MODULE IN GENERAL MICROBIOLOGY

Unit 1
Introduction to Microbiology

Lesson 3: The microscope – a microbiologist’s best friend

Introduction
What is the smallest living organism that you can see with your unaided eyes? In my case, I can see
bird mites, arthropods, which are very tiny. They can be about the size of the period at the end of
this sentence. Although I have not personally seen any of the giant bacteria we learned earlier, they
would have probably the size as the bird mites.
Don’t you know that the good, unaided eye can only separate objects that are about 0.10 – 0.15
millimeters (mm) apart? This would mean that two dots closer than 0.10 mm cannot be seen as
separate entities but these will blur into one dot. Many biological specimens, such as animal cells,
are much smaller than the resolution of the human eyes. For instance, the red blood cell is 6-8 µm
in size (10-6 mm) but we have learned that bacteria, fungi and protozoa are even much smaller, and
the viruses, many times much smaller. So how do we study these microbes? We need a tool to in
order to study them and this is the microscope. It is the most basic and most valuable tool of a
microbiologist.
This lesson provides an introduction to microscopy and the operation of a simple light microscope.

Learning Objectives
At the end of the lesson, you are expected to:
 identify and describe the parts of a bright-field microscope
 calculate total magnification for a compound microscope
 distinguish magnification and resolution
 describe the distinguishing features and typical uses for various types of light microscopes,
electron microscopes, and scanning probe electron microscopes

Activating Prior Learning

When was the last time you used a microscope? Was it when you were in high school? What
remarkable experiences have you acquired while using the microscope?
Figure 1.3.1 below shows you a schematic diagram of a light microscope. Recall your experience
in using the microscope by labeling the parts indicated by numbers? Write your answers on a
separate sheet.

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College of Arts and Sciences, CAGAYAN STATE UNIVERSITY
First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

 Activity 1.3.1.
How well can I remember the parts of a light microscope?

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3 10
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Figure 1.3.1. Parts of a light microscope.

(https://timvandevall.com/microscope-diagram-parts-of-a-microscope/)

How did you find the activity? Did it bring back fond memories of your high school life? To find
out if you remembered the parts of a microscope correctly, read on the following notes.

Core Content

PRINCIPLE OF LIGHT MICROSCOPY

Microbiology is a science that studies living organisms that are too small to be seen with the naked
eye. Needless to say, such a study must involve the use of a good compound microscope. There are
many types and variations, however, they all fundamentally consist of a two-lens system, a variable
but controllable light source, and mechanical adjustable parts for determining focal length between
the lenses and the specimen.

Components of the Microscope

STAGE A fixed platform with an opening in the center allows for the passage of light from an
illuminating source below to the lens system above the stage. This platform provides a surface for

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First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

the placement of a slide with its specimen over the central opening. Less sophisticated microscopes
have clips on the fixed stage and the slide must be positioned over the central opening.

ILLUMINATION The light source is positioned in the base of the instrument. Some microscopes
are provided with a built-in light source to provide direct illumination. Those that are not are
provided with a mirror, one side flat and the other concave. The flat side of the mirror is used for
artificial light, and the concave side for sunlight.

ABBE CONDENSER This is found directly under the stage and contains two sets of lenses that
collect and concentrate light passing upward from the light source into the lens systems. The
condenser is equipped with an iris diaphragm, which is a shutter controlled by a lever and used to
regulate the amount of light entering the lens system.

BODY TUBE Above the stage and attached to the arm of the microscope is the body tube. This
structure houses the lens system that magnifies the specimen. The upper end of the tube contains
the ocular or eyepiece lens. The lower portion consists of the movable nosepiece containing the
objective lenses. Rotation of the nosepiece positions objectives above the stage opening. The body
tube may be raised or lowered with the aid of coarse-adjustment and fine-adjustment knobs that
are located above or below the stage, depending on the type and make of instrument.

Now that you have learned about the components of the microscope, do you feel happy about the
results of your activity above? Congratulations if you are. If you are frustrated, then it is not
something to be worried about. Just go back to the lesson anytime to master the part that you missed.

So, let us move on to learn about the important properties of a microscope.

Theoretical Principles of Light Microscopy

To use the microscope with efficiency and minimal frustration, students should understand the basic
principles of microscopy: magnification, resolution, numerical aperture, illumination, and focusing.

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 plan 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 magnification of the specimen is obtained. Therefore,

Total Magnification = Ocular Magnification x Objective Magnification

For example, if a 40x objective lens is selected and the ocular lens is 10x, the total magnification
would be (40x) (10x) = 400x.

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College of Arts and Sciences, CAGAYAN STATE UNIVERSITY
First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

RESOLVING POWER OR RESOLUTION Although magnification is important, you must be

aware that unlimited enlargement is not possible by merely increasing the magnifying power of the
lenses or by the use of additional lenses, because lenses are limited by a property called resolving
power. Resolving power is the ability of the lenses to show two adjacent objects as discrete entities.
When a lens cannot discriminate, that is, when the two objects appear s one, it has lost resolution.
The resolving power of a lens is dependent on the wavelength of light used and a characteristic of
the lens called the numerical aperture. This is expressed by the formula:

Resolving power = Wavelength of light/Numerical aperture

With this formula it can be shown that the shorter the wavelength, the greater the resolving power
of the lens. Thus the short wavelengths of the electromagnetic spectrum are better suited than longer
wavelengths in terms of the numerical aperture.

The numerical aperture is defined as a function of the diameter of the objective lens in relation to
its focal length. It is doubled by the use of the condenser which illuminates the objects with rays of
light that pass through the specimen obliquely as well as directly. Thus the larger the numerical
aperture the greater the resolving power, which may be expressed mathematically, as follows:

Resolving power = Wavelength of light/2(numerical aperture)

The relationship between wavelength and numerical aperture is valid only for increased resolving
power when light rays are parallel. Therefore the resolving power is 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, and as light rays pass from
the glass into the air they are bent or refracted so that they do not pass into the objective lens. This
would cause loss of light, which would reduce the numerical aperture and diminish the resolving
power of the objective lens. Loss of refracted light can be compensated for 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.

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. The condenser should always be kept close to the stage, especially when using
the oil-immersion objective.

Between the light source and the condenser is the iris diaphragm that 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 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 object lens and slide, called working
distance, decreases and the angle of aperture of the objective increases.

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First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

Types of Light Microscope

The following are variations of the light microscope.

 Bright-field microscope
 Dark-field microscope
 Phase contrast microscope
 Differential interference contrast
 Fluorescence microscope
 Confocal scanning laser microscope

The compound microscope that we commonly use in the laboratory is a light microscope,
particularly a bright-field microscope because the specimen is lit from underneath. It is a
compound microscope which means that the specimen is magnified by two lenses: the objective
lens and the ocular lens, and it can be monocular or binocular. The diagram of a microscope that
you have met earlier is of the binocular type. A light microscope provides a magnification of up
to 1,000 times, which is considered to be high, although the resolution is low. The specimen
appears dark against a bright field.

You shall learn about the other variations of LM later on in the lesson.

 Self-Assessment Questions

1.3.1. Differentiate magnification and resolution. __________________________________

______________________________________________________________________
____________________________________________________________________

1.3.2. What are the components that impact resolution and in what way? ________________
______________________________________________________________________
____________________________________________________________________

1.3.3. What is the function of the immersion oil? ___________________________________

______________________________________________________________________
____________________________________________________________________

ELECTRON MICROSCOPY

In the preceding presentation you have learned that in light microscopy light passes through one or
more lenses to produce an enlarged image of a specimen and the maximum theoretical resolution of
images is ultimately limited by the wavelengths of visible light. As mentioned earlier, most light
microscopes can magnify 1000x, but some can magnify up to 1,500x. Resolution is the limiting
factor to a light microscope since the greater the magnification is, the less it is able to resolve the
image. Images may appear blurry beyond 2,000x. The limit of resolution for a LM is 0.2 um or 200
nm but most viruses are smaller than that. Algae, yeasts, molds, bacteria and archaea can be

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First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

observed using LM but viruses cannot be observed with it. Viruses are smaller than the limit of
resolution for a LM which is 0.2 um or 200 nm.

Electron microscopes (EM) are more powerful and produce sharper images than LM. They can
increase magnification and resolution through the use of short-wavelength electron beams rather
than light. Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of
1.23 nm (as opposed to the 530 nm wavelength of blue-green light), resolution increases to around
0.5 nm, with magnification over 150,000x. Hence, EM can produce much better resolution than
visible light.

There are two basic types of EM: the transmission electron microscope (TEM) and the scanning
electron microscope (SEM). The TEM uses a beam of electrons transmitted through a very thinly
sliced specimen (20-100 nm thick). EM uses magnetic lens (instead of glass lens) to guide the beam
of electrons toward the specimen, and a detector to capture the image to view. TEM can magnify
up to 200,000x. TEM also requires staining with heavy metals as these enhance the varying opacity
of the image as these materials are electron dense. TEM require that the beam and specimen be in a
vacuum and that the specimen be very thin and dehydrated.

SEMs provide detailed three-dimensional (3-D) image of the specimen. Unlike thinly sliced
specimen in TEM, SEM preparation involves spraying the specimen with a fine metal coating. As
the beam of electrons is passed over the specimen’s surface, the metal coating emits a shower of
electrons, and a 3-D image of the specimen’s surface is produced to view. Typically, specimens are
prepared with fixatives that reduce artifacts, such as shriveling, that can be produced by drying,
before sputter-coated with a thin layer of metal such as gold. SEM can magnify and image up to
2,000,000x.

Whereas TEM requires very thin sections and allows one to see internal structures such as organelles
and interior of membranes, SEM can be used to view surfaces of objects.

 Self-Assessment Questions

1.3.4. Compare and contrast transmission electron microscope (TEM) and scanning electron
microscope in terms of effective magnification, function and final products. Fill in the
table below.

Type Effective Function Final Product

Magnification

1. Transmission
Electron
Microscope
(TEM)

2. Scanning Electron
Microscope
(SEM)

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College of Arts and Sciences, CAGAYAN STATE UNIVERSITY
First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

Application
 Light Microscope vs. Electron Microscope. Extend your learning about microscopes by
describing more features that distinguish LM and EM. Fill in the table below.

Feature Light Microscope Electron Microscope

1. Illuminating beam

2. Wavelength

3. Medium

4. Lens

5. Resolving Power

6. Aperture Angle

7. Magnification

8. Focusing Contrast

9. Stains Used

10. Type of Image

produced

 Variations of Light Microscopes. Describe each microscope in terms of their key uses.

1. Bright-field –

2. Dark field –

3. Phase contrast –

4. Differential interference contrast –

5. Fluorescence –

6. Confocal –

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First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

Assessment

Modified True or False: Write True if the statement is correct. If not correct, copy the phrase that
made the statement false, then write the correct phrase beside it to make it true. Write your answer
on a separate sheet.

1. The resolving power of a microscope is a function of the numerical aperture and the refractive
index.
2. Preparation for TEM and SEM requires that specimen be dehydrated and stained.
3. The light microscope has a total magnification of about 1000X and can be used to study
viruses in the lab.
4. When using a light microscope, the light waves that pass through or bounce off a specimen
are focused and magnified by the lens.
5. Electron microscopes use beams of electrons focused by glass lenses rather than by magnetic
fields.
6. The working distance decreases as the magnification of the objective lens increases.
7. The stage contains the shutter that regulates the amount of light entering the lens system.
8. The phase-contrast microscope would be most effective in visualizing live and microbes.
9. SEM is most often used to reveal both surface and internal structures.
10. Light microscopes can be used to examine living cells, electron microscope cannot.

Reflection
Take a few minutes to reflect on the significance of the microscope. How do the various types of
microscope

UNIT 1 References
https://microbiologyonline.ifas.ufl.edu/about/articles/what-are-the-branches-of-microbiology/

https://upload.wikimedia.org/wikipedia/commons/0/06/Figure_04_02_02.jpg
Attribution: CNX OpenStax, CC BY 4.0 <https://creativecommons.org/licenses/by/4.0>, via
Wikimedia Commons
Levin, P. A., & Angert, E. R. (2015). Small but Mighty: Cell Size and Bacteria. Cold Spring Harbor
perspectives in biology, 7(7), a019216. https://doi.org/10.1101/cshperspect.a019216
https:www.micropia.nl. Use of microbes
(Yeasts) By Mogana Das Murtey and Patchamuthu Ramasamy - [1], CC BY-SA 3.0,
https://commons.wikimedia.org/w/index.php?curid=52254246
( E.coli) File:EscherichiaColi NIAID.jpg. (2021, July 29). Wikimedia Commons, the free media
repository. Retrieved 06:12, September 4, 2021
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College of Arts and Sciences, CAGAYAN STATE UNIVERSITY
First Semester, SY 2021-2022
MODULE IN GENERAL MICROBIOLOGY

from https://commons.wikimedia.org/w/index.php?title=File:EscherichiaColi_NIAID.jpg&oldid=
577388787.

Credit: Rocky Mountain Laboratories, NIAID, NIH, Public domain, via Wikimedia Commons
Giardia: http://phil.cdc.gov/PHIL_Images/8698/8698_lores.jpg

File:Alternaria alternata-5 copie.jpg. (2020, October 4). Wikimedia Commons, the free media
repository. Retrieved 06:47, September 4, 2021
from https://commons.wikimedia.org/w/index.php?title=File:Alternaria_alternata-
5_copie.jpg&oldid=480778126

Rhizopus zgospores. Curtis Clark, CC BY-SA 3.0 <https://creativecommons.org/licenses/by-

sa/3.0>, via Wikimedia Commons
Mucor: Photo Credit:Content Providers: CDC/Dr. Lucille K. Georg, Public domain, via Wikimedia
Commons
http://www2.hawaii.edu/~johnb/micro/m130/m130lect2.html#:~:text=Girolamo%20Fracastoro%2
0(1546)%20is%20credited,Fracastoro%20also%20named%20syphilis.&text=Many%20ancient%2
0accounts%20about%20where%20living%20organisms%20come%20from.

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College of Arts and Sciences, CAGAYAN STATE UNIVERSITY
First Semester, SY 2021-2022