MICROSCO

Y
 Chapter Preview

▰ Bright-field or Light Microscope

▰ Phase Contrast Microscope

▰ Electron Microscope

▰ Dark Field Microscope

▰ Fluorescence Microscope

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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
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Microscopes –Definition &
Different types
of Microscopes
Microscopy refers to the use of a specialized instrument— called
‘microscope’ to view objects and areas of objects that cannot be seen
with the naked eye (objects that are not within the resolution range of
the normal eye). There are various types of microscopes that are used
in diagnostic Microbiology
▰ Bright-field or light
microscope
▰ Dark field (or dark ground)
microscope
▰ Phase contrast microscope
▰ Fluorescence microscope
▰ Electron microscope. 4
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 0.5 nm
microscope 5
PROPERTIES OF A
MICROSCOPE (Cont..)
2. Good contrast:

▰ Improved by staining the specimen.

▰ When stains bind to the cells, the contrast is

increased

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PROPERTIES OF A
MICROSCOPE (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.

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BRIGHT-FIELD OR
LIGHT MICROSCOPE
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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

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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.

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Magnifying Parts

▰ Ocular lens

▰ Objective lens
▰ The total magnification of a field is the
product of the magnification of the
objective lens and ocular lens:
Scanning field (40x)
Low power field (100x)
High power field (400x) and
Oil immersion field (1000x). 11
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:

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Sharpen the image.
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

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DARK FIELD
MICROSCOPE
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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)

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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.

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Applications

▰ Used to identify the living, unstained cells and thin

bacteria like spirochetes which cannot be visualized by
light microscopy.

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PHASE
CONTRAST
MICROSCOPE 18
PHASE CONTRAST MICROSCOPE

▰ Contrast is enhanced.

▰ Visualizes the unstained living cells by creating

difference in contrast between cells and water.

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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 1/4th of a
wavelength

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Working principle

[Link] ground microscopic picture demonstrating spirally

coiled bacteria (spirochete); B. Phase-contrast microscopic
picture demonstrating Naegleria fowleri trophozoites (free-
livingamoeba). 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

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dark
Working principle (Cont..)

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

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FLUORESCENCE
MICROSCOPE
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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
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Working principle (Cont..)

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 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

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Applications (Cont..)

Confocal microscope: Advanced design, uses optical

sectioning to get better resolution of the fluorescent image.

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ELECTRON
MICROSCOP
E 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.

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ELECTRON MICROSCOPE
(Cont..)

It is of two types:

▰ Transmission electron microscope (TEM, most common

type)

▰ Scanning electron microscope (SEM)

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Differences between light
microscope and
electron microscope

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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.
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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).

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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.
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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.

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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.

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Questions:
I. Write short notes on:
1. Principle and uses of dark field microscope.
2. Principle and uses of fluorescence microscope.
3. Principle and uses of electron microscope.

II. Multiple Choice Questions (MCQs):

1. U naided human eye has a resolution power of:
a. 0.2 mm b. 0.2 μm
c. 0.2 nm d. 0.5 nm 39
2. Phase contrast microscopy is useful for studying:
▰ a. Microbial motility
▰ b. Determining the shape of living cells
▰ c. Detecting bacterial components, such as endospores
▰ and inclusion bodies
▰ d. All of the above

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3. Which of the following microscopes, the object
appears bright against a dark background?
▰ a. Simple light microscope
▰ b. Dark ground microscope
▰ c. Compound light microscope
▰ d. Electron microscope

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4. Electron microscope differs from light microscope in all,
except:
▰ a. Highest practical magnification
▰ b. Medium of travel
▰ c. Resolution
▰ d. No need for specimen preparation
5. Highest useful magnification available in light
microscope is____.
▰ a. 100X b. 400X
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▰ c. 1000X d. 2000X
6. T he correct direction of flow of light rays in
fluorescence microscope is:
▰ a. Specimen – excitation filter – barrier filter – dichromic mirror
▰ b. Dichromic mirror – excitation filter – specimen – barrier filter
▰ c. Excitation filter – specimen – dichromic mirror – barrier filter
▰ d. Specimen – barrier filter – dichromic mirror – excitation filter

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7. A 5-year child is having diarrhea. Microscope that can be
used to demonstrate the commonest pathogen directly from
stool sample is:
▰ a. Darkfield microscope
▰ b. Electron microscope
▰ c. Phase contrast microscope
▰ d. Fluorescence microscope
8. Best resolution obtained using electron microscope is:
▰ a. 0.5 cm b. 0.5 nm
▰ c. 0.5 μm d. 0.5 mm
Answers 1. a 2. d 3. b 4. d 5. c 6. c 7. b 8. b
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