to base as well as arm.
• 3. Arm (Limb):
• It is a curved metallic handle that connects with the arm by inclination joint. It supports stage and body tube.
• 4. Inclination Joint:
• It is used for tilting the microscope if required for observation in sitting position.
• 5. Stage:
• It is a metallic platform with a central hole fitted to the lower part of the arm. Microscopic slides held on the stage by either simple side clips or by a mechanical stage clip.
• 6. Body tube:
• It is meant for holding ocular and objective lenses at its two ends. The end holding ocular lens is called head while the end containing 3-4 objective lens is called nose piece. The body tube has an
internal pathway for the passage of light rays which form the enlarged image or microscopic objects.
• 7. Draw tube:
• It is a small tube that remains fixed at the upper end of the body tube. It holds eyepiece or ocular lens.
• 8. Rack and pinion:
• The microscope has a rack and pinion attached either to body tube or the stage for bringing the object under focus.
• 9. Adjustment screws:
• There are two pairs of screws for moving the body tube in relation to stage, larger for coarse adjustment and smaller for fine adjustment. In fine adjustment the body tube or stages moves for
extremely short distances. In coarse adjustment the body tube or stage can move up and distance. In coarse adjustment is meant for briefly objective lens at a proper distance from the object so as to
form image of the same at the ocular end. Fine adjustment is required to obtain sharp image.
• 10. Automatic Stop:
• It is a small screw fitted at lower end or rack and pinion. It is meant for stopping the downward sliding of the body tube so as to prevent the damage of objective lens and the slide.
• Optical Components:
• 11. Diaphragm:
• It is flitted just below the stage for regulating the amount of light failing on the object. Diaphragm is of two types, disc and iris.
• 12. Condenser:
• It is attached below the diaphragm. Condenser can be moved up and down to focus light on the object.
• 13. Reflector (Mirror):
• It is attached just above the base. Both its surface bear mirrors, plane on one side and concave on other side. Plane side is used in strong light and concave side in weak light. Reflector directs the light
on the object through the condenser and diaphragm system.
• 14. Objective Lenses:
• They are fitted over the nose piece. Objective lenses are of two 10 three types – low power (commonly 10X or 5X), high power (commonly 45X) and oil immersion (commonly 100X, can be more). An
objective lens is not a simple lens but compound lens. It forms real inverted image of the object inside the body tube.
• 15. Ocular Lens or Eyepiece:
• It is lens through which image of the microscopic object is observed. It also takes part in magnification. Depending upon magnification, the eye piece is of four types-5X, 10X, 15X, and 20 X. Advanced
microscope has two eye pieces so that both the eyes can be used (Fig. 1.4). Microscope head having device for using two eye pieces is called binocular head. It contains a number of internal mirrors and
prisms for the passage of light.
Working Principle
• A beam of visible light from the base is focused by a condenser lens onto the
specimen. The objective lens picks up the light transmitted by the specimen and
create a magnified image of the specimen called primary image inside the body
tube. This image is again magnified by the ocular lens or eye piece.

When you look through a simple light microscope or a magnifying glass, you are
looking through a biconvex lens (one that’s bent like the back of a spoon on both
sides) made of glass. The object being viewed is on the far side of the lens. Light
from the object passes through the lens and is bent (refracted) towards your eye,
so it seems as though it comes from a much bigger object.
Principle of Microscopy
• The objective of use of microscope is basically to find the large (Magnified) and Clear (resolution)
Image of Object.
• Magnification
• Simple/ Dissecting Microscope:
• dissecting microscope consists of a biconvex lens which is moved up and down by an adjustment screw
to bring the object in sharp focus. The object is placed on the platform and light is focused with the
help of a concave mirror fitted below.
• In simple microscope, convex lens of short focal length is used to see magnified image of a small object.
The object is placed between the optical centre and the focus of a convex lens, its image is virtual, erect
and magnified and on the same side as the object. The position of the object is so adjusted that the
image is formed at the least distance of distinct vision (D).
• Magnifying power (M) of a simple microscope is the ratio of the angle subtended by the image at the
eye to the angle subtended by the object seen directly, when both lie at the least distance of distinct
vision or the near point.
• M = 1 + D/ f Where D is the least distance of distinct vision and f is the focal length of the lens.
• The focal point F and focal length f of a positive (convex) lens, a negative (concave) lens, a concave
mirror, and a convex mirror.] In optometry, the least distance of distinct vision (LDDV) or the reference
seeing distance (RSD) is the closest someone with "normal" vision (20/20 vision) can comfortably look
at something. In other words, LDDV is the minimum comfortable distance between the naked human
eye and a visible object.
Vision 20/20
A Snellen chart is an eye chart that can be used to measure
visual acuity. Snellen charts are named after the Dutch
ophthalmologist Herman Snellen, who developed the chart in
1862. The normal Snellen chart is printed with eleven lines of
block letters. The first line consists of one very large letter,
which may be one of several letters, for example E, H, or N.
Subsequent rows have increasing numbers of letters that
decrease in size. A person taking the test covers one eye from
6 metres or 20 feet away, and reads aloud the letters of each
row, beginning at the top. The smallest row that can be read
accurately indicates the visual acuity in that specific eye. The
symbols on an acuity chart are formally known as
"optotypes". In the case of the traditional Snellen chart, the
optotypes have the appearance of block letters, and are
intended to be seen and read as letters. They are not,
however, letters from any ordinary typographer's font. They
have a particular, simple geometry in which:
the thickness of the lines equals the thickness of the white
spaces between lines and the thickness of the gap in the letter
the height and width of the optotype (letter) is five times the
thickness of the line.
• A Compound Microscope:
• A compound microscope consists of two set of convex lenses. A lens of short aperture
and short focal length facing the object is called objective. Another set of lens of
relatively moderate focal length and large aperture facing the eye is called the eye
piece. The objective and the eye piece are placed coaxially at the two end of a tube.
The object is placed between the centre of curvature and focus of the objective – it
forms real, inverted and magnified image on the other side of the objective. This
image acts as an object for the eye piece which then acts as a simple microscope to
produce virtual, erect and magnified image.
• Magnifying power (M) of a compound microscope will be
• M = L/ f0 (1+D/fe)
• Where f0and fe are focal length of objective and eye piece respectively, L is the length
a the microscope tube and D is the least distance of distinct vision. The least distance
of distinct vision for a young adult with normal vision is 25 cm
• Resolution -
• The magnification of small things is a necessary facet of biological research, but the
fine detail in cells and in subcellular components requires that any imaging system be
capable of providing spatial information across small distances. Resolution is defined
as the ability to distinguish two very small and closely-spaced objects as separate
entities. Resolution is best when the distance separating the two tiny objects is small.
Resolution is determined by certain physical parameters that include the wavelength
of light, and the light-gathering power of the objective and condenser lenses. A simple
mathematical equation defines the smallest distance (dmin) separating the two very
small objects:
• dmin = 1.22 x wavelength / N.A. objective + N.A. condenser

• This is the theoretical resolving power of a light microscope. In practice, specimen

quality usually limits dmin to something greater than its theoretical lower limit.
• N.A. (Numerical Aperture) is a mathematical calculation of the light-gathering
capabilities of a lens. The N.A. of each objective lens is inscribed in the metal tube,
and ranges from 0.25-1.4. The higher the N.A., the better the light-gathering
properties of the lens, and the better the resolution. Higher N.A. values also mean
shorter working distances (you have to get the lens closer to the object). N.A. values
above 1.0 also indicate that the lens is used with some immersion fluid, such as
immersion oil.
• Numerical Aperture
• Numerical Aperture (also termed Object-Side Aperture) is a value (often
symbolized by the abbreviation NA) originally defined by Abbe for microscope
objectives and condensers. It is given by the simple expression:
• Numerical Aperture (NA)=n×sin(µ) or n×sin(α)
• n the numerical aperture equation, n represents the refractive index of the
medium between the objective front lens and the specimen, and µ or α is the one-
half angular aperture of the objective.

• The angular aperture of a lens is the angular size of the lens aperture as seen from
the focal point:
• a = 2 arctan ( D / 2 f ) = 2 arctan ( D 2 f ) {\displaystyle a=2\arctan \left({\frac
{D/2}{f}}\right)=2\arctan \left({\frac {D}{2f}}\right)} where
• f {\displaystyle f} is the focal length D {\displaystyle D} is the diameter of the
• The numerical aperture of a microscope objective is a measure of its ability to
gather light and resolve fine specimen detail at a fixed object distance. Image-
forming light waves pass through the specimen and enter the objective in an
inverted cone . A longitudinal slice of this cone of light reveals the angular
aperture, a value that is
• In practice, it is difficult to achieve numerical aperture values above 0.95 with dry
objectives. Figure 1 illustrates a series of light cones derived from objectives of
varying focal length and numerical aperture. As the light cones grow larger, the
angular aperture (α) increases from 7° to 60°, with a resulting increase in the
numerical aperture from 0.12 to 0.87, nearing the limit when air is utilized as the
imaging medium. Higher numerical apertures can be obtained by increasing the
imaging medium refractive index (n) between the specimen and the objective
front lens. Microscope objectives are now available that allow imaging in
alternative media such as water (refractive index = 1.33), glycerin (refractive index
= 1.47), and immersion oil (refractive index = 1.51). The numerical aperture of an
objective is also dependent, to a certain degree, upon the amount of correction
for optical aberration. determined by the focal length of the objective.
• Illumination -
• An essential factor in producing a good image with the light microscope is obtaining adequate levels of
light in the specimen, or object plane. It is not only necessary to obtain bright light around the object,
but for optimal imaging, the light should be uniform across the field of view. The best way to illuminate
the specimen involves the use of yet another lens system, known as a condenser. The front element of
the condenser is usually a large, flattened lens that sits directly beneath the specimen. Its placement on
a movable rack provides you with the means to focus the light beam coming past the object and
maximixe the intensity and control the uniformity of illumination. Two apertures in the illumination
system allow you to regulate the diameter of the illumination beam by closing or opening iris
diaphragms. One of these diaphragms, housed within the brightfield condenser and known as the
condenser diaphragm, allows you to increase contrast, but at the cost of worsening resolution. The
second of these diaphragms, known as the field aperture diaphragm, does not affect resolution as
dramatically and is regularly adjusted for optimal illumination.
• Optimal illumination of a specimen with all microscopes currently manufactured is achieved by using a
variation of Kohler Illumination, where (for those of you are technophiles) the filament of the light
source is in focus at the rear focal plane of the objective lens. Operationally, it is easy to obtain optimal
illumination for brightfield (or phase contrast) by first placing any specimen on the stage and focusing on
the object. Next, turn the ring for the field aperture diaphragm (the lowest aperture on the microscope)
so that its edges obscure the periphery of the field of view. Next, raise or lower the condenser until the
edges of the field aperture diaphragm are clearly focused. Do not refocus the objective on the specimen
while you are adjusting the condenser. It may be necessary to center the field aperture diaphragm, using
the condenser centering screws. When the microscope is properly illuminated, both the object and the
edges of the field aperture diaphragm should be in the same plane of focus and the field iris diaphragm
should be centered in the field of view.
• Using light microscope
• 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
• To adjust and align the microscope, start by reading the manual. If no manual is
available, try using these guidelines. If 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
apprearance. 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
• 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.
• microscopes.
• 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
• 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
• 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
• 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.
• 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
– Moisten the lens paper with the cleaning
– Wipe gently the objective in circular
motion from inside out
– Wipe with dry tissue or lens cleaning
• Objectives should never be removed from the
• 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
Phase Contrast Microscope:
• In recent years, remarkable advances have been made in
the study of living cells (unstained) by the development of
special optical techniques such as phase contrast and
interference microscopy.
• The biological specimens are highly transparent to visible
light and they cause phase changes in transmitted
• The phase contrast microscope has the same resolving
power as the ordinary light microscope but it permits
visualization of different parts of the cell due to differences
in their refractive index (Refractive index is defined as the
ratio of the velocity of light in a vacuum to its velocity in a
transmitting medium).
• Because light is transmitted through a structure at a velocity
inversely proportional to the refractive index of the
structure, light waves emerging from structures with
different refractive index will be out of phase with one
• The phase contrast microscope is able to convert these
differences in phase to differences in light intensity,
producing an image with good contrast. The phase-contrast
microscope utilizes interference between two beams of
• In the phase contrast microscope, the small phase differences are intensified. The most lateral light
passing through the objective lens of the microscope is advanced or retarded by an additional l/4th
wavelength (1/4λ.) with respect to the central light passing through the medium around the object,
by an annular phase plate that introduces a 1/4 wavelength variation in the back focal plane of the
• In addition an annular diaphragm is placed in the substage condenser. The phase effect results from
the interference between the direct geometric image given by the central part of the objective and
the lateral diffracted image, which has been retarded or advanced to a total of 1/2 wavelength.
• In bright or negative contrast, the two sets of rays are added and the object appears brighter than the
surroundings. In dark or positive contrast, the two sets of rays are subtracted making the image of the
object darker than the surroundings. Because of this interference, the minute phase changes within
the object are amplified and intensified.
• A transparent object thus appears in various shades of gray, depending upon the thickness of the
object and the difference between the refractive indices of the object and the medium.
• Phase microscopy is used to observe living cells and tissues. It is particularly valuable for observing
the cells cultured in vitro during mitosis.
• Principle of the phase-contrast microscope:
• This is to convert small phase differences into differences in contrast that can be detected
visually. An annular phase plate is placed in the objective of the microscope and an annular
diaphragm is placed in the condenser as shown in the figure 2. As light is transmitted through
the lenses, some of the rays pass through in a direct path while others are diffracted laterally.
Diffracted light rays are, thus out of phase with the direct light, and an image of strong
contrasts is produced.
• The annular diaphragm illuminates the object with a narrow cone of light, and the annular
phase plate produces a variation of 1/4 A. between the diffracted lateral light and the direct
light. The phase effect is the result of interference between the direct image in the centre of
the objective and the diffracted lateral image.
• If the diffracted image is retarded, negative contrast results, whereas if it is advanced,
positive contrast results. When the refractive index of the medium is greater than that of the
object, the object is dark, and when the refractive index of the medium is less than that of
the object, the object is bright.
• Phase Contrast Microscopy The human eye can perceive changes in light amplitude
(intensity). Unstained biological specimens, such as living cells, are essentially transparent to
our eyes, but they interact with light in a fairly uniform way, by retarding (slowing) the
passage of a light beam by approximately 1/4 of a wavelength (). By slowing a light beam this
much relative to another light beam that had passed though the surrounding medium, the
biological specimen alters the phase of the beams. Intensity (amplitude) is additive and light
rays that are 1/2 out of phase are perceived as darkness. Zernicke realized that if he could
retard the light passing through biological specimens without affecting the light passing
through the surrounding medium, he could generate changes in amplitude within living cells.
The phase contrast microscope was invented by Zernicke in the 1930's as a means to
generate contrast in biological specimens, changing these invisible phase differences into
visible amplitude differences.
• Zernicke employed an optical trick to separate the light beams interacting with the specimen
from those that do not encounter the specimen. To separate the beams of light from each
other, he placed a transparent ring (known as an annulus) in an opaque disk and inserted this
disk into the optical path of the microscope, within the condenser. He placed a
complementary ring inside the objective lens. Nearly all of the light that passes through the
sample but misses the specimen then passes through the objective lens through this ring.
Most of the light that passes through the specimen is scattered and some of it enters the
objective lens in such a way that it will not pass through the objective lens ring, but will pass
this plane in some other location. He designed the glass plate holding the ring so that all light
missing the ring would encounter an additional 1/4 of retardation relative to the beams of
light that had not interacted with the specimen, placing the light rays that had interacted
with the specimen out of phase with rays that had not interacted with the specimen by 1/2.
He found that a reduction in intensity of the light that had not passed through the specimen
would create a grey background and increase contrast even more, with some parts of the
specimen darker and other parts of the specimen brighter than the background.
• The operation of any microscope in the phase contrast mode requires that you first set up
proper brightfield illumination, with a centered field iris diaphragm whose edges are in focus
in the specimen plane. Next, rotate the condenser turret cylinder until the number on the
condenser turret matches the number engraved on the objective lens. Under this condition,
the condenser annulus is matched to the phase ring present in the objective. Next, remove
one of the oculars and insert the Bertrand focusing telescope into the ocular hole. This lens
enables you to see the rear focal plane of the objective lens, the plane where the ring
resides. You will see a bright circle of light (the condenser annulus) and a dark ring (present
within the objective). The dark ring is stationary, but the bright annulus is not. You may need
to align the annulus with the ring so that the two are superimposed. On the back side of your
condenser, you will find two adjustment screws that permit this alignment to be performed.
When the ring and the annulus are aligned, place the ocular back into the microscope. The
difference between phase contrast and brightfield for the observation of living cells is
Fluorescence microscopy
• Power On the instrument 15 Min Before use
• Place the electrode in DW
• Adjust temperature using Temperature compensation control Most modern
meters and
• electrode systems have automatic
• temperature-compensation correction
• 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.

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 –
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
• 12 Plot calibration curve.
• Most modern instruments have a built-in
• automatic buffer recognition facility and will
• automatically identify and set to the
• appropriate temperature-corrected calibration
• values. This can be over-ridden in some
• instruments to allow free entry of the actual
• buffer pH.
• 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: r To dry the
electrode, use clean soft tissues and blot the liquid from the electrode. r Immerse
in pH 4 buffer for short-term storage. For 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
• This does not apply to combination or gel electrodes, as these must be stored
• in a concentrated solution of KCl only. Never store your electrode in water (see
• below). Always rinse thoroughly with deionised water after use. If the response
• of a glass electrode has become sluggish, the recommended treatment (which
• should only be performed when other measures have failed) consists of
• 1 minute in 20% ammonium bifluoride solution, followed by 15 seconds in
• 6 mol/L hydrochloric acid. Care should be exercised when carrying out this
• treatment as the risk of the formation of hydrofluoric acid is present. The
electrode should then be rinsed thoroughly and soaked for 24 hours in water or in
an acidic buffer solution. r 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.
• 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.
• 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.
• 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