University of Santo Tomas

College of Science
Department of Biological Sciences

Module I
Microscopy and the Microbial World

I.A. The Microscope and its Resolving Power

Microscopes have played an important role in the history of microbiology, having allowed
scientists over the centuries to view microorganisms. This is because our eyes cannot resolve
anything less than 0.1 mm, so most microbes are invisible to us. Its use has facilitated our
understanding of microbial structures and it remains to be indispensable in the identification of
microorganisms.

The type of microscope to be used in this exercise is the bright-field microscope, where the
specimen is often darker against a lighter background. This compound microscope has two kinds
of lenses, the objective and the ocular. Together, they can magnify a tiny specimen up to 1000x.
The first part of this module reviews the parts of the microscope together with their functions.
This is followed by the resolving power of the microscope.

LEARNING OBJECTIVES

Once you have completed the exercise, you should be able to…

1. Recognize the parts of a compound microscope and know their functions.

2. Execute the proper operation of the microscope using the LPO, HPO, and OIO objectives.
3. Compute the microscopic parameters including Total Magnification and Resolving
Power of each objective using Abbe’s equation and to know their practical application.
4. Know the proper care for the microscope.

MATERIALS

Equipment
• Brightfield microscope

Procedure

i. Proper use and care of the microscope

Transport of the microscope

1. Get your microscopes from the technician counter keeping the microscope in an upright
position.

2. Hold the microscope arm with your right hand and support the base of the microscope
with your left hand. Never hold the microscope like a bag or in any position other than
upright to prevent accidental damage of loose parts.

3. Do not drop the microscope or remove any of its parts. All parts of a microscope are
expensive and difficult to procure.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

4. Lost or damage parts of the microscope should be reported immediately to your

facilitators.

Cleaning of lenses and other parts:

1. Wipe the microscope lenses and its mechanical parts with clean dry lens paper.

2. Gently clean the oil immersion lens (objective marked 100X) with clean lens paper.
Xylene is the solvent of choice to clean hardened oil sticking from the Oil Immersion
Objective (OIO).

3. Do not use alcohol for cleaning as some lens adhesives are soluble in alcohol.

ii. Parts of the microscope and their functions – a review

1. Try locating each one on your microscope and as you go along, label the microscope in
Figure 1 of the worksheet.

a. Eyepiece or Ocular - the lens where you look through in a microscope.

b. Draw tube - the tube where the eyepiece is attached.
c. Dust shield - the mechanical part where the revolving nosepiece is attached; protects
the objectives from dust.
d. Revolving nosepiece - the mechanical part where the objectives are attached; can
be rotated to position the objective in use. Also called “Turret”.
e. Objectives - the lenses above the stage attached to the revolving nosepiece are
f. Scanner – 4X is optional for most microscopes
g. Low Power Objective (LPO) - marked 10X
h. High Power Objective (HPO) - marked 40X
i. Oil Immersion Objective (OIO) - marked 100x
j. Stage - the platform under the objectives where you place the specimen to be
viewed.
k. Translational control knobs – move the slide left or right, and forward or backward
l. Condenser - the lens located below the stage which collects light and delivers it to
the objective.
m. Iris diaphragm – located above the condenser and below the stage, it regulates the
amount of light reaching the specimen
n. Lamp – a built-in light source for electric microscopes. This is controlled by an
adjustment dial that regulates light intensity, and a power switch.
o. Base - the rectangular or U-shaped part that supports the body of the microscope.
p. Arm - the C-shaped part where the microscope is grasped when it is moved or
transported.
q. Fine adjustment - the knob at the body tube used to focus specimen under the HPO
and OIO.
r. Coarse adjustment - the knob at the body tube used to focus the specimen viewed
under the LPO.
s. Pillar - the part connecting the body of the microscope to the base.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

iii. Resolving power of the microscope

1. Look at the objectives and you will notice that there are numbers engraved on the
barrels. Using the scanner objective as an example (see diagram) the numbers represent
the following information:

i. 4 is the magnification of the objective

ii. 0.10 is the numerical aperture
iii. 160 is the tube length in mm
iv. 0.17 is the recommended thickness of the
coverslip in mm

2. Summarize the values for each of the objectives pictured above (LPO, HPO, OIO) by
filling out the first 3 rows of Table 1.
3. The resolving power or limit of resolution describes the ability of a microscope lens to
gather fine details of the specimen being observed. Two tiny objects separated by a
distance that is smaller than the limit of resolution cannot be resolved as two separate
objects. The limitations in both magnification and resolving power of the microscope lens
prevents us from seeing very small microbes such viruses and from observing the fine
details of a cell.
4. The limit of resolution is mathematically defined by Abbe’s Equation (after Ernst Abbe,
1840-1905), which states that resolution is defined by the wavelength of light and the
numerical aperture.

Limit of Resolution = 0.61 λ = 0.61 λ

η sin θ NA (Numerical Aperture)

• 0.61 is constant value derived from the properties of light

• 𝝺 (lambda) is the wavelength of visible light illuminating the microscope. The visible
light spectrum ranges from ~380 nanometers of nm (violet) to ~700 nm (red).

• η (eta) is the refractive index of the medium between objective lens and glass slide.
Values of η: Air = 1.00, Water = 1.33, Immersion oil = 1.5. When the oil immersion
objective is used, the presence of a drop of oil between the specimen and the lens,
instead of air, reduces light scattering and increases the entry of light into the objective,
thus increasing the numerical aperture and resolving power. Immersion oils can be
natural (cedar wood oil) or synthetic.

• θ (theta) is half the value of the angular aperture. The angular aperture is also known
as the cone angle; it is the cone of light that enters the objective lens.

• An objective with a higher numerical aperture will have a shorter working distance. The
working distance is the distance between the objective lens and the specimen when it is
well-focused, or when the image is at its sharpest.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

5. Using the tabulated values and Abbe’s equation, solve for the limit of resolution of each
of the objectives, assuming blue light as your light source. Write your answers on the 4th
row of Table 1 in the worksheet.

The computation for the scanner objective is given below to serve as your guide.

Scanner

Given:

• 0.61 (constant)
• 𝝺 or lambda (wavelength for blue light) = 475 nanometers (nm)
• Numerical aperture for scanner = 0.1

Limit of resolution = (0.61) (𝝺)

Numerical aperture 0.1

= (0.61) (475 nm) = 289.75nm

0.1 0.1

= 289.75 nm or 2.9 micrometers (μm)*

*One micrometer (μm) is equal to 1000 nm

I.B. Viewing the Microbial World

The microbial world is the largest assembly of a wide variety of minute organisms occurring
everywhere in nature. The major groups of microorganisms include the Bacteria and Archaea –
having a prokaryotic cell structure; and various eukaryotes such as the Fungi (yeasts and molds),
and the Protists (protozoa, algae, slime molds); and the non-cellular microbes - the Viruses.

In this exercise, you will learn the proper way of manipulating the microscope to view your
specimens using the different objectives lenses. In doing so you will observe a variety of microbes
with diverse shapes and sizes so that you can develop familiarity with their appearance or
morphology.

LEARNING OBJECTIVES

Once you have completed the activity, you should be able to…
1. Learn how to use the microscope and focus using the oil immersion objective
2. Prepare wet mounts of microorganisms and observe them in their living state.
3. Recognize and identify common examples of the major groups of cellular
microorganisms – bacteria, yeast, mold, algae, and protozoa.
4. Describe microbial morphology (cell shape, arrangement) and reproductive structures
using the correct terms.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

Materials:

Yeast culture Slides and cover slips

Pond water sample (Algae) Pipettes and aspirator bulbs
Hay Infusion (Protozoans) Immersion oil
Prepared slides of bacteria and molds Lens paper
Light microscope

Procedure:

a. Microorganisms in their Living State: Wet – Mount Preparation

1. Place a drop of hay infusion / pond water / yeast cell suspension on a clean glass slide.
You may clean your glass slide with alcohol and soft tissue paper.
2. Carefully cover the drop with a cover slip.
3. Examine the preparation under LPO and HPO. Reduce the amount of light entering the
microscope through the lamp control and iris diaphragm, as several live microorganisms
are more readily seen in dim light.
4. Identify, illustrate and describe all the microorganisms that you see in the worksheet
(Section I.B.).

b. Microorganisms in their Fixed State: Stained Prepared Slides

1. Secure prepared slides of bacteria, yeasts, molds, algae, and protozoa. Examine them
under the microscope using the HPO for molds, yeasts, algae, and protozoa and the oil
immersion objective (OIO) for the bacteria.
2. Mount the slide on the stage and check the illumination of the microscopic field by tilting
the mirror and opening the iris diaphragm. Get the brightest microscopic field as this will
get dimmer once the switch to the OIO is done.
3. Focusing under the OIO takes a little practice and care. With the nosepiece halfway
between the HPO and the OIO (100X), apply a small drop of immersion oil on the center
of the specimen on the slide. Gently swing the OIO into position over the drop. Refocus
by adjusting the fine knob.
4. Again, identify, illustrate and describe all the microorganisms that you see in the
worksheet (Section I.B.)

For the illustrations, make sure to take note of the following details:

a) Name (scientific name or common name, whichever is given)

b) Major microbial group to which it belongs (bacteria, fungi, protozoa, algae)
c) Type of preparation: Prepared slide or Wet mount
d) Cell shape and general appearance
e) Natural color (for live algae: which have natural pigments)
f) Motility (for live protozoa: motility structures)

IMPORTANT: Keep fingers off optical surfaces. Clean up lenses after a season of oil immersion
work using lens paper.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

A Simplified Guide to Microbial Morphology

1. Common shapes and arrangements of bacteria

These terms are generally to used describe bacterial cells, but some of these may also
be used to describe yeast cells, algal cells, and molds (filamentous).

2. Yeasts

These are eukaryotic microbes that comprise the unicellular fungi. They reproduce
asexually through budding or fission.

Ex. Baker’s yeast (Saccharomyces cerevisiae)

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 University of Santo Tomas
College of Science
Department of Biological Sciences

3. Molds

These are the filamentous fungi. They are composed of filamentous cells called hyphae
(singular: hypha). Masses of hyphae are collectively called mycelia. Molds reproduce
asexually by forming spores that are formed on the tips of aerial hyphae that grow vertically
from the medium. The aerial hyphae are anchored on the medium by substrate hyphae. Mold
filaments may or may not have cross walls.

Below are 3 examples of spore forming structures, represented by 3 species:

(1) (2) (3)

Aspergillus niger Rhizopus stolonifer Penicillium notatum

4. Protozoa

The protozoa are mostly unicellular eukaryotic microbes that are motile through motility structures
that include pseudopodia (for amoebae), cilia, and flagella. While many ciliated and flagellated
protozoans have a fixed cell shape, the amoeba and certain flagellates and ciliates may manifest
shape change as they move and feed.

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 University of Santo Tomas
College of Science
Department of Biological Sciences

5. Algal cells and colonies

The algae are a heterogeneous group of photosynthetic organisms. There are prokaryotic
and eukaryotic algae. Prokaryotic algae are composed of the cyanobacteria (blue-green
algae). The eukaryotic algae may be microscopic or macroscopic (seaweeds).

Examples of eukaryotic microalgae include diatoms, dinoflagellates, many green algae,

and other algal groups. Below are examples of cell morphologies and cell arrangements
of the microalgae.

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