By Dr. SARITA KUMARI SAHU ,MD Microbiology, SCB MEDICAL COLLEGE, ODISHA

1. MICROSCOPY
PRESENTED BY-
DR.SARITA KUMARI SAHU
PG 1ST YEAR
Dept. of MICROBIOLOGY

2. CONTENTS
Introduction
Timeline Of Microscope
Terms & Definitions
Properties Of Microscope
Types Of Microscope
Micrometry
Care Of Microscope

3. INTRODUCTION
 A Microscope (from the Ancient Greek:, mikrós, "small"
and, skopeîn, "to look" or "see") is an instrument used
to see objects that are too small to be seen by the
naked eye.
 Giovanni Faber coined the name microscope for the
compound microscope by Galileo .
 Since then microscope continued to play a important role
in every Branch of science.
 The only scientific equipment ,Charls Darwin took with
him on the Voyage Of Beagle, was a simple microscope.

4. TIMELINE OF MICROSCOPE
• 1590 :- Two Dutch eye
glass makers,
Zaccharias Janssen and
Hans Janssen
constructed the first
compound light
microscope.
• 1609 - Galileo Galilei
developed a compound
microscope with convex
and a concave lens.

5. Timeline contd….
• 1676 :- Antonie Philips Van Leeuwenhoek
built a simple microscope.
.

6. Timeline contd….
• 1931 :- Ernst Ruska and Max Knoll developed the
Electron Microscope for which
they won the Nobel Prize in
Physics in 1986.
• 1981 :- Gerd Binnig and Heinrich Rohrer
invented the
Scanning Tunnelling Microscope
Electron microscope
constructed by Ernst
Ruska in 1931

7. TERMS AND DEFINITIONS
RESOLUTION
• It is Also called Resolving Power.
• It refers to the ability to produce separate image of closely placed
objects,sothat they can be distinguished as two separate entities.
• It is calculated by Abbés equation.
NUMERICAL APERTURE
• It is the light gathering capacity of a lens.
NA = n X Sin θ
n – Refractive index of the medium between
the object & the objective lens
Θ – ½ of angular aperture of objective lens

8. Terms & Definition contd….
REFRACTIVE INDEX( RI)
RI = Sin i/Sin r
i = angle of incidence
r = angle of refraction.
Higher the density of the medium, greater the RI.
IMMERSION FLUID
• This is any liquid that occupies the space between the object and
objective lens.
• It can also be used between the condenser and the microscope slide.
• Use of immersion fluid produce a homogeneous optical pathway
which minimize the refraction & maximize the NA and improve the
resolution.

9. EFFECT OF OIL IMMERSION ON RESOLUTION

10. PROPERTIES OF MICROSCOPE
• A good microscope should have at least three properties:
Good Resolution
• The resolution power of Human eye - 0.2 mm
Light microscope - 0.2µm.
Electron microscope - 0.5nm.
Good Contrast
• This can be improved by staining the specimen.
Good Magnification
• It can achieve by lens
Ocular Lens, Objective Lens
• The total magnification of microscope =
Power of the objective lens X Power of ocular lens

11. TYPES OF MICROSCOPE
LIGHT MICROSCOPE
Uses sun light or artificial light as source of illumination.
1. Bright Field Microscope
2. Dark Field Microscope
3. Phase Contrast Microscope
4. Fluorescence Microscope
ELECTRON MICROSCOPE
• Uses accelerated electron as source of illumination.
1. Transmission Electron microscope
2. Scanning Electron Microscope

12. The bright field or light microscope forms a dark image against a bright
background ,hence the name is called so.
COMPONENTS
The bright field microscope is divided into 3 parts:
Mechanical Parts
Base , C-Shaped Arm, Mechanical Stage
Magnifying Part
Ocular Lens/Eyepiece, Objective lens
Illumination Part
Condenser, Iris Diaphragm, Light Source, Fine and Coarse adjustment
Knob.
BRIGHT-FIELD MICROSCOPE

13. Terms &Definition…..
.

14. Bright-field microscope contd…
WORKING PRINCIPLE
The image from in the microscope in two stage.
• First image (i1) formed by the objective lens(L1),Which is magnified,
inverted & real.
• The second image (i2) is formed by ocular lens (L2) which is more
magnified and virtual.

15. Bright-field microscope contd…
agnifying Part :- All these have 2
magnification
THE LIGHT PATHWAY OF A MICROSCOPE

16. Bright-field microscope contd…
PROPERTY LOW POWER HIGH POWER OIL IMMERSION
Magnification
of objective
10x 40x 100x
Magnification of
eyepiece
10x 10x 10x
Total magnification 100x 400x 1000x
Iris diaphragm Partially closed Partially opened Fully opened
Position of condenser Lowest Slightly raised Fully raised

17. Bright-field
microscope contd…
Bright-field microscope contd…
Gram Positive Cocci Gram Negative Bacilli

18. Bright-field microscope contd…
APPLICATION
• Used to visualize and study the morphologies, Identification of bacteria,
parasites etc.
ADVANTAGE
• It is simple to use with few adjustments involved while viewing the
image.
• The optics of the microscope do not alter the color of the specimen.
• The microscope can be modified to other type microscope
DISADVANTAGE
• The heat produce by the light source may damage the specimen.
• Biological specimens are often of low contrast and needs to be stained.
• Staining may destroy or introduce artifacts.
• Resolution is limited to 0.2µm .

19. • This is similar to the ordinary light
microscope; however, the condenser
system is modified.
• In dark-field microscope, the
specimen appear bright against a dark
back ground.
• The diaphragm blocks all light from
entering the objective lens except
peripheral light that is reflected from the
specimen .
• The resulting image is a brightly
illuminated specimen surrounded by a
dark (black) field.
DARK-FIELD MICROSCOPE

20. BRIGHT-FIELD vs DARK-FIELD

21. Dark-field microscope contd…
APPLICATION
• It identifies the living,unstained
cells.
• It is also useful for the
demonstration of the motility of
flagellated bacteria and protozoa.
• This can rapidly demonstrate
Treponema pallidum in clinical
specimens.
• It is more useful in examining
external details, such as outlines,
edges, grain boundaries and surface
defects than internal structures.
Darkfield image of Treponema pallidum,

22. PHASE CONTRAST MICROSCOPE
• Phase-contrast microscope was first described by Dutch
physicist Frits Zernike.
• When light passes through cells, small phase shifts occur,
which are invisible to the human eye. In a phase-contrast
microscope, these phase shifts are converted into changes in
wavelength, which can be observed as differences in image
contrast.
• COMPONENTS
• Annular condenser
• Phase Plate

23. Phase contrast microscope contd…

24. Phase contrast microscope contd…

25. Phase contrast microscope contd…
APPLICATION
• To see the microbial motility.
• Determine the shape of living cell.
ADVANTAGE
• Living cells can be observed in their natural state.
• No special preparation for fixation or staining etc. is needed to
study.
DISADVANTAGE
• Phase-contrast condensers and objective lenses add considerable
cost to a microscope.
• To use phase-contrast the light path must be aligned.

26. FLUORESCENCE MICROSCOPE
• Refers to any microscope, that uses fluorescence in addition to
reflection and absorption, to generate an image.
• It utilizes a powerful mercury vapor arc lamp for its light source.
• COMPONENTS
• Light Source
• Excitation Filter
• Dichroic mirror
• Emission/Barrier Filter

27. Fluorescence microscope contd…
WORKING PRINCIPLE
• Most cellular components are colourless and cannot be clearly
distinguished under a microscope. The basic principle of fluorescence
microscopy is to stain the components with dyes. Such dye are called
FLUOROCHROMES or FLUOROPHORES
• Fluorochromes, are molecules that absorb excitation light at a given
wavelength (generally UV), and after a short delay emit light at a longer
wavelength.
• The emission light can then be filtered from the excitation light to reveal
the location of the fluorophores.
• The image produced is based on the emission wavelength of the
fluorescent species .
• This result image consist of brightly shine area against the dark
background.

28. Fluorescence microscope contd…
FLUOROCHROMES EXCITATION
WAVELENGTH
EMISSION
WAVELENGTH
Acridine Orange 500 526
Auramine O, 460 550
Calcofluor white 440 505-520
Ethidium Bromide 545 605
Evans blue 550 610
FITC 490 525
TRITC 555 580

29. Fluorescence microscope

30. APPLICATION
• It is used to study some microbes which directly fluoresce under UV
lamps.
• Some microbes visible under the microscope by staining them with
fluorochromes.
• It is used to detect parasites by QBC method and Tubercle bacilli
ADVANTAGE
• Slides can be examined at a lower magnification, thus allowing the
examination of a much larger area per unit of time
DISADVANTAGE
• Fluorophores lose their ability to fluoresce as they are illuminated in a
process called photobleaching
• The mercury vapour lamp tends to loose their potency with time.
Fluorescence microscope contd…

31. ELECTRON MICROSCOPE
• The Electron microscope was invented by Max Knoll and Earnst Ruska
in1931.
• The electronI microscope uses accelerated electrons as light source
• As wavelength of electron 100,000 times shorter than the visible light,
so resolving power is much better than a light microscope
• The magnified image is visible on a fluorescent screen and can be
recorded on a photographic film.
• Images produced by EM lack color, always shades of black,
gray, and white.
• Electron microscopes are of two types:
1.TEM (Transmission electron microscope)
2.SEM (Scanning electron microscope)

32. EM Contd….
Transmission Electron Microscope (TEM)
• TEM are the method of choice for viewing the detailed structure of cells and
viruses.
• This microscope produces its image by transmitting electrons through
the specimen.
• Because electrons cannot readily penetrate thick specimen, the specimen
must be sectioned into extremely thin slices (20–100 nm) .
Specimen Preparation
To make a suitable specimen following steps are followed
1. Fixation
2. Dehydration
3. Embedding
4. Slicing

33. EM Contd….
Electron Pathway
• Electron generated by electron gun travels in vacuum path, with high
speed, passes through condenser lens bombard to specimen mount on
slide.
• Scatters Objective Ocular Enlarge
electrons lens lens Image
• The thick area of specimen scatters more electron & appears dark and
thin area scatter less & appear bright.
APPLICATION
The important use of EM is for virus detection. It can detect virus
i) Direct from clinical specimen
ii)Tissue culture
iii)After adding specific antiviral antibody to the specimen.

34. TEM Cont….

35. EM Contd….
PROTEUS IN TEM

36. EM Contd….
Scanning Electron Microscope (SEM)
• SEM has been used for examining the surface of microorganism in
greater details.
Staphylococcus aureus

37. LIGHT VS ELECTRON MICROSCOPE
FEATURES LIGHT MICROSCOPE ELECTRON
MICROSCOPE
Magnification 1000x – 1500x Over 100,000x
Resolution 0.2µm 0.5nm
Illumination source Visible light Electron beams
Medium of travel Air Vacuum
Specimen mount Glass slide Metal grid
Type of lens Glass Electromagnetic

38. MICROMETRY
• Micrometry refer to the measurement of dimensions of the desire
microorganisms under microscope.
• The most common procedure used in clinical laboratory is by using
a graduated scale (Reticles) located in one eyepiece.
• Reticles must be calibrated against a stage micrometre.

39. Micrometry contd….
CALIBRATION OF OCULAR MICROMETRY

40. CARE OF MICROSCOPE
TRANSPORT
•When carrying your microscope from one part
of the room to another, use both hands to hold the
instrument,
And, under no circumstances should one attempt
to carry two microscopes at one time.
WORKING PLACE
•Should be placed in flat fixed area.
•Away from source of water.
•Workstation should be uncluttered while
doing microscopy.
LENS CARE
•At the beginning of each use, make sure lenses
are clean.
•At the end of each lab session, wipe any
immersion oil from the immersion lens.
The microscope should be held firmly with
both hands while being carried.
When oculars are removed for cleaning,
cover the ocular opening with lens tissue. A blast from
an air syringe or gas canister removes dust and lint.

41. Care of microscope contd…
PUTTING IT AWAY
1. Remove the slide from the stage.
2. If immersion oil has been used, wipe it off the lens and stage .
3. Do not wipe oil off slides you wish to keep. Simply put them into a slide
box and let the oil drain off.
4. Rotate the low-power objective into position.
5. If the microscope has been inclined, return it to an erect position.
6. If the microscope has a built-in movable lamp, raise the lamp to its highest
position.
7. Set the light intensity adjustment of the microscope to the lowest while
switching it off.
8. If the microscope has a long attached electric cord, wrap it around the
base.
9. Adjust the mechanical stage so that it does not project too far on either
side.
10.Place the dust cover over the microscope at the end of use.