PRPM110L NOTES
MODULE 1
LESSON 1: Proper Care and Maintenance of
Microscope
Proper care and maintenance of microscope is of
paramount importance and can help extend its life.
Few tips for microscope care are shared to you as
accessed from Edu-Lab Scientific Resources (Feb
2017).
1. Handle with care
 Improper handling is a common cause of
many problems that occur with
microscopes. When carrying a microscope,
hold it by the base and the metal support
arm. The stage on a microscope is the flat
plate where the slides are placed for
observation. Avoid picking your microscope
up by the stage or the eyepiece holder, as
this can cause misalignment.
2. Look after lenses
 When using your microscope, the objective
lens is lowered to adjust the focus.
However, be careful not to let the lens touch
the slide you’re looking at, as this can
damage the lens. Furthermore, dirty lenses
are notoriously difficult to clean.
 Avoid touching the lenses of the
microscope.
3. Keep covered
 Microscopes should always be sold with
dust covers. Whether transporting or storing
your instrument, make the most of the
microscope bag and remember to keep your
microscope covered when not in use. The
microscope’s eye tubes also need to be
kept dust free. If the eyepieces need to be
removed, cover the tubes with caps and
store them with the microscope.
4. Store safely
 Ensure you store your microscope in a
clean, dry space with good ventilation. Salt
air or damp, for example, can cause
damage to equipment over time. Expensive,
precision equipment should not be stored
next to solutions that may leak. Similarly,
keep your microscope away from areas with
potentially corrosive chemical fumes. Such
fumes can destroy lenses or corrode metal
parts.
5. Keep clean
 Oil immersion is a technique used to
increase the resolving power of a
microscope. Both the objective lens and
sample are immersed in a transparent oil of
high refractive index so that high
magnifications can be achieved while still
maintaining good resolution. It is essential
to ensure careful cleaning takes place
immediately after using immersion oil and
do not use damaging solvents.
6. Take care of bulbs
 After using your microscope, turn off the
illuminator and wait for it to cool down
before putting it away. Allowing the bulb to
cool will extend its life and avoid the
unnecessary cost of expensive
replacements. Similarly, if used constantly
on full power, the bulb will overheat and
blow. Remember too, to turn the illuminator
off when not in use.
7. Clean carefully
 Microscope lenses are delicate. Treat them
carefully to avoid any scratches. Use an
aspirator to remove dust. Moisten special
lens paper with distilled water or appropriate
cleaning solution. Rubbing gently in a
circular motion will remove any sticky
residue. Never use anything abrasive on
microscope lenses.
 Rotate the nosepiece of the microscope all
the way down to its lowest level when you
have finished using the microscope.
8. Refer to the user’s manual
 Your microscope should be sold with a
user’s manual and specialist spanners as
required. Always refer to the manual when
making any adjustments to the microscope
and use the supplied spanners. Never use
force, inappropriate tools or over-tighten
when making adjustments to your
microscope, as this will only result in
equipment damage.
9. Maintain your microscope
 An annual maintenance check of
microscopes is always a good idea. Moving
parts should be cleaned and lubricated.
Similarly, inspect the power cords and plugs
for safety.
10. Consider a professional service
 The company where microscope was
bought can be contacted for a range of
products to help keep your laboratory
microscopy equipment in tip top condition.
LESSON 2: Parts of the Microscope and Its
Uses
The origin of the word microscope according to the
Online Etymology Dictionary is as follows: 1656,
from Mod.L. microscopium, lit. "an instrument for
viewing what is small," from Gk. micro- (q.v.) +
-skopion. "means of viewing," from skopein "look
at." Microscopic "of minute size" is attested from
1760s.
The following are elemental parts of a typical
microscope used in the laboratory classes:
1. The Eyepiece Lens
 Eyepiece contains the ocular lens which you
will be looking through to see the magnified
specimens with magnification raging from
5X to 30X, but 10X or 15X is the most
common in use. The ocular lens provides a
re-magnified image to see when light enters
through the objective lens.
2. The Eyepiece tube
 It connects the eyepiece and ocular lens to
the objective lenses.
3. The Microscope Arm
 It connects the eyepiece tube to the base
where you should hold when carrying the
microscope.
4. The Microscope Base
 It provides stability and support for the
microscope in its upright position. Typically,
it holds the source of light or illuminator.
5. The Microscope Illuminator
 It is a light source which can come in a form
of a built-in, low voltage illuminator light, or
a mirror that reflects an external light source
like sunlight.
6. The Stage and Stage Clips
 It serves as the platform for slides which
hold the specimen in place through stage
clip on either side. Some has mechanical
stage with adjustment knobs that allow
movement of slides to achieve more precise
positioning.
7. The Microscope Nosepiece
 It contains the objective lenses. You can
rotate this part in switching objective lenses
and adjust the magnification power.
8. The Objective Lenses
 Generally, microscope feature three or four
objective lenses, with magnification levels
ranging 4X to 100X. Objective lenses are
combining with the eyepiece lens to
increase magnification levels. Objective
lenses are the lenses that protrude
downward over the specimen.
a) Scanning lens – 4X
b) LPO – 10X
c) HPO – 40X
d) OIO – 100X
9. The Rack Stop
 It prevents users from moving the objectives
too close to the slide.
10. Control Focus Knobs
 Turning the knobs adjusts the distance
between the stage and the lens. The
coarse adjustment knob is used to bring
the specimen into initial focus -- visible but
not sharp. The fine adjustment knob is
then turned to bring the specimen into sharp
focus.
11. The Condenser Lens and Diaphragm
 These parts are located under the
microscopic stage. Condenser concentrates
the light on the specimen whereas
diaphragm with a small movable lever is
adjusted that regulates the entry of light.
Parts of Compound Microscope
A. Mechanical parts
 Used to support and adjust the parts
 Base
 Arm
 Revolving Nosepiece
 Stage
 Stage clips
 Rack Stop
 Eyepiece tube
 Coarse Adjustment Knob
 Fine Adjustment Knob
B. Illuminating parts
 Used to provide light
 Diaphragm
 Condenser
  Illuminator LESSON 3: Mounting of Specimen on
Microscopic Slide
C. Magnifying parts
 Used to enlarged the specimen Preparation of Microscopic Slide
 Objective lenses
 Mounting of specimen on slides can be done in
 Eyepiece
three different ways as discussed below in
simple ways.

A. Dry mount slide

 Place sample seed of sesame on a clean
glass slide at the center and cover it.

B. Wet mount slide

1. Place a drop of water at the center of the
slide.
2. Using tweezer, place pollen of gumamela
flower or any flower with pollen in the middle
of the drop.
3. While holding the cover slip upright and by
the edges, carefully place one edge of the
cover slip next to the water.
4. Set one edge against the slide and lower it
until it contacts the liquid. Slowly lower the
upper edge of the cover slip onto the water.
5. Absorbent towel can be placed at the edge
of the cover slip to draw out some of the
water, further flattening the wet mount slide.

C. Staining a Slide
 Onion cell will be mounted on a slide. Pull a
thin membrane from an onion layer then lay
it at the center of microscopic slide.
 If starch slurry will be mounted, prepare it by
mixing 2 parts of cold water with 1 part of
starch/ all-purpose flour.
1. Place one drop of methylene blue stain
or iodine solution on one edge of the
cover slip and the flat edge of a piece of
paper towel on the other edge of the
coverslip. (The paper towel will draw the
water out from under the coverslip and
the cohesion of water will draw the stain
under the coverslip.)
2. If the stain has covered the area
containing the specimen, you are done.
(The stain does not need to be under the
entire coverslip. If the stain does not
cover the area needed, get a new piece
of paper towel and add more stain until it
covers.)
3. Wipe off the excess stain with a paper
towel so it will not mess up with
objective lenses.
4. The slide can now be placed on the
microscopic stage.
LESSON 4: How to Focus Image Under
Microscope
1. Place the microscope on a flat, level, firm table
free from vibration seeing to it that the arm is
towards you while the stage is going away from
you. Do not place it in front of a brightly lit
window.
2. Turn on the light source and adjust the optimum
light setting to ensure correct level of brightness
by turning or sliding the brightness adjustment
knob at the base.
3. Turn the revolving nosepiece (turret) so that the
lowest power objective lens is clicked into
position.
4. Place the slide (dry mount, wet mount, prepared
mount) on the mechanical stage secured with
clips.
5. Look through the eyepiece and move the focus
knob until the image comes into focus.
6. Adjust the condenser and light intensity for the
greatest amount of light.
7. Move the microscope slide around until the
sample is in the center of the field of view.
8. Manipulate the focus knob and readjust the
condenser and light intensity to obtain the
clearest image possible.
9. Once the image is sharp with LPO, move to
HPO and do minor adjustments using fine
adjustment knob. (You might need to readjust
the sample into focus and/or readjust the
condenser and light intensity).
 Do not let the objective lens touch the slide!
 Both eyes should be open when viewing
through the microscope.
10. Complete your drawings (image seen under
LPO, HPO).
11. When finished, lower the stage, click the low
power lens into position and remove the slide.
12. Wash and dry the slides and keep it on your
storage rack or tray.
MODULE 2
INTRODUCTION AND ILOS - MICROSCOPIC
CALCULATIONS
Introduction
          What we are commonly using in the
undergraduate college laboratory are light
microscopes though in advance science courses
and programs other sophisticated and forms of
microscopes are housed in
laboratory/instrumentation room and utilized. Most
light microscopes used in our college biosci lab can
magnify cells up to approximately 400 times and
have a resolution of about 200 nanometers.
          Two important parameters in microscopy
are magnification and resolving power. Changes in
the orientation of the image that you see owe it to
the optical system of microscope, where I am
referring to the lenses.
LESSON 1: Field of View
Before proceeding to calculations, let me
emphasize some important definition of terms to
remember and here are the following:
Power of resolution – is the measure for the
ability to tell two points apart. It describes whether
two adjoining points can still be perceived as
separate.
Magnification of a microscope – is the product of
Vobjective    X     Vocular
Numerical aperture – is the sine of half the angle
of the cone of light from each point of the object
that can be accepted by the objective multiplied by
the index of refraction of the medium in which the
object is immersed. The N.A. represents a
performance number which can be compared to the
relative aperture (f-number) of a camera lens. The
N.A. values can be used for directly comparing the
resolving powers of all types of objectives. The
larger the N.A., the higher resolving power is.
Working distance – refers to the distance from the
cover glass to the nearest point of the objective.
Focal depth - refers to the distance between the
upper and lower limits of sharpness in the image
formed by an optical system. As you stop down the
aperture iris diaphragm, the focal depth becomes
larger. The larger the N.A. of an objective the
shallower the focal depth is.
Field number – This is a number that represents
the diameter in mm of the image of the field
diaphragm that is formed by the lens in front of it.
Field of view diameter - The actual size of the
field of view in mm on the object surface     What
did you see when you look into the microscope?
Did you see a polygonal or circular lit area? Of
course, better focus should reflect a circular one.
To calculate the dFOV illustrated on the slide
above, you will need to place a plastic transparent
ruler on the stage, focus the ruler until you see the
field of view, then measure the diameter of field of
view in millimeters. If we continue to increase the
magnification of your microscope, what do you
think will happen to the field of view? Will
it decrease or increase?
          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
nose-piece. 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 possessing different degrees
of magnification. When these are combined with
the magnification of the ocular lens, the total or
overall linear magnification of the specimen is
obtained.
          Magnification does not describe the quality
of the image. Magnifying an object without good
resolution is called empty magnification, as the
image appears larger but no greater detail can be
seen.
          The microscope magnification depends on
both ocular lens and objective lens magnifications.
Microscope maginification = Vobjective   X    Vocular
           For example, the magnification of ocular
lens is 10X with magnification of lenses of
objectives as follows: 4X, 10X, 40X, and 100X. So
what will be the magnifications at low, medium,
high power and oil immersion? 
magnification at low power (SO)= 10   x   4   =   
40 times
magnification at medium power (LPO)=   10   x   
10   =   100 times
magnification at high power (HPO)=   10   x   40   
=   400 times
magnification at oil immersion type of power 
(OIO)=   10   x   100   =   1,000 times
How do you now calculate the field of view in
your microscope?
          1. If using an eyepiece only?
Please check the specifications of the eyepiece,
let's say you have wide-field (WF) 10X/20. The
magnification of the eyepiece is 10X with a field
number of 20. Therefore, the FOV here is 10
divided by 20 is 0.5 or an FOV diameter of 0.5
millimeters.
          2. If eyepiece and objective lens are
used?
Let us use the specification of the eyepiece in the
example above, WF 10X/20 and you set the
objective lens at 40.  Multiply 10 (Vocular ) by 40
(Vobjective ) to get 400. An FOV diameter of 0.05 mm
will be obtained in dividing 20 by 400.
If you know the dFOV for low power lens, FOV of
higher power lenses can be calculated using the
ratio as follows and vice versa:   
Low Power Magnification × Low Power Field of
View = Higher Power Magnification × Higher Power
Field of View
Example: 
The Vocular of 10× with the Vobjective of 10× produced  the  true size of the object? I am referring
the field of view of 800 µm. Compute for the field of
view when the Vocular is 40× and the Vobjective is 100×.
Solution:
The magnification at lower power is 10 × 10 = 100
The magnification at higher power is 40 × 100 =
4000
Field of view = 100 × 800 / 4000 = 20 µm
LESSON 2: Object (Actual) Size
How would you find the size of the object you are
observing? Knowing the dFOV for instance will help
you out. Then estimate how many times the object
fits across the dFOV. Refer to the diagram below.
Let's say for example, using medium power (10X
objective), the diameter of field of view is measured
as 2 mm. Looking across the diameter are cells
aligned. Counting it gives you 8 cells. Therefore,
there are 8 cells visible across the dFOV of 2 mm.
You are determining the approximate size of each
cell, dividing the dFOV by number of cells (2 mm /
8) will give you 0.25 mm. The size of each cell is
therefore 0.25 mm or 250 µm. 
LESSON 3: Drawing Magnification
For some time, you need to draw the image you've
seen under microscope or attached camera to take
photos of the image. The question is how many
times your drawing has been enlarged relative to
to drawing magnification. Below is the equation for
calculating the drawing magnification.
Magnification size of drawn object in μm
=
of drawing   object’s actual size in μm
          Let us say the drawing is roughly 7 cm long.
This equates to 70 mm or 70,000µm.  The actual
size of the specimen is about 500 µm. What is the
drawing magnification?
Drawing magnification = drawing size / actual size.
70,000 µm / 500 µm = 140 X
Drawing Magnification = 140 X
          Let us say the corpse flower (Rafflesia
arnoldi) below measure around 1 meter in
diameter. You are instructed to draw in  a paper in
such that the diameter will measure only 10
centimeters. Applying the formula above will give
you 0.1 X . So, what does this means?
Scale bars are a useful way to indicate the size of a
cell or object of study.How do you calculate the
magnification of an image using scale bar?
A. use ruler to measure the length of the scale
bar in mm.
B. convert into the same units as the scale bar.
C. divide the IMAGE scale bar length by the
ACTUAL object scale bar length.
ACTIVITIES