The Microbial

Observing
World and You
Microorganisms
Through a
Microscope

Source:
Source:Tortora
Tortora1313 Edition
th th
Edition
Microbes and Microscopy
1. Microorganisms are much too small to be seen with the unaided
eye; they must be observed with a microscope.

2. The word microscope is derived from the Latin word micro (small)
and the Greek word skopos (to look at).

3. Modern microbiologists use microscopes that produce, with great

clarity, magnifications that range from ten to thousands of times
greater than those of van Leeuwenhoek’s single lens.

4. This chapter describes how different types of microscopes function

and why one type might be used in preference to another.
Units of Measurement
1. Microorganisms are measured in even smaller units, such as
micrometers and nanometers. A micrometer (μm) equals 0.000001
m (10-6 m).

2. The prefix micro indicates the unit following it should be divided

by 1 million, or 106

3. A nanometer (nm) equals 0.000000001 m (10-9 m). Angstrom (Å)

was previously used for 10-10 m, or 0.1 nm.
Microscopy: The Instruments
1. A simple microscope consists of one lens; a compound
microscope has multiple lenses.

2. The simple microscope used by van Leeuwenhoek in the

seventeenth century had only one lens and was similar to a
magnifying glass.

3. However, van Leeuwenhoek was the best lens grinder in the world
in his day.

4. His lenses were ground with such precision that a single lens could
magnify a microbe 300X. His simple microscopes enabled him to
be the first person to see bacteria.
Microscopes and Magnification

If a bacterium is 1 micrometer long and your index finger is 6.5 cm long, how many of the
bacteria can you place end-to-end on your finger?
Answer: 65,000.
Microscopy: The Instruments

1. Light microscope

2. Electron microscope
Microscopy: Types
Light Microscopy

Bright field
Dark field
Phase contrast
Differential
interference
contrast
Polarized light
Fluorescence
Confocal Electron Microscopy
Transmission Electron
Two-Photon Microscopy Scanning Electron
Scanning Microscopy
Acoustic
Scanned-Probe
Microscopy Scanning
Light Microscope
Light Microscope
1. The most common microscope used in microbiology is the
compound light microscope (LM).

2. The total magnification of an object is calculated by multiplying

the magnification of the objective lens by the magnification of the
ocular lens.

3. The compound light microscope uses visible light.

4. The maximum resolution, or resolving power (the ability to

distinguish two points) of a compound light microscope is 0.2 μm;
maximum magnification is 1500X.

5. Specimens are stained to increase the difference between the

refractive indexes of the specimen and the medium.
Light Microscope

(a) Principal parts and functions (b) The path of light (bottom to top)
Compound Microscope
Objective Lens
Clear photo

Chromatic aberration
Light Microscope
Resolution (also called resolving power) is the ability of the lenses to
distinguish fine detail and structure.

Specifically, it refers to the ability of the lenses to distinguish two

points that are a specified distance apart.

For example, if a microscope has a resolving power of 0.4 nm, it can

distinguish two points if they are at least 0.4 nm apart.

A general principle of microscopy is that the shorter the wavelength

of light used in the instrument, the greater the resolution.

The white light used in a compound light microscope has a relatively

long wavelength and cannot resolve structures smaller than about 0.2
μm.
Light Microscope
To obtain a clear, finely detailed image under a compound light microscope,
specimens must contrast sharply with their medium (the substance in which they are
suspended).

To attain such contrast, we must change the refractive index of specimens from that
of their medium.

The refractive index is a measure of the light-bending ability of a medium.

We change the refractive index of specimens by staining them.

Light may refract after passing through a specimen to an extent that it does not pass
through the objective lens.

Immersion oil is used to keep light from refracting


Numerical Aperture (N.A.): This is a number
that expresses the ability of a lens to resolve
fine detail in an object being observed. It is
derived by a mathematical formula (n sine u)
and is related to the angular aperture of the
lens and the index of refraction of the medium
found between the lens and the specimen.
An Airy disk is the central bright circular
region of the pattern produced by light
diffracted when passing through a small
circular aperture. The central disk is
surrounded by less intense concentric rings, so
light intensity takes local maxima and mimina
while it decreases away from the center.
Usually only two or three of the
circular luminous rings are visible in
the microscope (this number is
dependent upon the objective
numerical aperture), because the
higher orders are absorbed by stray
light and are not visible.
 The left-hand position of the slider shows
the pattern at the lowest objective
numerical aperture (0.20), and the right-
hand position illustrates the highest degree
As the slider is moved from left to right, of resolution (numerical aperture = 1.30).
the objective numerical aperture increases
and the complex Airy pattern in the view
port shrinks, demonstrating a progressively
increased resolution of image detail.
The resolving power of an objective determines the size of the Airy
diffraction pattern formed, and the radius of the central disk is
determined by the combined numerical apertures of the objective and
condenser. When the condenser and objective have equivalent
numerical apertures, the Airy pattern radius from the central peak to the
first minimum is given by the equation:

Where
r(Airy) is the Airy radius, 

λ is the wavelength of illuminating light, and 

NA(Obj) is the objective (and condenser) numerical

aperture. 
The numerical aperture is dependent upon the angle of the
inverted cone of illumination entering the objective aperture,
as well as the refractive index of the imaging medium:

where 
θ is the objective angular aperture and 

n is the refractive index of the medium

(air, water, or oil) between the objective
and the specimen.
Figure 3.3 Refraction in
the compound
microscope using an
oil immersion objective
lens.

Because the refractive

indexes of the glass
microscope slide and
immersion oil are the
same, the light rays do
not refract when
passing from one to the
other when an oil
immersion objective
lens is used.

Use of immersion oil is

necessary at
magnifications greater
than 900X.
A dramatic increase in numerical
aperture is observed when the objective is
designed to operate with an immersion
medium such as oil, glycerin, or water
between the front lens and the specimen
cover glass.
 1) By using an immersion medium with a
refractive index similar to that of the glass
coverslip, image degradation due to
thickness variations of the cover glass are
practically eliminated whereby rays of
wide obliquity no longer undergo
refraction and are more readily grasped by
the objective.

2) Typical immersion oils have a refractive

index of 1.51 and a dispersion similar to
that of glass coverslips.

3) Light rays passing through the specimen

encounter a homogeneous medium between the
coverslip and immersion oil and are not
refracted as they enter the lens, but only as they
leave its upper surface. 4) It follows that if the specimen is placed at
the aplanatic point of the first objective lens,
imaging by this portion of the lens system is
totally free of spherical aberration.
Light Microscope
6. Immersion oil is used with the oil immersion lens to reduce light
loss between the slide and the lens.

7. Bright field illumination is used for stained smears.

Numerical Aperture
Task

What is the total magnification of a compound light

microscope with objective lens magnification of 40X
and ocular lens of 10X?

Why is immersion oil necessary at 1000X but not

with the lower power objectives?
Dark Field Microscope

8. Unstained cells are more productively observed using dark field,

phase-contrast, or DIC (Differential interference contrast)
microscopy.

9. The dark field microscope shows a light silhouette of an organism

against a dark background. It is most useful for detecting the
presence of extremely small organisms.
Dark Field Microscope
Light Microscope
10. A phase-contrast microscope brings direct and reflected or
diffracted light rays together (in phase) to form an image of the
specimen on the ocular lens. It allows the detailed observation of
living organisms.

11. The DIC microscope provides a colored, three-dimensional image

of living cells.

12. In fluorescence microscopy, specimens are first stained with

fluorochromes and then viewed through a compound microscope by
using an ultraviolet light source. The microorganisms appear as
bright objects against a dark background.
Light Microscope
13. Fluorescence microscopy is used primarily in a diagnostic
procedure called fluorescent-antibody (FA) technique, or
immunofluorescence.

14. In confocal microscopy, a specimen is stained with a fluorescent

dye and illuminated with short-wavelength light.

15. Frits Zernike's development of phase contrast optical theory is an

excellent example of how research results from a highly
specialized field (in this case, theoretical physics) can yield
innovative new developments in seemingly unrelated disciplines,
such as biology and medicine.
Phase-Contrast Microscopy
PCM is especially useful because the internal structures of a cell become
more sharply defined, permitting detailed examination of living
microorganisms.

In addition, it isn’t necessary to fix (attach the microbes to the microscope

slide) or stain the specimen—procedures that could distort or kill the
microorganisms.

In a PCM, one set of light rays comes directly from the light source.

The other set comes from light that is reflected or diffracted from a
particular structure in the specimen.

PCM can be utilized to produce high-contrast images of transparent

specimens, such as living cells (usually in culture), microorganisms, thin
tissue slices, fibers, and subcellular particles (including nuclei and other
organelles).
Phase-Contrast Microscopy Inventor: Dutch physicist Frits Zernike
(1934) 

Diffraction is the scattering of light rays as they “touch” a specimen’s edge. The
diffracted rays are bent away from the parallel light rays that pass farther from the
specimen.
Phase-Contrast Microscopy

Bright field microscope PC microscope

Phase-Contrast Microscopy

When the two sets of light rays: direct rays and reflected or diffracted
rays: are brought together, they form an image of the specimen on the
ocular lens, containing areas that are relatively light (in phase), through
shades of gray, to black (out of phase).

Interference microscope enhance contrast between

objects and background by superimposing a
reference beam of light and light emerging from a
sample, as shown in the figure.

Since light from background and objects differ in

phase (Amplitude), there will be different amounts
of constructive and destructive interference,
producing the desired contrast in the final intensity.
Phase-Contrast Microscopy

surround (S) wave

diffracted (D) wave

Particle (P) wave : After leaving the specimen plane, surround and diffracted light waves
enter the objective front lens element and are subsequently focused at the intermediate image
plane where they combine through interference to produce a resultant particle wave (often
referred to as a P-wave).
Phase-Contrast Microscopy
P=S+D

constructive
surround (S) wave interference

diffracted (D) wave
destructive
interference

Particle (P) wave : After leaving the specimen plane, surround and diffracted light waves
enter the objective front lens element and are subsequently focused at the intermediate image
plane where they combine through interference to produce a resultant particle wave (often
referred to as a P-wave).
Phase-Contrast Microscopy
P=S+D

surround (S) wave

Wave peak comes upon wave peak : Amplification

diffracted (D) wave

Wave peak comes upon wave trough : Extinction

Particle (P) wave : After leaving the specimen plane, surround and diffracted light waves
enter the objective front lens element and are subsequently focused at the intermediate image
plane where they combine through interference to produce a resultant particle wave (often
referred to as a P-wave).
Figure 3.4 Brightfield, darkfield, and phase-contrast microscopy. The
photographs compare the protozoan Paramecium using these three
different microscopy techniques.

TASK:
What are the advantages of bright-field, dark-field, and phase-contrast
microscopy?
Bright-field (A), polarized-light (B), differential interference contrast (C), and phase-contrast (D)
micrographs of the same living newt cell in metaphase of mitosis. (E) Electron microscopy
Differential Interference Contrast (DIC) Microscopy

Similar to PCM in that it uses differences in refractive indexes.

However, a DIC microscope uses two beams of light instead of one.

In addition, prisms split each light beam, adding contrasting colors to

the specimen.

Therefore, the resolution of a DIC microscope is higher than that of a

standard PCM.

Also, the image is brightly colored and appears nearly three-

dimensional.
Differential Interference Contrast (DIC) Microscopy

Figure 3.5 Differential interference contrast (DIC) microscopy.

Like phase-contrast, DIC uses differences in refractive indexes to produce an
image, in this case a Paramecium. The colors in the image are produced by
prisms that split the two light beams used in this process.
PCM DIC

Aurelia Ephyra-Stage Jellyfish
PCM DIC

Blue-Green (Spirulina) Algae
PCM DIC

Diatom Frustule
PCM DIC

Human Erythrocytes

https://www.microscopyu.com/galleries/dic-phase-contrast
Polarized Light Microscopy

A light wave that is vibrating in more than one plane is referred to as unpolarized
light.

(Eg. Light emitted by the sun, by a lamp in the classroom, or by a candle flame is
unpolarized light. Such light waves are created by electric charges that vibrate in a
variety of directions, thus creating an electromagnetic wave that vibrates in a variety
of directions.)

Polarized light is a contrast-enhancing technique that improves the quality of the

image obtained with birefringent materials.
Polarized Light Microscopy

Polarization, property of certain electromagnetic (light, radiation, or magnetism)

radiations, in which the direction and magnitude of the vibrating electric field are
related in a specified way.

Polarized light microscopy: involving polarized light.

Simple techniques include illumination of the sample with polarized light. Directly
transmitted light can, optionally, be blocked with a polarizer orientated at 90o to the
illumination.
Polarization (also polarisation) is a property applying to transverse waves that specifies the
geometrical orientation of the oscillations. In a transverse wave, the direction of the
oscillation is perpendicular to the direction of motion of the wave.

In a transverse wave, the particles are

displaced perpendicular to the direction
the wave travels. Examples of
transverse waves include vibrations on
a string and ripples on the surface of Illustration of a simple (plane) transverse
water. wave propagating through an elastic medium
in the horizontal direction, with particles being
displaced in the vertical direction.
Polarized Light Microscopy Birefringence is the
optical property of a
material having a
refractive index that
depends on the
polarization and
propagation
direction of light.
These materials are
said to be
birefringent.
 In polarized light microscopy, two
polarization filters are introduced
into the beam path. The polarizer
converts white light (as a mixture of
all polarization directions) into linearly
polarized light.

If an analyzer with 90° orientation

with respect to the polarizer is used
(crossed polarizers), only light can
reach the camera, which has changed
its polarization direction upon
Non-polarized interaction with the sample. In this
light image contrast, amorphous parts of
the sample appear dark, whereas the
contrast of, e.g., certain crystals and
small particles is enhanced, which can
change the light polarization due to
birefringence or light scattering,
respectively.
Polarized Light Microscopy
Fluorescence Microscopy
A fluorescence microscope is an optical microscope that uses fluorescence
instead of, or in addition to, scattering, reflection, and attenuation or
absorption, to study the properties of organic or inorganic substances.

FM is often used to image specific features of small specimens such as

microbes. It is also used to visually enhance 3-D features at small scales.

This can be accomplished by attaching fluorescent tags to anti-bodies that in

turn attach to targeted features, or by staining in a less specific manner.

Many compounds absorb UV or visible light and undergo electronic transition

from low electronic energy levels to high electronic, energy levels. This
absorption of light requires 10-15 sec.
This instant re-emission of the absorbed energy is called Fluorescence.
While delayed re-emission of the absorbed energy is called
Phosphorescence
Fluorescence Microscopy
Fluorescence Microscopy
Fluorescence Microscopy

Figure 3.6 The principle of immunofluorescence. (a) A type of fluorochrome is combined

with antibodies against a specific type of bacterium. When the preparation is added to bacterial
cells on a microscope slide, the antibodies attach to the bacterial cells, and the cells fluoresce
when illuminated with ultraviolet light. (b) In the fluorescent treponemal antibody absorption
(FTA-ABS) test for syphilis shown here, Treponema pallidum shows up as green cells against a
darker background.
Fluorescence Microscopy

Bovine pulmonary artery endothelial

(BPAE) cells under the microscope.

Nuclei are stained blue with DAPI

(4′,6-diamidino-2-phenylindole),

Microtubules are marked green by an

antibody bound to FITC (Fluorescein
isothiocyanate) and

Actin filaments are labeled red with

phalloidin bound to TRITC
(Tetramethylrhodamine).
Fluorescence Microscopy

Impact factor: 2.8

Fluorescence Microscopy

Figure 7. Fluorescence microscopy (at X100) of Pseudomonas aeruginosa PAO1

biofilms in the absence (a) and presence of Pb-AgNPs at the concentration of 6
µg/ml (b) and 8 µg/ml (c).

Shah et al., 2019

Fluorescence Microscopy

Impact factor: 3.5

Fluorescence Microscopy

Figure 2. Light (upper panels) and fluorescence (bottom panels)

imaging of P. aeruginosa biofilms. A) Untreated, B) 0.62% of PEO
and C) 1.25% of PEO. Mansuri et al., 2020
Confocal Fluorescence Microscopy

Confocal microscopy is a technique in light microscopy used to reconstruct

3-D images.

Like fluorescent microscopy, specimens are stained with fluorochromes so they

will emit, or return, light.

But instead of illuminating the entire field, one plane of a small region of a
specimen is illuminated with a short-wavelength (blue) light which passes the
returned light through an aperture aligned with the illuminated region.

Each plane corresponds to an image of a fine slice that has been physically cut
from a specimen. Successive planes and regions are illuminated until the entire
specimen has been scanned.
Confocal Fluorescence Microscopy
Confocal Fluorescence Microscopy

Figure 3.7 Confocal microscopy. Confocal microscopy produces three-dimensional

images and can be used to look inside cells. Shown here is the nucleus in
Paramecium tetraurelia.

Tortora 13th Edition

Confocal Fluorescence Microscopy

In these multiple images of P. aeruginosa biofilm a bacterial community that takes

the form of mushroom-like structures. The main, upper left image looks down at the
biofilm, the lower, right-hand images show cross-sections. This biofilm was cultured
from the airways of a patient with cystic fibrosis.
Jacobson, 2015
Two-Photon Microscopy
Two-Photon Microscopy

Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging

technique that allows imaging of living tissue up to about one millimeter in thickness.

Unlike traditional fluorescence microscopy, in which the excitation wavelength is

shorter than the emission wavelength, two-photon excitation requires simultaneous
excitation by two photons with longer wavelength than the emitted light.

Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light

which can also excite fluorescent dyes.

However, for each excitation, two photons of NIR light are absorbed. Using infrared
light minimizes scattering in the tissue.
Two-Photon Microscopy

Due to the multiphoton absorption, the background signal is strongly suppressed. Both
effects lead to an increased penetration depth for this technique.

(Background fluorescence, sometimes referred to as noise, is the fluorescent signal

that you see but you don't want.)

Two-photon excitation can be a superior alternative to confocal microscopy due to its

deeper tissue penetration, efficient light detection, and reduced photobleaching.

Photobleaching: (sometimes termed fading) is the photochemical alteration of a dye

or a fluorophore molecule such that it permanently is unable to fluoresce. This is
caused by cleaving of covalent bonds or non-specific reactions between the
fluorophore and surrounding molecules.
Two-Photon Microscopy
Scanning acoustic microscope (SAM)
SAM is a device which uses focused sound to investigate, measure, or image an object (a
process called scanning acoustic tomography). It is commonly used in failure analysis and non-
destructive evaluation. It also has applications in biological and medical research. The
semiconductor industry has found the SAM useful in detecting voids, cracks, and delaminations
within microelectronic packages.

Principle: SAM works by directing focused sound from a transducer at a small point on a target
object.

Sound hitting the object is either scattered, absorbed, reflected (scattered at 180°) or transmitted
(scattered at 0°). It is possible to detect the scattered pulses travelling in a particular direction.

A detected pulse informs of the presence of a boundary or object. The `time of flight' of the
pulse is defined as the time taken for it to be emitted by an acoustic source, scattered by an
object and received by the detector, which is usually coincident with the source.

The time of flight can be used to determine the distance of the inhomogeneity from the source
given knowledge of the speed through the medium.
The working principle of the technique is based on the reflection that acoustic waves experience
at media interfaces and density irregularities. The technique makes use of the high penetration
depth of acoustic waves, in comparison to light, to image the internal structure of the specimen.
Thus, in scanning acoustic microscopy either reflected or transmitted acoustic waves are
processed to analyze the internal features.
Scanning acoustic microscope (SAM)

Acoustic microscopy techniques analyze the intensity and phase of both the reflected and
transmitted waves to create visual images reflecting the variations in the acoustic impedance
of the specimen, thereby disclosing internal flaws and defects such as delamination and
voids.
Scanning acoustic microscope (SAM)
Medicine and biology

SAM can provide data on the elasticity of cells and tissues, which can give useful information
on the physical forces holding structures in a particular shape and the mechanics of structures
such as the cytoskeleton. These studies are particularly valuable in investigating processes such
as cell motility.

Some work has also been performed to assess penetration depth of particles injected into skin
using needle-free injection.

Another promising direction was initiated by different groups to design and build portable hand-
held SAM for subsurface diagnostics of soft and hard tissues and this direction currently in the
commercialization process in clinical and cosmetology practice.
Scanning acoustic microscope (SAM)