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
