Activity 1: Basic Microscopy

ACTIVITY 1
BASIC MICROSCOPY

Most microorganisms like protozoa, algae, fungi, and bacteria are so small that they
cannot be seen by the naked eye. The smallest object that the eye can see at a distance of
250mm is about 0.07-0.14 mm. The use of the microscope allows us to see these minute
organisms since an object can be magnified a few hundred times to hundreds of thousand
times.
There are two general categories of microscopes: the light microscope which uses
light waves and lenses that are associated with the light microscope, and the electron type
which employs electron beams and magnetic fields to produce the image.

Light microscopes can be classified as simple if they have a short focal length are
held close to the eye and magnify objects only up to 300x. On the other hand, the compound
type employs two separate lenses, an ocular, and an objective, to achieve 2-5 times greater
magnification.

In this course, the compound microscope will be used in many of the exercises so
the student should be able to use it proficiently and care for it properly. Constant and proper
use of the microscope will increase one’s proficiency.

By the end of this topic, you will be able to:

1. identify the major parts and sections of the microscope and their function;
2. list the theoretical principles of brightfield microscopy;
3. identify how to maintain a microscope;
4. describe the process to correctly focus on the appropriate field of view;
5. use an ocular micrometer to measure an object under the microscope; and
6. demonstrate the ability to troubleshoot encountered problems with the
microscope.

Parts of the Microscope

The microscope consists of three main parts: the head, the arm, and the base. Each
part contains several important sections.

• Microscope Head
• The first part of the microscope is the
head. The head contains the oculars and
the nosepiece, which are connected to
the objective lens.
• The oculars are the eyepieces of the
microscope. They are connected to
lenses that have a magnification of 10X.
In most microscopes, they are binocular.
• You can adjust the eyepieces to match
the distance between your eyes
(interpupillary distance) so that you can
see one image.
• It is important to look at the image under
the microscope with both eyes because

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 Activity 1: Basic Microscopy

it improves the field of view.

• Contains the oculars and nosepiece, which connects to multiple objective
lenses of different magnifications such as 10x, 20x, 40x, 100x.
• Microscope Arm
• The microscope arm contains the stage,
stage controls, coarse and fine
adjustment knobs, and condenser.
• The stage, located underneath the
objectives, is a platform with clips to hold
the slide in place.
• To move the specimen in the field of
view, you may need to move the stage
right or left, backward or forward using
the stage control knobs. These knobs are
located at the side and beneath the
stage.
• Coarse and fine adjustment knobs,
located just above the base, move the stage up and down and are used to
bring a specimen into focus.
• Holds the stage, stage controls, coarse and fine adjustment knobs, and
condenser diaphragms. Mechanical clips located on the stage hold the
slide.
• Microscope Base
• At the bottom of the microscope is the base.
• The microscope base is used for support.
• The base of the microscope houses the
field diaphragm, illuminator, and the on/off
switch for the illuminator. The illuminator
may also be called the light source.
• Contains the field diaphragm, illuminator
(light source), and on/off switch for
illuminator.

* Please note that the location of the sections on the

arm and base can vary based on the type of
microscope used.

What is Microscopy?

Microscopy is the use of the microscope to view objects that cannot be seen with the
naked eye. It is the first step in the examination of specimens. Microscopy plays an
important role in helping laboratory professionals view specimens clearly.

Brightfield Microscope

To use your microscope effectively and efficiently in your daily routine, it is

necessary that you become familiar with the major parts of the microscope.
Once you are familiar with these parts, you should routinely inspect your microscope
and perform maintenance as required, to keep the microscope clean and functional.

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 Activity 1: Basic Microscopy

Theoretical Principles of Microscopy

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.

Table 1.1 Overall Linear Magnification

TOTAL
MAGNIFICATION
MAGNIFICATION
OBJECTIVE
OBJECTIVE
OCULAR LENS MULTIPLIED BY
LENSES
OCULAR
Scanner 4x 10x 40x
Low power 10x 10x 100x
High power 40x 10x 400x
Oil immersion 100x 10x 1000x

Resolving power or resolution - 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 blur the object. The resolving power of
a lens is dependent on the wavelength of light used and on the numerical aperture,
which is a characteristic of each lens and is imprinted on each objective. The
numerical aperture is 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:

Resolving power = wavelength of light

2 x numerical aperture

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 Activity 1: Basic Microscopy

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. This 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. We can compensate for loss
of refracted light 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 1.2. Refractive index in air and 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

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 Activity 1: Basic Microscopy

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 1.c. Relationship between objective, working distance and diaphragm opening

Care and Maintenance of the Microscope

1. Keep the microscope clean, dry and free from dust

2. When carrying the microscope grasp it arm with one hand and the bottom of
the base with the other hand.
3. Avoid jarring the microscope
4. Never touch the lenses with your fingers. Wipe dirty lenses gently with lens
paper.
5. Clean the stage as well as the objectives when you use a new slide

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6. Do not allow the objectives to come in contact with any liquid except the
immersion oil for the 1.8 mm objective.
7. Clean the oil immersion objective immediately after it has been used by
removing the oil with lens paper. Dried oil on the objective is removed by
moistening the lens with xylol and then drying immediately with lens paper.
Xylol is corrosive so the lens should be wiped dry right away.
8. Never force adjustments on the microscope. All adjustments should work
freely and easily.
9. Avoid unnecessary turning of the coarse and fine focusing knobs.
10. Do not allow alcohol or xylol to come in contact with the lacquered parts.
11. Before you begin routine cleaning of your microscope, make sure that you
have the necessary supplies on hand.
12. Your microscope should be cleaned as often as needed; however, yearly
maintenance is essential to keep the microscope in optimal working order.
13. All maintenance should be recorded.
14. It is also good practice to cover your microscope after use and cleaning. This
will prevent fine dust and particles from filtering into the internal components
of the microscope.

Moving the Microscope

If you need to move your microscope to a different location, it is essential to know

how to properly carry it to avoid any damage to components.
1. Unplug and secure the power cord.
2. If the microscope has a rotatable head, turn the head in before moving.
3. Grasp the arm with one hand and support the base with your other hand
4. Always carry the microscope with two hands
5. Carry the microscope close to your body, or use a cart for long distances.

Procedure to Clean the Microscope

1. When cleaning your microscope, start at the top of the microscope and work
down.
2. Clean the oculars by moistening the tip of a cotton swab or lens paper with
lens cleaning solution.
3. Working from the inside to the outside, gently clean each eyepiece in a
circular motion.
4. With a clean piece of lens paper, use circular motion to polish and remove
any cotton fibers.
5. Using the stage controls, lower the stage to reach the lenses on the
condenser diaphragm.
6. Clean each lens with lens paper.
7. Clean the stage with a suitable cleaner, as recommended in your facility's
SOP for maintenance (this helps to prevent the dragging of a slide across the
stage if there is oil on the stage).

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 Activity 1: Basic Microscopy

Focusing the Microscope

Specimens are viewed clearly when the microscope is in focus. This process is
performed using the fine and coarse adjustment knobs. It is important to proceed
carefully to avoid damage to the slide or objective.

Parfocal is the term used to describe the minimal or non-existent change in the
focus as a result of moving from one objective to another. Microscope objectives are
parfocal, where focus is achieved by going from a lower to a higher objective

Instructions

1. Put a slide on the

microscope stage, with
the coverslip side up.
2. Bring the 10X or the
40X objective over the
slide.
3. Using the coarse
adjustment knob, raise
or lower the stage until
you see an object come
into view.
4. Use the fine adjustment
knob to obtain a clearer
view of the object.
5. Manipulate the light
source to obtain the
correct amount of light.
6. If you are using the
100X (oil immersion)
objective to view your
specimen, a good
practice is to focus on
the specimen using the
10X or 40X objective
and then move to the
100X oil immersion lens
after adding oil.
7. If the scope is parfocal,
you should only need to
use the fine adjustment
knob to focus.

Note: Use oil immersion with 100X objective only and do not drag the 40x objective
through the oil.

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 Activity 1: Basic Microscopy

Ocular/Stage Micrometer

The stage and ocular micrometer are used to measure small specimens not easily
measured by a traditional ruler.

An ocular micrometer is used when it is necessary to measure the size of an

organism viewed under the microscope. Ensuring proper calibration when using the
ocular micrometer is beneficial to confirm measurements are accurate.

An ocular micrometer is a ruler embedded in a round glass used for measuring

objects under the microscope. In conjunction with the ocular micrometer, you will
need a stage micrometer for the calibration process.

The Ocular Micrometer Calibration Procedure

This is a simple and precise method

for measuring objects seen in the
microscope. Ocular micrometers are
calibrated by comparing the ocular
micrometer scale with a calibrated
stage micrometer. By aligning and
calibrating the ocular micrometer under
the microscope the measurements can
be used to determine the size of the
specimen. A calibration procedure
must be completed to determine the
calibration factor for each objective
and each microscope

1. Insert the ocular micrometer into

a 10X eyepiece. The ocular
micrometer is divided into ocular
divisions (OD).
2. Place the calibrated stage
micrometer slide on the stage
and focus on the scale. The
stage micrometer has a
calibrated scale which is divided
into 0.1 millimeters (mm) and
0.01 mm units.
3. Adjust the field so the 0 line of
the ocular micrometer (OM)
scale is exactly superimposed
upon the 0.0 line of the stage
micrometer (SM) scale.
4. Without moving the stage
micrometer, locate the point as
far to the extreme right as
possible where any two lines
are exactly superimposed upon

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 Activity 1: Basic Microscopy

each other.
5. Count the number of divisions (mm) on the stage micrometer between the 0.0
line and the superimposed line to the far right.
6. Count the number of ocular divisions on the ocular micrometer between the 0
line and the superimposed line to the far right.
7. Divide the distance determined in step 5 by the number of ocular divisions in
step 6 and multiply by 1000 to give the ocular micrometer units in μm.
8. Repeat steps 3 through 7 for each objective on the microscope.
9. If at any time the ocular micrometer is moved to a different microscope or a
new objective is added to the microscope, the calibration procedure must be
completed again.

Example

1. Add the ocular micrometer to the eyepiece and pre-calibrated stage

micrometer to the stage, on top of the specimen to be measured. For this
example we are using a 10X objective.

2. Align the ocular micrometer with the stage micrometer so that both meet at
the 0 mark.

3. Identify the farthest point to the right where both the stage and ocular
micrometer rulers align. This should look something like the image below.

As you can see, the last measurement where both rulers align is at 60mm
(millimeters). The larger the number on the ocular micrometer, the more
accurate the calculation.

4. For this example, the stage micrometer is 1mm long with 100 divisions, which
means that each division is 0.01mm (millimeters). By counting the lines on the
stage micrometer up to where it aligns with the ocular micrometer at 60mm,
you get 0.4mm (40 divisions x 0.01mm=0.4mm) for the stage micrometer.

5. Take the 0.4mm and divide it by 60mm, which should look like
0.4mm (stage micrometer)/60mm (ocular micrometer) = .00667mm

6. Finally, convert millimeters (mm) into micrometers (μm) by multiplying by

1000.
0.00667mm x 1000 = 6.67µm (micrometers)

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 Activity 1: Basic Microscopy

This means that with the 10X objective each ocular division (OD) is equal to 6.67µm
(micrometers)

Measuring Objects Using an Ocular Micrometer

Now that you have calibrated your ocular micrometer, you are ready to measure the
size of objects/organisms seen under the microscope using the ocular micrometer as
a ruler.

1. Align the left side of the object with the 0 of the ocular micrometer.
2. Count the ocular divisions (OD) to the end of the specimen.
3. Multiply the OD by the calibrated micrometer unit.
4. To calculate the size of the specimen, multiply the ocular micrometer
calibration by the number of ocular divisions (OD) the specimen covers. The
formula should look like this:

ocular μm (micrometer) x OD (ocular divisions) = size of the specimen.

To calculate the size of the specimen to the right using our previous ocular
micrometer calibration, multiply 6.67μm by 11 which is the number of ocular
divisions (OD) the specimen covers.
6.67μm x 11 OD = 73.37μm.
This means the size of the specimen is 73.37μm.

Troubleshooting Microscope Issues

In your daily use of the microscope, you may encounter issues such as not seeing
the specimen clearly, dimming, or a burned out bulb.

Black Field in the Oculars

If you put the slide on the stage and look through the
oculars but all you see is a black field, the possible
causes could be:
• The microscope is unplugged
• Power is not available at outlet
• The objective is not clicked into place
• The bulb is burned out or not inserted correctly
• The condenser is too low and the diaphragm is
closed

Blurred Image

If you look into the oculars after attempting to focus and

have a blurred image, the possible causes could be:
• Dirty objective
• Oil has leaked into the objective
• Dirty coverslip
• Dirty microscope slide
• Slide is upside down

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Problem Focusing

If you place a slide on the stage and move the objective

into place but still have problems focusing, the possible
causes could be:
• Slide is not seated properly on the stage
• Microscope needs to be aligned
• Parfocal problems
• Jammed focus mechanism
• Oil on the stage

Light Flickers

If you turn on the microscope's light and it flickers or surges, the possible causes
could be:
• Wiring could be damaged in the cord
• Bulb is getting ready to burn out
• Bulb is not inserted correctly

Partial Illumination

If you look through the ocular and you see a field that is
partially illuminated, the possible causes could be:
• Objective is not clicked into place
• Condenser is not centered correctly
• Condenser is too low
• Field diaphragm is not open enough

Troubleshooting Microscope Issues

When troubleshooting issues with the microscope, here are a few things you can do
to minimize the likelihood of having issues while viewing a specimen.
Make sure that the:
• Microscope is clean.
• Objective is clicked in place.
• Condenser is centered correctly.
• Condenser is positioned at the correct height.
• Field diaphragm is open appropriately.

References:

Cappucino, J. & Welsh, C. 2020. Microbiology: a laboratory manual, 12th ed.,

Pearson Education, Inc., New York

The Centers for Disease Control and Prevention, Division of Laboratory Systems.
(n.d.-b). CDC Train:Basic Microscopy: Microbiology Curriculum.
Www.Train.Org. Retrieved August 28, 2020, from
https://www.train.org/cdctrain/course/1044412/

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