Microscopic Observation of

Microorganisms
Dr. Mohd. Raeed Jamiruddin, PhD
Associate Professor
School of Pharmacy
B R A C University

“I have had several gentlewomen in my house, who were keen on seeing the little eels in vinegar; but some
of ‘em were so disgusted at the spectacle that they vowed they’d never use vinegar again. But what if one
should tell such people in [the] future that there are more animals living in the scum on the teeth in a man’s
mouth, than there are men in a whole kingdom?”
—Anton van Leeuwenhoek, 1683
1
 Chapter Overview

▪ Different types of Microscopy such as Bright field, dark

field, fluorescence and phase contrast microscopy,
electron microscopy.
▪ Preparations of microscopic examinations, wet mount
and hanging drop techniques, fixed and stained smears.
▪ Microbiological stains: simple and differential staining
methods
How many bacteria are there on the tip of a
small pin?
Scale of living organisms

4
 Discovery of Microorganisms
Antonie van Leeuwenhoek (1632-1723)
first person to observe and describe microorganisms
accurately

(a) A replica of one of

Leeuwenhoek’s microscopes.
(b) The proper way of looking
through Leeuwenhoek’s
microscope.
 Properties of Light:
Wavelength and Resolution
 Properties of Light:
Wavelength and Resolution
 Lenses and the Bending of Light

▶ Light is refracted (bent) when passing from one medium

to another.
Refractive index
▶ A measure of how greatly a substance slows the velocity
of light
▶ Direction and magnitude of bending is determined by
the refractive indexes of the two media forming the
interface

8
 Lenses

▪ Focus light rays at a specific place called the focal point

▪ Distance between center of lens and focal point is the
focal length

▪ Strength of lens related to focal length

short focal length more magnification

9
Figure 2.2 1
0
 Types of Microscope

▶ Microscopes are of two categories, depending upon

the principle on which magnification is based:

➢ Light (optical) microscope

➢ Electron microscope

11
 The Light Microscope

▪ Light microscope is the type of microscope in which

magnification is obtained by a system of optical lenses using
light waves.
▪ Different types
❖ Bright-field microscope

❖ Dark-field microscope

❖ Phase-contrast microscope

❖ Fluorescence microscopes

▪ Compound microscopes
▪ Image formed by action of 2 lenses 12
 The Bright-Field Microscope

▪ Produces a dark image against a brighter background

▪ Has several objective lenses
▪ Parfocal microscopes remain in focus when
objectives are changed
▪ Total magnification
Product of the magnifications of the ocular lens and
the objective lens

▪ Bright-field microscope produces a useful

magnification of about X1000 to X2000.
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15
 Concepts in Light Compound Microscopy:
Microscope Resolution
▪ Resolution is the ability of a lens to separate or
distinguish small objects that are close together
▪ Wavelength of light used is a major factor in the resolution
shorter wavelength  greater resolution

Which one is
clearer image
and why?
1
6
17
 Concepts in Light Compound Microscopy:
Working Distance

Working distance
Distance between the front surface of lens and
surface of cover glass or specimen

18
 Concepts in Light Compound Microscopy:
Numerical aperture
The numerical aperture (NA) is the
product of the refractive index
of the medium (µ) between the
front lens of the objective and
the object being examined, and
the sine of half the angle (θ)
formed by the rays of light
entering the front lens from a
point on the object

NA = µsinθ
Numerical Aperture
 Concepts in Light Compound Microscopy:
Resolving power
Resolving power:
It is the ability to distinguish between two separate objects.
1
Resolving power =
Limit of resolution

Limit of Resolution (d):

It is the smallest distance by which two objects can be
separated and still be distinguishable as two separate
objects.
λ λ
Limit of resolution (d) = =
2NA 2µsinθ
 Concepts in Light Compound Microscopy:
Resolving power (continued)

Resolving power depends on:

i) Wavelength (λ)
ii) Refractive index of the medium between the object and
objective lens (µ)
iii) Half angle of the cone of light from one of the objects (θ)
How can resolving
power be
increased??
The resolving power can be increased in two ways:
1. Decreasing the wavelength, λ (i.e. by using filters)
2. Increasing the NA. As stated earlier, NA = µsinθ.
Thus, NA can be increased the following ways:
▶Increasing the refraction index, µ (this can be
done by adding oil to the object)
▶Increasing the angle of light coming from the
object, θ.
 Why oil is used in oil
immersion microscopy?

Adapted from:
Microbiology: An
Introduction- Tortora,
Berdell R. Funkee & Case.
12th Edition, Prentice Hall

24
Explanation:
When light passes from a material of one refractive index to another (for
example: from glass to air), it bends. In the space between the microscope
objective lens and the slide (where air is), light is refracted, scattered and lost.
The refractive index of air is approximately 1.0, while the refractive index of
glass is approximately 1.5. When light passes through both glass and air, it is
refracted. Light of different wavelengths bends at different angles, so as objects
are magnified more, images become less distinct.

If we can reduce the amount of light refraction, more light passing through the
microscope slide will be directed through the very narrow diameter of a higher
power objective lens. In microscopy, more light = clear and crisp images. A
substance such as immersion oil is placed between the glass slide and the oil
immersion objective lens. Immersion oil has the same refractive index as the
glass slide (approx. 1.5), so it helps in directing more light through the
objective and a clearer image is observed.

The oil has the same effect as increasing the objective lens diameter; therefore, it
improves the resolving power of the lenses. If oil is not used with an oil
immersion objective lens, the image has poor resolution and 25becomes fuzzy.
How immersion oil can increase refractive index, µ?

Numerical aperture, NA = µsinθ

The value of sinθ is always less than or equal to unity (the

number ‘1’, sin90° is 1 which is the maximum obtainable sinθ
value), so the numerical aperture can never be greater than unity
for an objective lens in air. If the space between the objective lens
and the specimen is filled with immersion oil however, the
numerical aperture can obtain values greater than unity. This is
because oil has a refractive index greater than 1 (approx. 1.5,
same as glass).
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 Why oil is used in oil immersion
microscopy (continued)

Oil used in oil immersion microscopy:

1. Cedar tree oil (R.I. = 1.516)
2. Synthetic immersion oil

Problems with Cedar tree oil:

1. Absorbs blue and ultraviolet light.
2. Yellows with age.
3. Has sufficient acidity to potentially damage objectives
with repeated use.
4. Diluting it with solvent changes its viscosity.
5. It can harden and get stick with objective lens, tough
2
to clean 7
Magnification calculation

Brain storming:

For tube length L = 10 cm,

objective focal length fo = 40 mm and
eyepiece focal length fe = 30 mm.

What will be the linear, angular and total

magnification of the microscope?
 Brain storming! (5 minutes)

1. Light of frequency 4.6 × 1014 Hz travels at a speed of 1.24 ×

108 ms-1 in diamond. Calculate R.I.
2. What would be the maximum resolving power of a light
microscope? Given that visible wave length 400mµ - 700mµ ,
N.A. = 1.0
3. What would be the maximum resolving power of a light
microscope in case of oil-immersion microscopy? Given that
N.A. in case of oil-immersion is 1.4
 Brain storming!

1. Which color of light would give you better resolution when using a
microscope: red (wavelength 0.68 μm) or blue (wavelength 0.48
μm)? Why?
2. If you built a light microscope having a total magnification of
5,000X, would it give you better, worse, or the same resolution as
one that has a magnification of 1,000X? Why?
3. Why would radio waves and microwaves be unsuitable for
• examining microbes?
4. Would standard immersion oil placed on a plastic slide
prevent refraction? Why or why not?
 The Dark-Field Microscope
▪ Produces a bright image of the object against a
dark background
▪ Used to observe living, unstained preparations

31
A comparison of the illumination in bright-field and dark-
32

field microscopy.
 Uses of Dark-Field Microscope

▪ Initial examination of suspensions of cells such as yeast,

bacteria, small protists, or cell and tissue fractions including
cheek epithelial cells, chloroplasts, mitochondria, even blood
cells (small diameter of pigmented cells makes it tricky to find
them sometimes despite the color).
▪ Initial survey and observation at low powers of pond water
samples, hay or soil infusions, purchased protist or metazoan
cultures.
▪ Examination of lightly stained prepared slides. Initial location
of any specimen of very small size for later viewing at higher
power.
▪ Determination of motility in cultures
 The Phase-Contrast Microscope

▪ Enhances the contrast

between intracellular
structures having slight
differences in refractive
index
▪ Excellent way to observe
living cells

A phase-contrast image.
Amoeba, a protozoan (160X).
(Biophoto Associates/Science
Source) 34
Application of Phase-contrast microscope
Specimens that can be observed and studied include live
microorganisms such as
▪ Protozoa
▪ Erythrocytes
▪ Bacteria
▪ Molds and sperm
▪ Thin tissue slices
▪ Lithographic patterns
▪ Fibers
▪ Glass fragments and
▪ Sub-cellular particles such as nuclei and organelles.
 Advantages of Phase contrast microscope
The advantages of the phase contrast microscope include:
▪ The capacity to observe living cells and, as such, the ability to
examine cells in a natural state
▪ Observing a living organism in its natural state and/or environment
can provide far more information than specimens that need to be
killed, fixed or stain to view under a microscope
▪ High-contrast, high-resolution images
▪ Ideal for studying and interpreting thin specimens
▪ Ability to combine with other means of observation, such as
fluorescence
▪ Modern phase contrast microscopes, with CCD or CMOS computer
devices, can capture photo and/or video images
37
38
Image Comparison: Bright-Field vs. Phase-Contrast
Microscopy

(a) (b)

Living Cells in Bright-field (a) and Phase Contrast (b)

Image Comparison Between Different Microscopy

Images of the same organism (Paramecium, 338X) produced by four different

techniques.
(a) Bright-field microscopy, (b) dark-field microscopy, (c) phase-contrast
microscopy, (d) Nomarski microscopy. One microscope can have the optics for all
four techniques. (a, b, c, d) Wim van Egmond/Visuals Unlimited/Corbis)
 The Differential Interference Contrast (DIC)
Microscope/ Nomarski Microscopy
▪ Creates image by detecting differences
in refractive indices and thickness of
different parts of specimen
▪ Excellent way to observe living cells

A Nomarski image. The

protozoan Paracineta is
attached by a long stalk to the
green alga Spongomorpha
(magnified 400X). 41
(Biological Photo Service)
 The Fluorescence Microscope

▪ Exposes specimen to
ultraviolet, violet, or blue
light
▪ Specimens usually stained
with fluorochromes
▪ Shows a bright image of
the object resulting from
Fluorescent antibody staining.
the fluorescent light The fluorescent
emitted by the specimen dye-tagged antibodies clearly show
live bacterial cells (green) and
dead (red) cells (854X). (David
Phillips/Science Source)

42
The Fluorescence Microscope

4
3
The Fluorescence Microscope

44
The Fluorescence Microscope( Contd)
 Electron Microscopy

▶ Beams of electrons are

used to produce
images
▶ Wavelength of electron
beam is much shorter
than light, resulting in
much higher
resolution

45
 The Scanning Electron Microscope (SEM)

▪ Uses electrons reflected from

the surface of a specimen to
create image
▪ Produces a 3-dimensional
image of specimen’s surface
features

Light and electron microscopy images compared.

(a) Light (160X) (Eric V. Grave/Science Source) and
(b)electron (425X) microscope images of a Didinium
eating a Paramecium. Notice how much more detail is
revealed by the scanning electron micrograph
(Biophoto Associates/Science46source)
Scanning Electron Microscope (SEM)

4
7
 Transmission Electron Microscope (TEM)

▪ Electrons scatter when they pass through thin sections of a

specimen.
▪ Transmitted electrons (those that do not scatter) are
used to produce image.
▪ Denser regions in specimen, scatter more electrons and
appear darker.

48
Transmission Electron Microscope (TEM)

4
9
Comparison between SEM and TEM

SEM TEM
Comparison between SEM and TEM

TEM and SEM compared. Colorized electron

micrographs of Escherichia coli produced by (a) transmission
electron microscopy (66,952X) (Dennis Kunkel/Phototake) and
(b) scanning electron microscopy (39,487X). (Eye of Science/Science source)
1. What does the electron microscope use
instead of light beams and glass lenses?
What does the fluorescence microscope
use?
2. How can you distinguish between TEM
and SEM micrographs?
3. Which type is the Figure?
4. Why are colored photos of electron
micrographs referred to as “colorized” or
‘‘false color’’?
5. Rank the following types of microscopes
according to the wavelength of illuminating
beam they use, beginning with the longest
wavelength: fluorescent, TEM, bright-field.
What effect does wavelength have on the
resolving power of these microscopes?
 Newer Techniques in Microscopy

▶ Confocal microscopy
and scanning probe
microscopy
▶ have extremely high
resolution
▶ can be used to observe
individual atoms

53
 Confocal Microscopy

▶ Confocal scanning laser microscope

▶ Laser beam used to illuminate spots on specimen
▶ Computer compiles images created from each point
to generate a 3-dimensional image

54
Confocal Microscopy

(a) A confocal microscope system

manufactured

(b) standard fluorescent microscopy,

and

(c) confocal microscopy.

(a, b, c: Courtesy Olympus Corporation,
Scientific Equipment Division)
Confocal Microscopy

5
6
Confocal Microscopy

5
7
 Scanning Probe Microscopy

▶ Scanning tunneling microscope

▶ Steady current (tunneling current) maintained
between microscope probe and specimen
▶ Up and down movement of probe as it
maintains current is detected and used to
create image of surface of specimen

58
 Scanning Probe Microscopy

▶ Atomic force microscope

▶ Sharp probe moves over surface of specimen at
constant distance
▶ Up and down movement of probe as it maintains
constant distance is detected and used to create
image

59
 Sample preparations for observing under
microscope

To observe live organism:

▪ Wet mount technique
▪ Hanging drop technique

To observe non-living microorganism:

▪ Fixing and staining methods
 Techniques to observe specimen
Wet mount techniques:
▪ Place a drop of water on the center
of the slide.
▪ Place the specimen into the drop of
water and if the specimen floats, add
another drop of water on top of it.
▪ Carefully lower the cover glass so
that it touches with one side the
drop of water.
▪ Then lower the cover slip
completely. Placing the cover slip at
an angle prevents the formation of
air-bubbles.
▪ Remove excess water with a filter
paper or tissue paper
 Techniques to observe specimen

Hanging drop technique:

▪ Place a drop stool in the center
of a coverslip.
▪ Place a drop of water/ Vaseline
at each corner of the cover slip.
▪ Invert a slide with a central
depression over the coverslip.
▪ The coverslip will stick to the
slide and when the slide
inverted the drop of bacterial
culture will be suspended in the
central depression of the slide.
▪ Examine microscopically (100X)
for motile organisms.
 Preparation and Staining of
Specimens

▪ Increases visibility of specimen

▪ Accentuates specific morphological features
▪ Preserves specimens

63
 Fixation
▪ Process by which internal and external structures are preserved and
fixed in position
▪ Process by which organism is killed and firmly attached to microscope
slide.

Heat fixing
➢ It kills the organisms.
➢ it causes the organisms to adhere to the slide,
➢ it alters the organisms so that they more readily accept stains
(dyes).

Chemical fixing
➢ protects fine cellular substructure and morphology of larger, more
delicate organisms 64
 Dyes and Simple Staining
Dyes
▪ Make internal and external structures of cell more
visible by increasing contrast with background
▪ Have two common features
▶Chromophore groups
❖ Chemical groups with conjugated double bonds
❖ Give dye its color
▶Ability to bind cells

65
 Dyes and Simple Staining

Simple staining
▪ A single staining agent is used
▪ Basic dyes are frequently used
❖ dyes with positive charges

❖ e.g., crystal violet

66
 Differential Staining

Divides microorganisms into groups based on their

staining properties
❖ e.g., Gram stain

❖ e.g., The Ziehl-Neelsen Acid-Fast Stain

67
 Gram staining

▪ Most widely used differential staining procedure

▪ Divides Bacteria into two groups based on
differences in cell wall structure

68
Gram staining

Primary
stain

Mordant

Decolorization/selective
treatment

Counterstain

Positive 69
Negative
 Gram staining

Escherichia coli – a gram-negative r7o0 d

 Acid-fast staining
▪ particularly useful for staining
members of the genus
Mycobacterium
e.g., Mycobacterium
tuberculosis – causes
tuberculosis
e.g., Mycobacterium leprae –
causes leprosy
▶ high lipid content in cell walls The Ziehl-Neelsen acid-fast stain.
This stain produces vivid red color in
is responsible for their
acid-fast organisms such as
staining characteristics Mycobacterium leprae (magnified
3,844X), the cause of leprosy.
(John D. Cunningham/Getty Images,
Inc.) 7
1
 Staining Specific Structures

Negative staining
▪ Often used to visualize capsules surrounding
bacteria
▪ Capsules are colorless against a stained
background.
▪ The acidic dye with its negatively charged
chromogen will not penetrate the cell due to
repulsion with the negatively charged bacterial
surface (Do not confuse it with grams negative Negative staining.
bacteria!) For capsules reveals a clear area (the
capsule, which does not accept stain)
▪ As a result the bacterial cell remain unstained in a dark pink background of India
but easily discernible against the colored back ink and crystal violet counterstain.
ground. So it is also called indirect staining. The cells themselves are stained
deep purple with the counterstain.
▪ Since no heat is used negative stains are used to The bacteria are Streptococcus
visualize the cells that are too delicate to be pneumoniae7 (3,399X), which are
heat fixed. arranged in2 pairs.
The Ziehl-Neelsen Acid-Fast Staining

• 1. Air dry and heat fix a thin film of microorganisms. Allow the slide
to cool.
• 2. Flood the slide with Carbolfuchsin. Steam the slide with a Bunsen
burner over the sink. Let the slide set for 5 minutes. Rinse with water.
• 3. Flood slide with Acid Alcohol for 30 seconds. Rinse with water.
• 4. Counterstain by flooding the slide with Methylene Blue for 30
seconds. Rinse with water.
• 5. Dry the slide by putting it between the pages of a book of Bibulous
paper.
• 6. View organisms using the oil immersion objective of your
microscope.
 Staining Specific Structures

▪ Spore staining
❖ Double staining technique
❖ Bacterial endospore is one
color and vegetative cell is
a different color
▪ Flagella staining
❖ Mordant applied to
increase thickness of
flagella

73
 Specimen Preparation

▪ Analogous to procedures used for light

microscopy
▪ For transmission electron microscopy,
specimens must be cut very thin
▪ Specimens are chemically fixed and stained
with electron dense material

74
 Other preparation methods

▶ Shadowing
▶ Coating specimen with a thin film of a heavy metal

▶ Freeze-etching
▶ Freeze specimen then fracture along lines of greatest
weakness (e.g., membranes)

75
Shadowing and Freeze-etching

7
6
Ebola
77
SEM of a fly head 78