MICROSCOPY

Dr Praseetha MJ
1st year PG
MD Microbiology
BMCRI
 MICROSCOPE

 A microscope (Greek: mikron = small and scopeos = to look).

- MICROSCOPE: Is an instrument for viewing objects that are too small to be seen
by the naked or unaided eye.

 - MICROSCOPY: The science of investigating small objects using such an

instrument .

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 Antonie van Leeuwenhoek(1676)

 Used single-lens
microscope

 Little animalcules

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 First microscope used by Leeuwenhoek

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 TYPES OF MICROSCOPE

 Bright field or light microscope

 Dark field/dark ground microscope
 Phase contrast microscope
 Fluorescence microscope
 Electron microscope
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 PROPERTIES

 Good resolution
-ability to produce separate images of closely placed objects they
can be distinguished as separate entities.
Resolution power of:
-unaided human eye:0.2 mm
-Light microscope:0.2µm
-Electron microscope:0.5nm
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 RESOLUTION

-Depends on the reftactive index of the medium.

-Oil has higher refractive index, so higher resolution power.

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  Good contrast
-Differences in intensity between two objects, or between an object and
background.
-staining increases contrast

 Good magnification
-ocular lens
-objective lenses
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 Magnification power of lenses & total
magnification

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 BRIGHT-FIELD /LIGHT MICROSCOPE

 Forms dark image against brighter background

 Structure
1. Mechanical parts
 Base – Bottommost portion that supports the entire/lower microscope
 C shaped arm-holds the microscope. connects ocular lens to objective lens
 Stage-consist of stage clips, stage control knobs, aperture at the centre.

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 Magnifying parts

-Eye piece with

ocular lens

-Revolving nose
piece with
objective lens

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 3.Illuminating parts
-condenser: focus cone of light on the slide
-iris diaphragm: controls the light
-light source: mirror/electric bulb
-fine & coarse adjustment knobs
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 HOW IT WORKS?

 Light is transmitted and focused by mirror and condenser.

 Focused light illuminate the object or specimen.
 The refracted light is collected by an objective where primary image of the
object is formed.
 The eyepiece further magnifies this primary image into
 virtual,
 erect
 enlarged image,
 this is the final image that lies above the stage.
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 APPLICATIONS

 Observation of morphology of microorganisms.

 Detection of cell structures.
 Observation of intracellular structures.
 Detection of motility.
 Measurement of size.
 Observation of blood smears.
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 ADJUSTMENTS

LOW POWER HIGH POWER OIL IMMERSION

CONDENSER LOW MIDDLE HIGH

DIAPHRAGM MINIMALLY HALF OPENED FULLY OPENED

OPENED
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 Object appears bright against dark background

DARK FIELD MICROSCOPY

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 HOW IT WORKS?

 Darkfield condenser has a central opaque area & peripheral annular

hollow.
 A hollow cone of light is focused on the specimen in such a way that
unreflected and unrefracted rays do not enter the objective.
 Only light that has been reflected or refracted by the specimen
forms the image
 The field surrounding specimen appears dark while the object
brightly illuminated.
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 APPLICATIONS

 Living unstained cells

 spirochetes

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 Enhancing contrast by phase difference of light

PHASE CONTRAST MICROSCOPE

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 HOW IT WORKS?

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 APPLICATIONS

 To visualize unstained living cells

 Microbial motility
 To determine the shape of living cells
 To visualize internal structures

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 Uses fluorescence property to generate an
image

FLUORESCENCE MICROSCOPY

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 HOW IT WORKS?

 Fluorescent dyes exposed to UV light become excited

and fluoresce.
 Source of light emits rays which pass through an
excitation filter which allows only short wavelength
UV light.
 These rays get reflected by dichromatic mirror to fall
on the fluorescent dyed specimen focussed under the
microscope
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  Fluorescent dye absorbs the exciting rays of short
wavelength & emits rays of higher wavelength
 A barrier prevents remaining harmful uv rays reaching
the viewers eye.

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 APPLICATIONS

1. Epifluorescence microscope: simplest form

2. Auto fluorescence: Cyclospora
3. Using fluorescent dye:
 Auramine-phenol: tubercle bacilli
 Acridine orange: Plasmodium, Filarialparasites(QBC examination)
4. Immunofluorescence: fluorescent coated immunoglobulins to
detect surface antigens.
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 Cyclospora

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 ELECTRON MICROSCOPE

 Source of illumination: accelerated electrons.

 Ernst Ruska & Max Knoll in 1931
 Electron microscope in Bengaluru- NIMHANS and IISc
Types:
1. Transmission electron microscope
2. Scanning electron microscope
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 Transmission electron microscope

 Specimen preparation
 Fixation: Glutaraldehyde/osmium
tetroxide
 Dehydration: Acetone/ethanol
 Embedding
 Slicing
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 HOW IT WORKS?


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 Scanning electron microscope

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 APPLICATIONS

 Virus detection: Eg ,rota

viruses
 To study the internal
structures of a cell, details
of flagella, fimbriae

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 SARS-CoV-2 infected cell

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 THANK YOU

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