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