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Microscopes

1. Different types of microscopes like light, dark field, phase contrast, fluorescence, and electron microscopes are used to view bacteria, viruses, parasites, and fungi that are too small to be seen with the naked eye. 2. Light microscopes can magnify objects up to 1000x but have a resolution of only 0.2 microns, while electron microscopes can achieve magnifications of 100,000x and resolutions of 0.5 nm. 3. Specialized microscopes like fluorescence and electron microscopes require specific preparation of samples, use of stains or electron beams, and operating in a vacuum to achieve their high magnifications and resolutions for viewing intracellular structures and organelles.

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8 views29 pages

Microscopes

1. Different types of microscopes like light, dark field, phase contrast, fluorescence, and electron microscopes are used to view bacteria, viruses, parasites, and fungi that are too small to be seen with the naked eye. 2. Light microscopes can magnify objects up to 1000x but have a resolution of only 0.2 microns, while electron microscopes can achieve magnifications of 100,000x and resolutions of 0.5 nm. 3. Specialized microscopes like fluorescence and electron microscopes require specific preparation of samples, use of stains or electron beams, and operating in a vacuum to achieve their high magnifications and resolutions for viewing intracellular structures and organelles.

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atharva sawant
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MICROSCOPY

Dr Krishan Ruhela
WHY... ?

Bacteria :- 0.2 - 1.5 micron in diameter


3 - 5 micron in length

Viruses :- 20 - 300 nm in diameter

Parasites :- Protozoans in microns


Helminths in few mm to meters

Fungi :- 2 - 10 microns in diameter


Cm in length
TYPES OF MICROSCOPES

Light Microscope

Dark field Microscope

Phase Contrast Microscope

Fluorescence Microscope

Electron Microscope
# PROPERTIES OF
A MICROSCOPE
1. Resolution :~
Ability to produce separate images of closely placed objects
- distinguished as two separate entities

Refractive index = Oil > Air

Oil enhances the resolution power

Resolution

Human eye 0.2 mm

Light Microscope 0.2 micron

Electron Microscope 0.5 nm


2. Contrast :~

By using Dyes

Improved by staining the specimen

When stains bind to the cell, the contrast is increased


3. Magnification :~

Achieved by using lenses


Ocular lens / Eye piece ~ 10 x

Objective lens ~

Scanning - 4x
Low power - 10 x
High power - 40 x
Oil immersion - 100 x

Total Magnification = Objective lens X Ocular lens


# LIGHT MICROSCOPE
WORKING PRINCIPLE :~
APPLICATIONS
# DARK FIELD MICROSCOPE
APPLICATIONS

1. Used to identify the living unstained cells.

2. Used for thin structures -

a) Flagella

b) Spirochetes - Leptospira & Treponema palladium


# PHASE CONTRAST MICROSCOPE

1. Contrast is enhanced
2. Visualises the unstained living cells by creating difference
in contrast between cells & water.
WORKING PRINCIPLE :~
APPLICATIONS

1. Microbial motility

2. Determining the shape of living cells

3. Detecting microbial internal cellular components

Cell membranes Chromosomes


Nuclei Golgi apparatus
Mitochondria Endospores
Spindles, Inclusion bodies

These becomes clearly visible because they have


refractive indices markedly different from that of water
# INTERFERENCE CONTRAST
MICROSCOPE

1. Reveals cell organelles


2. Measurement of chemical constituents of a cell, such as :
Lipids
Proteins
Nucleic acids
# FLUORESCENCE MICROSCOPE
Light source :- Ultraviolet light

uses :- Cases of TB / Malaria / Kidney biopsy

Fluorescent Dyes :-

TB - Auramine & Rhodamine

Malaria - Acridine Orange

Fungus - Calcofluor white

Flow cytometry - FITC - Fluoro Isothiocyanate


WORKING PRINCIPLE
APPLICATIONS
a) Auto fluorescence :-
Certain microbes directly fluorescence when placed under
UV lamp
1. Cyclospora
2. Isospora

3. Formalin
Skin biopsy for immunofluorescence in NS & not
Formalin
4. NADPH - Used in fluorescent spot test in G6PD
deficiency
b) Microbes coated with fluorescent dye -

TB - Auramine & Rhodamine

Malaria - Acridine Orange

Fungus - Calcofluor white

Flow cytometry - FITC - Fluoro Isothiocyanate


# ELECTRON MICROSCOPE
Invented by - Ernst Ruska

Source of illumination -

Accelerated Electrons

No light is used, a beam of


electrons is used

Medium - Vaccum

Slides - Copper metal slides


TYPES :-
1. Transmission Electron Microscope
2. Scanning Electron Microscope

Features Scanning Transmission

Principle Scattered electrons Transmitted electrons

Dimensional 3D 2D

View & Time More sample viewed Less sample viewed


in less time in same time

Details Surface details Internal details


# TRANSMISSION ELECTRON MICROSCOPE
Very thin specimens ( 20 - 100 nm ) - suitable for EM

STEPS TO PREPARE THIN SPECIMEN :~


Fixation :- 2 - 2.5 % Glutaraldehyde
Osmium tetraoxide
Dehydration :-
Acetone or Ethanol

Embedding :-
Resin or Plastic polymer

Slicing :- Ultramicrotome Knife

Slides :-
Copper metal slides
WORKING PRINCIPLE
APPLICATIONS
Mainly used in research, to study :

Intracellular structures

Virus structures

Pathogen - host structural interactions

Freeze etching techniques :


Alternate method for specimen preparation, which helps to
disclose the shape organelles within the microorganisms

Deep freezing and & forming carbon platinum replicas of


the material - used in study of cellular ultrastructure
LIGHT MICROSCOPE ELECTRON MICROSCOPE

Magnification 1000 X 100,000 X

Resolution 0.2 microns 0.5 nm

Source Visible light Electron beam

Medium Air High Vaccum

Slide Glass slide Copper slides

Lens Glass lens Electromagnet

Fixation 10 % Neutral 2 - 2.5 %


Buffered Formalin Glutaraldehyde

Embedding Paraffin wax Resin / Plastic polymer

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