BIO 100 – INTRODUCTORY BIOLOGY

LAB (2022)

Week 2 –Microscopy

Aishah Bilal, Esha Amir, Ghulam Hassan, Hina Ahmad, Hira

Sarfaraz, Hadeeqa Raza, Khalida Mazhar

Muhammad Tariq

Department of Biology
School of Science & Engineering
 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Day 2: Microscopy

Basics of Simple & Fluorescent Microscopy

A microscope is typically required to view the cellular morphology of microorganisms.
The compound light microscopes are most frequently used by microbiologists for observing cells.
The four commonly used varieties of light microscopes are the brightfield, phase contrast,
fluorescent and the dark field. More advanced microscopes can also be used to view more details
associated with cells and structures. These include the scanning and transmission electron
microscope, atomic force microscope, and the confocal laser microscope. This laboratory is going
to focus on the compound light microscopy techniques.
All light microscopes utilize the same basic principle. Light is produced by a light source
and it is focused into a specimen on a glass slide. An image is produced as light is diffracted by
the specimen. The image is magnified by a series of lenses, focused and sent to the eye.
Magnification refers to an increase in the apparent size of the object displayed by the microscope
and resolution refers to the ability to discern two objects as separate and distinct. Most light
microscopes have a limit of magnification of 1000X. Lenses that focus light are not capable of
producing a clear image of an object with high resolution when the object is magnified higher than
1500X. When the magnification produced by a light microscope approaches 1500X the resolution
is often too low to produce a reliable image. This is due to the fact that glass or quartz lenses
cannot gather enough light to produce a clear image. The ability of a lens to gather light is called
its numerical aperture. Quality resolution is dependent on two things: the wavelength of the
light (λ) and the numerical aperture (NA) of the lens. A simple formula applies when determining
the smallest diameter (SD) of an object that is resolvable by a lens:
SD=0.5λ/NA
You should try to remember these rules of thumb:
• The higher the numerical aperture of the lens the greater the resolving power of the
lens.
• Higher power lenses need to have a higher numerical aperture to produce resolvable
images.
• The smaller the λ of light the better the resolving power. Because of this most light
microscopes use a blue filter over the light source or condenser.
• The smallest object that can be easily discerned by a light microscope is 0.2 μm.
Most microscopes have several different objective lenses and one ocular lens. Four
common objective lens are; 4X, 10X, 40X and 100X. The ocular lens is usually fixed at 10X. The
total magnification of an image is dependent on the magnification of the ocular and objective
lenses. For instance, when using the 4X lens to view a specimen the total magnification would be
40X.
Most 100X lenses are special lenses called oil immersion lenses. When viewing a
specimen at a total magnification of 1000X, the 100X objective lens often has difficulty gathering
the light coming from the specimen. As light exits the glass slide it bends when it reaches the air
and often bends away from the objective lens. Immersion oil has the same refractive index as
glass. When using oil the light will exit the glass slide containing the specimen and will travel
through the oil at the same speed as it was traveling through the glass and will not bend. This
allows more light into the 100X lens and produces a much clearer image (increases the resolution

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

of the 100X lens). It is always recommended to use oil with oil immersion lenses to have
maximum resolution. Oil should not be applied to other lenses as it can damage them.
The brightfield microscope is used to view specimens that have contrast or color associated
with them. Most cells have to be killed, fixed to slides and stained prior to viewing them using
the brightfield scope. Phototrophs that have pigments do not need to be stained. White light
generated by the light source is focused by the condenser and passed through the colored specimen
on the slide. The image produced by the specimen is then magnified by the lenses and appears to
the user as a darkened specimen against a bright background.
Besides the lenses, brightfield microscopes have several parts.

Parts of the Brightfield Microscope and their Function

Part Function
Ocular lens Magnifies the image 10X.
Nosepiece Contains the objective lenses.
Objective lenses Magnifies the image 4, 10, 40 or 100X.
Stage Holds the slide and usually has clips to hold slide stationary.
Diaphragm Adjusts the amount of light to the lens.
Condenser Focuses light into the specimen.
Light Source Produces and adjusts the intensity of the light.
Rheostat Adjusts the intensity of the light.
Course focus Focuses image when using the 4 and 10X lenses.
Fine Focus Focuses the image when using the 40 and 100X lenses
Mechanical Stage Moves the slide on the stage
Adjuster

The phase contrast microscope was invented in 1936 by Frits Zernike. It is very similar to
the brightfield microscope. White light is used to produce the image and the lenses are the same
and most of the structures are the same. The phase contrast scope can be used to view specimens
that are transparent and is especially useful to view live cells. The phase contrast microscope has
one part that the brightfield scope lacks and that is the phase ring. This ring can be used manipulate
the light to produce an image from a transparent specimen.
To understand how a phase contrast scope works it is important to recall the properties of
light. When light passes through a colored object the rays emerge with diminished amplitude, thus
producing an image that is dark against a white background. When light passes through a
transparent object most of the light emerges with the same amplitude and images are “washed out.”
After light passes through a transparent object it emerges as either direct or diffracted rays. Direct
rays are those that pass straight through the specimen with no change in amplitude or wavelength
and diffracted rays emerge from the object with the same amplitude, but the wavelength (λ) is ¼
less than the λ of the direct rays. The phase ring works to create an image by reducing the
wavelength of the direct rays by ¼ so that the direct rays will be brought into coincidence with the
diffracted rays. When this occurs the amplitude of the combined rays (direct and diffracted) will
increase, thus producing a bright image against a dark background.
In this laboratory you will use a brightfield scope to view dead/live cells.You will view
prokaryotic cells (please review the various morphologies possible with prokaryotic cells) and
eukaryotic cells. You will also use a phase contrast scope to view live cells.

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

A fluorescence microscope is basically a conventional light microscope with added features and
components that extend its capabilities. A fluorescence microscope uses a higher intensity light
to illuminate the sample. This light excites fluorescence species in the sample, which then emit
light of a longer wavelength. A fluorescent microscope also produces a magnified image of the
sample, but the image is based on the second light source -- the light emiting from the fluorescent
species -- rather than from the light originally used to illuminate, and excite, the sample.

The basic task of the fluorescence microscope is to permit excitation light to irradiate the specimen
and then to separate the much weaker emitted fluorescent light from the brighter excitation light.
Thus, only the emission light from the specimen reaches the eye or other detector (usually a digital
or conventional film camera). The resulting fluorescing areas shine brightly against a dark
background with sufficient contrast to permit detection.

To become visible, the emitted light is separated from the much brighter excitation light in a second
filter. Here, the fact that the emitted light is of lower energy and has a longer wavelength is used.
The fluorescing areas can be observed in the microscope and shine out against a dark background
with high contrast.

There are specific conditions that may affect the re-radiation of light by an excited fluorophore,
and thus reduce the intensity of fluorescence. This reduction of emission intensity is generally
called fading or photo bleaching. Bleaching is irreversible decomposition of the fluorescent
molecules because of light intensity in the presence of molecular oxygen. Quenching also results
in reduced fluorescence intensity and frequently is brought about as a result of oxidizing agents or
the presence of salts of heavy metals or halogen compounds. To reduce the degree of fading in
some specimens, one can change the pH of the mounting medium or use anti-bleaching agents

In this laboratory you will use a bright field scope to view dead/live cells. You will view
prokaryotic cells (please review the various morphologies possible with prokaryotic cells) and
eukaryotic cells. You will also use a phase contrast and fluorescent microscope to view live cells.

Ref: http://micro.magnet.fsu.edu/primer/techniques

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Principle of excitation and emission

In order to determine the emission spectrum of a particular fluorochrome, the wavelength of

maximum absorption (usually the same as the excitation maximum) is determined and the
fluorochrome is excited at that wavelength. The absorption and emission spectra of a typical
fluorochrome is illustrated in Fig.2.3 where the relative intensity of absorption and emission is
plotted against the measured wavelength and the resulting profiles are overlapped. The emission
maximum is chosen and only emission light at that wavelength is allowed to pass to the detector.

Absorption and emission spectra

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Activity 2A: Mitosis

Prepare slides of onion root tips demonstrating the stages of mitosis (somatic cell division) and
observe various cells undergoing mitosis.

Background:

Mitosis is the process of cell division that occurs in somatic (body) cells. In mitosis, a cell
divides to give two daughter cells, essentially identical to the parent cell. Mitosis results in an
equal distribution of hereditary material and usually an equal distribution of the cell contents. All
of us began life as single cells. These cells divided by mitosis to become 2, then 4, then 8, then 16
cells and so on. At some point some of these cells started to differentiate into other cell types to
form our various tissues.

In cancer, the process of cell division has gone awry. To begin to understand the kinds of
changes that occur that allow cells to grow and divide past their normal "checkpoints", it is crucial
to understand the process of division in normal cells.

Materials:

Onion root tips

Acetocarmine stain (1% in 45% acetic acid, boiled 30 min. and filtered)
1 M HCl
Surgical blades
Forceps
Slides, coverslips
Compound microscope
Mitosis slides: Allium (onion) root tip

Procedure
Prepare slides of onion root tip.
1. Cut the terminal 1 cm of the root tip from a growing onion bulb.
2. Place several drops of 1 M HCl in a watch glass.
3. Place the terminal of the onion root in the HCl solution.
4. Incubate at room T for 5 min.
5. Using a forceps, transfer the root tip to a drop of acetocarmine stain on a clean slide.
6. Using a razor blade or sharp scalpel, cut off and retain the tip-most 1 mm of the root.
Chop the root tip into many pieces with the razor blade. Incubate at room temperature for
3-5min.
7. Apply a clean cover glass to the slide. Cover the slide with a paper towel and push
downward firmly with your thumb over the cover glass.
8. Seal the cover slip by sealant. Leave for a while to dry sealant.
9. Examine with the light microscope at low power and high power. Look for cells where
the nuclei have stained red-purple.

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Observe mitosis on prepared slides of onion root tip. Identify cells in the stages of interphase,
prophase, metaphase, anaphase, and telophase.

• Interphase: Chromatin appears dispersed, DNA replication occurs.

• Prophase: Chromatin condenses, chromosomes become visible, nuclear membrane
breaks down, and spindle starts to form.
• Metaphase: Chromosomes line up on the spindle in the center of the cell (on metaphase
plate).
• Anaphase: Chromosomes are separated at their centromeres; spindle pulls them toward
opposite poles.
• Telophase: Chromosomes recondense, new cell wall forms between daughter cells
(plant cells) or cell membrane pinches off (animal cells)

Interphase

Label: Nucleus, chromatin, nuclear envelope, cytoplasm

Prophase

Label: Chromosomes, spindle

Metaphase

Label: Chromosomes, spindle, metaphase plate

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Anaphase

Label: Chromosomes, microtubules

Telophase

Label: Nuclear membrane, cell membrane, chromosomes

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Activity 2B: Temporary Mount

Preparation of temporary slides (pond water, cheek cells, onion epidermis etc)

Material:

Glass slides
Cover slips
50% glycerol
Sealant
Pond water, bacteria and fungi etc.

Procedure:

1. Carefully wipe the slide and cover glass clean, if needed.

2. Take a thin piece of the specimen and use the forceps to place it in the center of the
microscope slide.
3. Use a pipette to put one small drop of water or 50% glycerine on the sample, and then
gently lay the cover glass on it, taking care not to trap any air bubbles.
4. Remove any excess glycerine by placing the edge of a piece of paper towel, tissue, or cloth
at the edge of the cover glass and allowing the paper or cloth to soak up any excess. You
have removed enough glycerine when the specimen is trapped between the slide and the
cover glass and the glycerine doesn't bulge out from under the cover glass. This surface
tension of the water will hold the cover glass securely to the microscope slide so that
you can observe at any angle needed for good viewing.
Note: do not place cover slip for pond water specimen.
5. Seal the cover slip by sealent provided (nail enamel).
6. Observe under microscope.

• Observe and focus on the sample using the lowest power objective. Adjust the light source
as needed; often lower light optimizes viewing by increasing contrast. The 4x objective is
good for scanning a large number of cells. The 10x is good for general observations.
• Focus first at 10x.
• Leave the course focus knob in position and shift to the 40x while observing that it does
not come in contact with the suspension or the cover slip.
• Use only the FINE FOCUS knob with the 40x objective.

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 BIO 100 – INTRODUCTORY BIOLOGY LAB (2022)

Activity 2C: Observation of Bacteria

Observe different types of bacteria by compound microscope

What are bacteria?

Bacteria are tiny living beings (microorganisms) - they are neither plants nor animals. Average
bacteria 0.5 ‐ 2.0 um in diameter.
Bacteria are microscopic single-celled organisms that thrive in diverse environments. They can
live within soil, in the ocean and inside the human gut. Humans' relationship with bacteria is
complex. Sometimes they lend a helping hand, by curdling milk into yogurt, or helping with our
digestion. At other times they are destructive, causing diseases like pneumonia.

Shapes of Bacteria:
1) Spherical (like a ball)

These are usually the simplest ones. Bacteria shaped like this are called cocci (singular coccus).

2) Rod shaped

These are known as bacilli (singular bacillus).

Some of the rod-shaped bacteria are curved; these are known as vibrio.

3) Spiral

These known are as spirilla (singular spirillus).

If their coil is very tight they are known as spirochetes.

Types of bacteria:
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Danish scientist Hans Christian Gram devised a method to differentiate two types of bacteria based
on the structural differences in their cell walls. In his test, bacteria that retain the crystal violet dye
do so because of a thick layer of peptidoglycan and are called Gram-positive bacteria. In contrast,
Gram-negative bacteria do not retain the violet dye and are colored red or pink. Compared with
Gram-positive bacteria, Gram-negative bacteria are more resistant against antibodies because of
their impenetrable cell wall.

GRAM STAIN PROTOCOL

Purpose
This technique is used to observe and identify the bacterial microflora based on their gram stain
reaction.

Material Required:
• Microscopic slides
• Bunsen burner
• Inoculation loop
• Bacterial culture
• Gram staining kit
• Microscope

Method:
• Put 10-ul drop of culture containing bacteria on slide.
• “Heat-fix” the slide with the specimen by passing it over a flame. The slide should be passed very
quickly through the flame and not be heated excessively. Place slide on the staining tray.
• Flood the fixed smear with crystal violet solution (#1) and allow to remain for 1 minute.
• Rinse off the crystal violet with distilled or tap water.
• Flood the slide with iodine solution (#2). Allow to remain for one minute.
• Rinse off the iodine solution with distilled or tap water.
• Flood the slide with decolorizer (#3) for one to five seconds.
• Rinse off the decolorizer with distilled or tap water.
• Flood the slide with safranin (#4). Allow to remain for 30 seconds.
• Rinse off the safranin with distilled or tap water.
• Dry the slide by placing in an upright position.

Microscopically examine the slide for bacterial organisms under a 100X objective. Observe
several fields on the slide for bacterial organisms. Gram-positive bacteria stain deep violet to
blue and gram-negative bacteria stain pink.

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Use the above space to sketch six different specimens as observed under the
microscope.

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