Microscopy Handout
©2012 Jerald D. Hendrix
The Microscope
Cells are the basic structural and functional units of living organisms. To understand the
organization and mechanisms of life, one must be able to observe and study cells. However, their
small size makes direct observation with the unaided eye impossible. Most cells are between
about 1 to 100 micrometers (m) in diameter, but the human eye can see objects only down to a
size of about 200 m. There are 1000 micrometers in a millimeter, the smallest unit on a typical
metric ruler.
Biologists use devices called microscopes to examine the details of cell structure. There are
several types of microscopes, each of which has its own advantages for particular observations.
Microbiologists use the compound light microscope for routine observation of bacteria in the
laboratory. This instrument uses a combination of lens to bend light rays into an enlarged image.
There are two properties of a microscope that determine the quality of the image produced:
magnification and resolution. Magnification is the number of times larger the image appears as
compared to the actual sample. Each objective has a different magnification. Total magnification
is the objective magnification times the eyepiece magnification. Resolution is the ability of a
microscope to separate, or resolve, two closely spaced objects. Imagine two small, closely
spaced dots being viewed under a microscope. With good resolution, the dots will appear as two
separate objects. With poor resolution, the images will blur together and appear as a single dot.
Without good resolution, an image will appear blurred and out of focus even at high
magnification. A microscope that has high magnification but poor resolution is of little value for
serious microbiological work.
Cleaning the microscope:
The instructor will demonstrate the proper technique for cleaning the lenses of the
microscope. The proper technique is recommended by the manufacturer of the microscope, and
may vary with different models. Failure to follow the recommended procedure for cleaning the
lenses can result in permanent damage to the microscope.
Carrying the microscope:
Grasp the microscope by its arm, and place your other hand under its base. Do not tilt the
microscope, because the eyepiece lenses will fall out and break.
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Parts of the microscope:

Eyepiece (Ocular lens):
Most microscopes are equipped with binocular eyepieces having an eyepiece magnification of
10X. Between the two eyepieces, there is a thumbscrew to adjust the distance between the lenses
for comfortable viewing. The eyepiece lenses often become soiled with oil or other material from
the eyelashes. If this happens, simply clean the lenses as demonstrated by the instructor.

Nosepiece:
Also called the turret, this is a rotating wheel on which the objective lenses are mounted. When
changing from one objective to another, make sure that the lenses “clicks” completely into place.

Objective lenses:
Most microscopes are equipped with four different objective lenses: the scanner lens (usually
with an objective magnification of 4X); the low-power lens (usually 10X); the high-dry lens
(usually 40X); and the oil immersion lens (usually 100X). A small number, like “10/1.25,” is
stamped on each lens. The first number (10) is the objective magnification, and the second
number (1.25) is the numerical aperture (used to calculate the resolution). Some older
microscopes may not have a scanner lens.
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
Mechanical stage:
Microscopes used in microbiology are usually equipped with mechanical stages, rather than
stage clips. The mechanical stage acts like a spring-loaded “pincher,” with the slide fitting
between the jaws of the pincher and sitting flat on the stage. Do not attempt to pry the stage and
jam the slide under the pinchers; to do so will damage the stage. Underneath the stage you will
find two thumbscrews used to move the slide horizontally and vertically. Use these to move the
object on the slide over the hole in the center of the stage.

Condenser and condenser focus:
The condenser is a system of lenses underneath the stage that focuses light on the specimen. The
condenser focus knob is used to move the condenser up and down. For work in microbiology, the
best light is obtained when the condenser is focused all the way up.

Iris diaphragm:
The iris diaphragm is a small lever located on the front of the condenser. It is used to adjust the
amount of light during observations. It is like the contrast knob on a television set; use it to get
the best image of the object after you have focused the microscope. Generally, it is best to use
the least amount of light necessary for the best contrast. As you go from low to high power, you
will have to increase the amount of light, using the iris diaphragm control.

Light switch:
Some microscopes have a knob for a light switch that allows the amount of light source to be
increased or decreased. For microbiological work, turn the light switch knob all the way up to the
maximum position, then use the iris diaphragm control to adjust the amount of light.

Coarse focus:
The coarse focus is the outer wheel of the focus knob. It moves the stage platform up and down.
The focal distance for most professional microscopes is set at the “stop position,” where the
stage is focused as close as possible to the objective lens. Therefore, to focus the microscope
start with the scanner lens and use the coarse focus to move the stage all the way up until it stops.
Then, use the fine focus knob to achieve sharp focus. (On some older microscopes, the coarse
focus knob moves the nosepiece, not the stage. In this case, use the coarse focus to move the
nosepiece all the way down until it stops.)

Fine focus:
The fine focus is the inner wheel of the focus knob. Once the stage has been moved all the way
up with the coarse focus, the fine focus knob is used to achieve sharp focus.
Calculating total magnification:
Calculate the total magnification for each objective by multiplying the objective magnification
by the eyepiece magnification.
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Calculating resolution:
Resolution can be expressed as the shortest distance between two objects that a microscope can
resolve. It depends on two factors: the wavelength of the light used (), and a property of the
objective lens called the numerical aperture (NA). Resolution is calculated using the following
formula.
Resolution 
λ
2(NA)
The wavelength of light produced by most microscopes is 5000 Angstrom units (Å). There are
10 Angstroms per nanometer and 10,000 Angstroms per micrometer. The numerical aperture, a
measurement of the light-gathering ability of the objective, is printed next to or below the
magnification on the barrel of the objective.
To calculate the resolution in Angstroms for an objective, divide 5000 by the numerical aperture
for the lens, then divide the result by 2. To convert the resolution to micrometers, divide the
resolution in Angstroms by 10,000.
Observation with scanner, low-power, and high-dry objectives:

Place the slide on the stage. Use the knobs of the mechanical stage to move the slide,
centering the object in the hole in the center of the stage.

Select the scanner (4X) objective. You should begin all microscopic observations on
low power. This is the only way to properly focus the microscope. Grasp the nosepiece
(not the objectives) and rotate it until the low power objective clicks into place. (If your
microscope does not have a scanner lens, then begin with the low-power lens.)

Focus the microscope on the scanner power. With the coarse focus knob, raise the
stage until it stops. With the fine focus, bring the object into sharp focus. You should be
able to focus with only a slight movement of the fine focus.

Observe the slide under the scanner power. Use the mechanical stage knobs to find the
object and center it in the field of view.

Set the proper illumination. Adjust the iris diaphragm until you can see the maximum
amount of detail. Use the minimum amount of light necessary to get the best contrast and
resolution.

Switch from scanner to low-power (10X) objective. Beginners often have trouble
changing powers. After you have focused the object on the scanner, rotate the lowpower lens into place. Do not change the focus knobs before moving the nosepiece.
Microscopes used in microbiology are parfocal, which means that they keep their focus
from one objective lens to the next. Once you have moved the low-power lens into place,
bring the object into sharp focus with the fine focus knob. Use the iris control to set the
best illumination.
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
Switch from low-power to high-dry. After you have focused the object on the lowpower, rotate the high-dry lens into place. Do not change the focus knobs before
moving the nosepiece. Once you have moved the high-dry lens into place, bring the
object into sharp focus with the fine focus knob. Use the iris control to set the best
illumination.
Simple Staining of Bacteria
Microscopic examination of a bacterial species can reveal its characteristic cell morphology, or
appearance of its cells. Cell morphology includes cell shape, arrangement, special structures, and
other features.
Because bacteria are so small, they usually must be stained to determine their morphology.
Simple staining is a technique in which only a single stain is used to color the bacterial cells.
Very little cellular structure can be seen in a simple stain, but the method is useful for
determining the shape and arrangement of bacterial cells. Differential staining techniques employ
a combination of stains, and other agents that react differently with different organisms. These
methods are useful to distinguish between bacteria and to visualize cellular structures such as
spores.
Microbiologists use the following terms to describe the shape and arrangement of bacterial cells.
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Coccus (plural, cocci): Cells that are spherical in shape. The picture shows cocci arranged
singly.
Streptococcus: A bacterium with spherical cells arranged in chains, like beads on a string.
Staphylococcus: A bacterium with spherical cells arranged in clusters, like clusters of grapes.
Diplococcus: A bacterium with spherical cells arranged in pairs.
Tetrad: Spherical bacterial cells arranged in a group of four. A tetrad arrangement looks almost
like a square under the microscope.
Sarcina (plural, sarcinae): Spherical bacterial cells arranged in a group of eight. Sarcinae look
like small cubes and may be difficult to distinguish from tetrads.
Bacillus (plural, bacilli): A bacterium with rod-shaped cells. The picture shows bacilli arranged
singly.
Diplobacillus: A bacterium with rod-shaped cells arranged in pairs.
Streptobacillus: A bacterium with rod-shaped cells arranged in end-to-end chains. Streptobacilli
often resemble link sausages.
Coryneform bacillus: A bacterium with irregularly rod-shaped cells arranged at angles to form
V- and L-shaped arrangements.
Spirillum (plural, spirilla): A bacterium with cells that are rigid and spiral in shape.
Vibrio: A bacterium with curved or comma-shaped cells.
Spirochete: A bacterium with flexible, spiral-shaped cells. Spirochetes often appear helical or
corkscrew-shaped with tapered ends.
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In addition to shape and arrangement, you may be able to see cytoplasmic inclusions within the
cells of some bacterial species. For example, members of the genus Corynebacterium contain
metachromatic granules, which are crystals of phosphate inside the cell. Metachromatic granules
are best observed in a smear stained with methylene blue. Members of the genus Bacillus may
contain starch granules inside their cells.
Heat-fixed bacterial smears:
To stain bacteria, a heat-fixed smear is prepared on a microscope slide. Make a bacterial smear
using the following method.

When Starting From A Broth Culture: Place a small drop of the culture on a clean
microscope slide, using a sterilized inoculating loop. Use the loop to spread the drop into
an area about the size of a dime.
When Starting From A Plate Culture: Using the loop, place a drop of water on a clean
microscope slide. Flame the loop, and take a small amount of growth from the plate. Use
only a tiny bit of material on the edge of the wire. Mix the bacteria into the water drop,
and spread the drop into an area about the size of a dime.

Allow the smear to dry completely. Do not try to speed up the drying process by heating
the slide, or you could destroy the bacterial cell structure.

After the smear has completely dried, heat-fix the smear by passing the slide several
times through the flame of a Bunsen burner. The slide should become warm, but not hot.
The purpose of heat-fixing is to remove any traces of moisture so the cells will adhere
tightly to the glass.
Simple staining:
The staining should be done over a staining tray (a rectangular plastic kitchen container works
well) to catch the drips of stain. Place a drop of an appropriate bacterial stain, such as methylene
blue, on a heat-fixed smear. Leave the stain on for an appropriate period of time (usually 60
seconds), and rinse the stain off (into the stain tray) with deionized or distilled water. Used stain
waste must be saved and chemically treated for proper disposal.
Drying the stained smear:
After rinsing off the stain, place the slide between the pages of a blotting pad or bibulous paper
pad. Gently dry the slide, taking care not to wipe away the bacteria.
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Observing the stained smear with the scanner, low-power, and high-dry objectives:
Place the slide on the stage, and observe under scanner, low-power, and high-dry objectives
(using the same method as with the wet mount, above). Please note that you do not use a cover
glass on the slide when you look at a bacterial smear. Notice that you can see very little detail
with these powers, because the size of most bacteria is barely within the resolution of these
objectives. To achieve the best resolution for bacterial work requires the use of the oil immersion
lens.
Using the oil immersion lens:

First, you must focus the microscope using the scanner, low-power, and high-dry
objectives.

With the microscope sharply focused with the high-dry lens, rotate the nosepiece halfway to
the oil immersion objective, and place a small drop of immersion oil directly on the
specimen. Continue to rotate the nosepiece until the oil immersion objective clicks into place.
The oil should touch the bottom of the lens and fill the gap between the lens and the object.
Focus with the fine focus only until the object comes into view.

Never use immersion oil with any lens other than the oil immersion lens. The low-power
and high-dry lenses do not have the proper gaskets, so oil can seep into the objective
and cause it to become permanently blurred.

Clean the oil immersion lens after each use, using the technique demonstrated by the
instructor.
You will use the oil immersion lens, the objective with the highest magnification, for most of
your work. It is 100X on most microscopes, and it works properly only when used with
immersion oil. The oil acts to reduce the amount of light lost by scattering, or refraction. This
greatly improves the resolution at the highest magnification.
Gram Staining
Gram staining is an example of a differential staining technique, because different types of
bacteria react differently when gram-stained. There are four stages to this technique.

Crystal violet: This is the primary stain in the gram technique. Most bacteria will absorb at
least some crystal violet in this stage.

Iodine: Iodine serves as a mordant. This means that it intensifies the staining reaction of the
primary stain.

Acetone-alcohol: This is the decolorizer. Some bacterial cells bind the crystal violet so
tightly that they resist decolorization by acetone-alcohol and remain violet after this step.
Other bacteria lose the violet color when they are treated with the decolorizer.
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
Safranin: This is the counterstain. Safranin stains bacterial cells a light pink or red color.
Bacteria that have been decolorized by acetone-alcohol will appear pink after this step, but
bacteria that retained the crystal violet will still be violet or blue in color. Bacteria that retain
the crystal violet, and appear blue or violet in color, are called gram-positive. Bacteria that
lose the crystal violet stain and appear pink or red after the counterstain are called gramnegative.
Gram-positive and gram-negative bacteria differ with respect to their cell wall structures. Grampositive bacteria have a cell wall with a thick outer layer of peptidoglycan, a polysaccharide that
forms a loose mesh-like layer around the cell. Microbiologists believe that crystal violet can
penetrate the peptidoglycan layer easily. When the iodine is added, it causes the crystal violet
molecules to aggregate together, making it difficult to decolorize the cells. Gram-negative
bacteria vary with respect to their cell wall structures, but most of them have an outer layer rich
in a fatty substance called lipopolysaccharide. Crystal violet most likely does not penetrate this
lipid but only binds to its outer surface. This makes the gram-negative cells easy to decolorize.
The gram staining technique is one of the most important steps in identifying an unknown
bacterium. To ensure that the method is properly done, the technician must run a control smear
containing a mixture of a known gram-positive coccus (Staphylococcus aureus) and a gramnegative rod (Escherichia coli).
It is important to remember that not all of the cells in a smear may give the expected reaction,
especially when large numbers of cells are piled on top of each other in thick layers. Also,
cultural factors may influence the success of a gram stain. The culture should be no more than 36
hr old, because the cell wall properties of some bacteria may change with age and give false
results.
Steps in Gram staining:

Prepare a heat-fixed bacterial smear, using the same method as for simple staining.

Cover the smear with crystal violet for 60 seconds.

Wash away the excess crystal violet with a stream of water.

Cover the smear with Gram’s iodine for 30 seconds.

Wash away the excess iodine with a stream of water.

Decolorize the smear with acetone-alcohol (Gram’s decolorizer) for a few seconds.
Decolorizing is the hardest and most critical step in the gram procedure. The best method for
decolorizing is this: Place a few drops of decolorizer on the smear, tilt the slide twice, and
flick off the decolorizer. Do this a total of three times. After the third time, rinse the
decolorizer off with water immediately. If the first attempt results in an overdecolorized
smear, then reduce the number of “drop, tilt, and flick” to two. If the result is an
underdecolorized smear, then increase the number to four.

Cover the smear with safranin for 60 sec.

Wash the smear with water, and blot it dry.

Focus the smear on low power, high-dry, and oil immersion objectives. Pay attention to the
proper technique for using the oil immersion objective.

Observe the gram reaction under oil immersion.