Chapter 2 - Micros

MICROSCOP
Y
Learning objectives
At the end of the session, the students will be able to know:
▰ Types and properties of microscope
▰ Principle and uses of light microscope, dark field microscope, phase

contrast microscope, fluorescence microscope and electron
microscope
3
Essentials of Medical Microbiology
INTRODUCTION
Size:
▰ Bacteria- 0.2–1.5 μm in diameter; 3–5 μm in length
▰ Viruses- 20–300 nm in diameter
▰ Parasites- Protozoans measured in µm; helminths - few mm to meters.
▰ Fungi- Grow as hyphae - thread-like structures 2–10 µm in diameter

and up to several cm in length
4
Different types of Microscopes
▰ Bright-field or light microscope
▰ Dark field (or dark ground) microscope
▰ Phase contrast microscope
▰ Fluorescence microscope
▰ Electron microscope.
5
PROPERTIES OF A MICROSCOPE
1. Good resolution: ability to produce separate images of closely placed
objects - distinguished as two separate entities.
▰ Refractive Index – Oil>Air (oil enhances the resolution power)

Object Resolution power
Human eye 200 µm
Light microscope 0.2 µm
Electron microscope 0.5 nm
6
(Cont..)
2. Good contrast:
▰ Improved by staining the specimen.
▰ When stains bind to the cells, the contrast is increased
7
(Cont..)
3. Good magnification: Achieved by use of lenses.
▰ Ocular lens - magnification power of 10x
▰ Objective lens-scanning (4x), low power (10x), high power (40x) and oil
immersion (100x)
▰ Total magnification = magnification of objective lens * ocular lens.
8
BRIGHT-FIELD OR LIGHT
MICROSCOPE
9
BRIGHT-FIELD OR LIGHT
MICROSCOPE
▰ Forms a dark image against a brighter
background.
Structure
▰ The parts are divided into three groups:
Mechanical Parts
Magnifying Parts
Illuminating Parts
10
Mechanical Parts
▰ Base: Holds various parts of the microscope
▰ C-shaped arm: Holds the microscope, connects -

ocular lens to objective lens
▰ Mechanical stage: Hold the slides.
11
Magnifying Parts
▰ Ocular lens
▰ Objective lens
12
Illuminating Parts
▰ Condenser: Focuses light on the slide (beneath the

stage)
▰ Iris diaphragm: Controls the light that passes through

the condenser
▰ Light source: mirror or an electric bulb
▰ Fine and coarse adjustment knobs: Sharpen the

13
image. Essentials of Medical Microbiology
Working Principle
Rays from the light source pass through the iris and
fall on the specimen
Light rays are gathered by the objective and a

magnified image is formed
14
DARK FIELD
MICROSCOPE
15
DARK FIELD MICROSCOPE
Working principle: Object appears bright against a dark

background - by use of a special dark field condenser.
o Central opaque area (blocks light from entering the
objective lens)
o Peripheral annular hollow area (allows the light to
pass through and focus on the specimen obliquely)
16
DARK FIELD MICROSCOPE
▰ Light - reflected by specimen enters the objective

lens
▰ Unreflected light does not enter the objective.
▰ So the specimen is bright; and the background

appears dark.
17
Applications
▰ Used to identify the living, unstained cells and thin bacteria like
spirochetes which cannot be visualized by light microscopy.
18
PHASE CONTRAST
MICROSCOPE
19
PHASE CONTRAST MICROSCOPE
▰ Contrast is enhanced.
▰ Visualizes the unstained living cells by creating difference in

contrast between cells and water.
20
Working principle
▰ Condenser is similar to dark field microscope.
Cone of light passes through a cell
some light rays are bent due to variations in density and refractive
index within the specimen and are retarded by 1/4 th of a wavelength
21
Working principle (Cont..)
Undeviated light rays strike a phase ring
Deviated rays miss the ring and pass through the rest of the plate
Background is bright, while the unstained object appears dark
22
The light rays go through → condenser → specimen (e.g. bacteria) →

phase ring → objective lens → ocular lens.
23
Applications
For studying:
▰ Microbial motility
▰ Determining the shape of living cells
▰ Detecting microbial internal cellular components
24
FLUORESCENCE
MICROSCOPE
25
FLUORESCENCE MICROSCOPE
Working principle:
Fluorescent dyes - exposed to UV rays, become excited and fluoresce
i.e. they convert short wavelength rays into longer wavelengths
Mercury lamp emits rays that pass through an excitation filter
26
Excitation filter allows only short
wavelength UV light to pass through
Exciting rays get reflected by a

dichromatic mirror - fall on the specimen
stained by fluorescent dye.
Barrier filter – removes remaining UV

light or blue and violet light
27
Applications
Epifluorescence microscope: Simplest form of fluorescence microscope. Has
following applications:
▰ Auto fluorescence (eg. Cyclospora)
▰ Microbes coated with fluorescent dye (eg. Acridine orange dye)
▰ Immunofluorescence
28
Applications (Cont..)
Confocal microscope: Advanced design, uses optical sectioning to get better
resolution of the fluorescent image.
29
ELECTRON
MICROSCOPE
30
ELECTRON MICROSCOPE
▰ Invented by German physicist - Ernst Ruska in 1931.
▰ Source of illumination - Accelerated electrons
▰ Wavelength of electrons 100,000 times shorter than visible light

photons - better resolving power than a light microscope.
31
ELECTRON MICROSCOPE
(Cont..)
It is of two types:
▰ Transmission electron microscope (TEM, most common type)
▰ Scanning electron microscope (SEM)
32
Differences between light microscope and
electron microscope
Features Light microscope Electron microscope
Highest practical About 1,000–1,500 Over 100,000

magnification
Best resolution 0.2 µm 0.5 nm
Radiation source Visible light Electron beam
Medium of travel Air High vacuum
Specimen mount Glass slide Metal grid (usually copper)
Type of lens Glass Electromagnet
33
TEM – Specimen Preperation
▰ Very thin specimens (20–100 nm thickness) -

suitable for EM.
▰ Steps to prepare thin specimen:
1. Fixation: Cells are fixed by using
glutaraldehyde or osmium tetroxide for
stabilization
2. Dehydration: Specimen is then dehydrated
with acetone or ethanol.
34
Transmission Electron Microscope
(Cont..)
3. Embedding: Specimen is embedded in plastic polymer.
4. Slicing: Specimen is then cut into thin slices by an ultramicrotome knife.

Such thin slices of the specimen are mounted on a metal slide (copper).
35
Electron Pathway
▰ Electrons - generated by electron gun-travel in

high speed.
▰ Medium of travel - fully vacuum path - because

in air path - electrons get deflected by collisions
with air molecules.
36
Applications and Modifications of EM
▰ Virus detection
▰ The contrast of EM - increased by —
(i) Negative staining with heavy metals (phosphotungstic acid)
(ii) Shadowing.
▰ Freeze-etching technique - alternative method for specimen preparation.
37
Scanning Electron Microscope
▰ Examine the surfaces of microorganisms in detail.
▰ Resolution of 7 nm or less.
▰ Produces image from electrons emitted by object’s surface.
38
ATOMIC FORCE
MICROSCOPY
39
ATOMIC FORCE MICROSCOPY
▰ Advanced microscope - uses scanning probe technology to study the

cellular structure.
▰ Has a resolution power in fraction of nanometer.
40
Questions:
Q1. Electrons are used as a source of illumination in?
a. Light microscope
b. Phase contrast microscope
c. Electron microscope
d. Fluorescence microscope
41
Questions (Cont..) :
Q2. Scanning probe technology is used in ?
a. Light microscope
d. Atomic force microscope
42
Q3. Microscope used for studying microbial motility?
a. Light microscope
d. Atomic force microscope
43
Q4.Resolution power of microscope is improved by using?
a. Oil
b. Stain
c. Lenses
d. Condenser
44

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MICROSCOP

At the end of the session, the students will be able to know:

▰ Types and properties of microscope

▰ Principle and uses of light microscope, dark field microscope, phase

▰ Bacteria- 0.2–1.5 μm in diameter; 3–5 μm in length

▰ Viruses- 20–300 nm in diameter

▰ Parasites- Protozoans measured in µm; helminths - few mm to meters.

▰ Fungi- Grow as hyphae - thread-like structures 2–10 µm in diameter

▰ Bright-field or light microscope

▰ Dark field (or dark ground) microscope

▰ Phase contrast microscope

▰ Refractive Index – Oil>Air (oil enhances the resolution power)

▰ Improved by staining the specimen.

▰ When stains bind to the cells, the contrast is increased

3. Good magnification: Achieved by use of lenses.

▰ Ocular lens - magnification power of 10x

▰ Total magnification = magnification of objective lens * ocular lens.

▰ Base: Holds various parts of the microscope

▰ C-shaped arm: Holds the microscope, connects -

▰ Mechanical stage: Hold the slides.

▰ Condenser: Focuses light on the slide (beneath the

▰ Iris diaphragm: Controls the light that passes through

▰ Light source: mirror or an electric bulb

▰ Fine and coarse adjustment knobs: Sharpen the

Light rays are gathered by the objective and a

Working principle: Object appears bright against a dark

▰ Light - reflected by specimen enters the objective

▰ Unreflected light does not enter the objective.

▰ So the specimen is bright; and the background

▰ Visualizes the unstained living cells by creating difference in

Background is bright, while the unstained object appears dark

The light rays go through → condenser → specimen (e.g. bacteria) →

▰ Determining the shape of living cells

▰ Detecting microbial internal cellular components

i.e. they convert short wavelength rays into longer wavelengths

Mercury lamp emits rays that pass through an excitation filter

Exciting rays get reflected by a

Barrier filter – removes remaining UV

▰ Auto fluorescence (eg. Cyclospora)

▰ Microbes coated with fluorescent dye (eg. Acridine orange dye)

▰ Source of illumination - Accelerated electrons

▰ Wavelength of electrons 100,000 times shorter than visible light

▰ Transmission electron microscope (TEM, most common type)

▰ Scanning electron microscope (SEM)

Highest practical About 1,000–1,500 Over 100,000

Radiation source Visible light Electron beam

Medium of travel Air High vacuum

Specimen mount Glass slide Metal grid (usually copper)

Type of lens Glass Electromagnet

▰ Very thin specimens (20–100 nm thickness) -

3. Embedding: Specimen is embedded in plastic polymer.

4. Slicing: Specimen is then cut into thin slices by an ultramicrotome knife.

▰ Electrons - generated by electron gun-travel in

▰ Medium of travel - fully vacuum path - because

▰ Examine the surfaces of microorganisms in detail.

▰ Produces image from electrons emitted by object’s surface.

▰ Advanced microscope - uses scanning probe technology to study the

▰ Has a resolution power in fraction of nanometer.

Q1. Electrons are used as a source of illumination in?

b. Phase contrast microscope

Q2. Scanning probe technology is used in ?

b. Phase contrast microscope

d. Atomic force microscope