Lab Module 1: Microscopy

LABORATORY MODULE 1
MICROSCOPY

INTRODUCTION
In 1673, with the aid of a crude microscope consisting of a biconcave lens
enclosed in two metal plates, Antoni van Leeuwenhoek, the man known as the
Father of Microbiology, introduced the world to the existence of microbial forms
of life. Over the years, microscopes have evolved from the simple, single-lens
instrument of Leeuwenhoek, with a magnification of 300X, to the present-day
electron microscopes capable of magnifications greater than 250,000X.

OBJECTIVES

Once this module has been completed, the student should be able to:
 Determine the diversity of microscopic instruments.
 List the theoretical principles of brightfield microscopy.
 Identify the components parts of a compound microscope and determine
the use of each.
 Discuss the proper use and care of a compound microscope.

DISCUSSION

Microscopy is the most common method used both for the detection of
microorganisms directly in clinical specimens and for the characterization of
organisms grown in culture. It is defined as the use of a microscope to magnify
(i.e., visually enlarge) objects too small to be visualized with the naked eye so that
their characteristics are readily observable. Because most infectious agents cannot
be detected with the unaided eye, microscopy plays a pivotal role in the
laboratory.

Microscopes are designated as either light microscopes or electron microscopes.

The former use visible light or ultraviolet rays to illuminate specimens and
include:
 Brightfield Microscope – contains two lens systems for magnifying
specimens: the ocular lens in the eyepiece and the objective lens located in
the nosepiece. The specimen is illuminated by a beam of tungsten light
focused on it by a substage lens called a condenser; the result is a
specimen that appears dark against a bright background. A major
limitation of this system is the absence of contrast between the specimen
and the surrounding medium, which makes it difficult to observe living

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cells. Therefore, most brightfield observations are performed on

nonviable, stained preparations.

 Darkfield Microscope – similar to the ordinary light microscope;

however, the condenser system is modified so that the specimen is not
illuminated directly. The condenser directs the light obliquely so that the
light is deflected or scattered from the specimen, which then appears
bright against a dark background. Living specimens may be observed
more readily with darkfield than with brightfield microscopy.

 Phase-Contrast Microscope – allows us to observe microorganisms in an

unstained state. The optics include special objectives and a condenser that
make visible cellular components that differ only slightly in their
refractive indexes. As light is transmitted through a specimen with a
refractive index different from that of the surrounding medium, a portion
of the light is refracted (bent) due to slight variations in density and
thickness of the cellular components. The special optics convert the
difference between transmitted light and refracted rays, resulting in a
significant variation in the intensity of light and thereby producing a
discernible image of the structure under study. The image appears dark
against a light background.

 Fluorescent Microscope – used most frequently to visualize specimens

that are chemically tagged with a fluorescent dye. The source of
illumination is an ultraviolet (UV) light obtained from a high-pressure
mercury lamp or hydrogen quartz lamp. The ocular lens is fitted with a
filter that permits the longer ultraviolet wavelengths to pass, while the
shorter wavelengths are blocked or eliminated. Ultraviolet radiations are
absorbed by the fluorescent label, and the energy is re-emitted in the form
of a different wavelength in the visible light range. The fluorescent dyes
absorb at wavelengths between 230 and 350 nanometers (nm) and emit
orange, yellow, or greenish light. This microscope is used primarily to
detect antigen–antibody reactions. Antibodies are conjugated with a
fluorescent dye that becomes excited in the presence of ultraviolet light,
and the fluorescent portion of the dye becomes visible against a black
background.

On the other hand, an electron microscope provides a revolutionary method of

microscopy, with magnifications up to 1 million. This permits visualization of
submicroscopic cellular particles as well as viral agents. In the electron
microscope, the specimen is illuminated by a beam of electrons rather than by
light, and electromagnets— instead of a set of optics—focus on the specimen.
These components are sealed in a tube in which a complete vacuum is established.
Transmission electron microscopes require specimens that are prepared as thin
filaments, fixed and dehydrated for the electron beam to pass freely through them.
As the electrons pass through the specimen, images are formed by directing the

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electrons onto photographic film, thus making internal cellular structures visible.
Scanning electron microscopes are used for visualizing surface characteristics
rather than intracellular structures. A narrow beam of electrons scans back and
forth, producing a three – dimensional image as the electrons are reflected off the
specimen’s surface.

While scientists have a variety of optical instruments with which to perform

routine laboratory procedures and sophisticated research, the compound
brightfield microscope is the “workhorse” and is commonly found in all
biological laboratories. Although you should be familiar with the basic principles
of microscopy, you probably have not been exposed to this diverse array of
complex and expensive equipment. Therefore, only the compound brightfield
microscope will be discussed in depth.

Figure 1.1. Compound microscope

Components of the Microscope

 Stage – a fixed platform with an opening in the center allows light to pass
from an illumination source below to the lens system above. This platform
provides a surface on which to place a specimen slide over the central
opening. In addition to the fixed stage, most microscopes have a
mechanical stage that can be moved vertically or horizontally by means
of adjustment controls. Less sophisticated microscopes have clips on the
fixed stage, and the slide must be positioned manually over the central
opening.

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 Illumination – the light source is positioned in the base of the instrument.

Some microscopes have a built-in light source to provide direct
illumination. Others have a reversible mirror that has one side flat and the
other concave. An external light source, such as a lamp, is placed in front
of the mirror to direct the light upward into the lens system. The flat side
of the mirror is used for artificial light, and the concave side for sunlight.

 Abbé condenser – this component is found directly under the stage and
contains two sets of lenses that collect and concentrate light as it passes
upward from the light source into the lens systems. The condenser is
equipped with an iris diaphragm, a shutter controlled by a lever that is
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 lens or eyepiece
lens lens. The lower portion consists of a 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 the instrument.

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 LENSES OCULAR LENS MULTIPLIED BY
OCULAR
Scanner 4x 10x 40x
Low power 10x 10x 100x
High power 40x 10x 400x

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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

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.

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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 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.

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

Use and Care of the Microscope

When transferring the microscope from one place to the other, grip the
microscope arm firmly with one hand and the base with your other hand to lift the
instrument. Carry it close to the body and gently place it on the laboratory bench.
This will prevent collision with furniture or coworkers and will protect the
instrument against damage.

Once the microscope is placed on the laboratory bench, observe the following
rules:
1. Remove all unnecessary materials (including books, papers, purses, and hats)
from the laboratory bench.
2. Uncoil the microscope’s electric cord and plug it into an electrical outlet.
3. Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will decrease
the efficiency of the microscope. The ocular, scanning, low power, and high
power lenses may be cleaned by wiping several times with acceptable lens tissue.
Never use a paper towel or cloth on a lens surface. If the oil immersion lens is
gummy or tacky, use a piece of lens paper moistened with xylol to wipe it clean.
Remove the xylol immediately with a tissue moistened with 95% alcohol, and
wipe the lens dry with lens paper. Note: This xylol cleansing procedure should be
performed only by the instructor and only if necessary; consistent use of xylol
may loosen the lens.

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The following routine procedures must be followed to ensure correct and efficient
use of the microscope.
1. Place the microscope slide with the specimen within the stage clips on the fixed
stage. Move the slide to center the specimen over the opening in the stage directly
over the light source.
2. Raise the microscope stage up as far as it will go. Rotate the scanning lens or
low-power lens into position. Lower the body tube with the coarse-adjustment
knob to its lowest position. Note: Never lower the body tube while looking
through the ocular lens; this may allow for an impact with the slide and damage
to the slide or the microscope.
3. While looking through the ocular lens, use the fine-adjustment knob, rotating it
back and forth slightly, to bring the specimen into sharp focus.
4. Adjust the substage condenser to achieve optimal focus.
5. Routinely adjust the light source by means of the light-source transformer
setting, and/or the iris diaphragm, for optimum illumination for each new slide
and for each change in magnification.
6. Most microscopes are parfocal, which means that when one lens is in focus,
other lenses will also have the same focal length and can be rotated into position
without further major adjustment. In practice, however, usually a half-turn of the
fine-adjustment knob in one direction or the other is necessary for sharp focus.
7. Once you have brought the specimen into sharp focus with a low-powered lens,
prepare to visualize the specimen under oil immersion. Place a drop of oil on the
slide directly over the viewing area. Rotate the nosepiece until the oil-immersion
objective locks into position. Note: Care should be taken not to allow the high-
power objective to touch the drop of oil. Observe the slide from the side as the
objective is rotated slowly into position. This will ensure that the objective will be
properly immersed in the oil. Readjust the fine-adjustment knob to bring the
image into sharp focus.
8. During microscopic examination of microbial organisms, it is always necessary
to observe several areas of the preparation. To do so, scan the slide without the
application of additional immersion oil. Note: This will require continuous, very
fine adjustments by the slow, back-and-forth rotation of the fine adjustment knob
only.

On completion of the laboratory exercise, return the microscope to its cabinet in

its original condition. The following steps are recommended:
1. Clean all lenses with dry, clean lens paper.
Note: Use xylol to remove oil from the stage only.
2. Place the low-power objective in position and lower the body tube completely.
3. Center the mechanical stage.
4. Coil the electric cord around the body tube and the stage.
5. Carry the microscope to its position in its cabinet carefully, as previously
described.

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REFERENCES
Cappucino, J. & Welsh, C. 2020. Microbiology: a laboratory manual, 12th ed.,
Pearson Education, Inc., New York

Granato, P., Morton, V. & Morello, J. 2019. Laboratory manual and workbook in
microbiology: applications to patient care, 12th ed., McGraw – Hill
Education, New York

Tille, P. 2014. Bailey & Scott’s diagnostic microbiology, 13th ed., Elsevier, Inc.,
Missouri