Microscopy and use of different Microscopes in Microbiology

1. Dr. Mahendra Swamy
MBBS,
MD(Microbiology)
Microscopy

2. Introduction
 Definition: Use of Microscope to magnify the objects
that are too small to be visible to naked eye.
 Used to detect for the presence of Microorganisms (
Bacteria, Fungus, Parasites, Viruses) in the
specimen.
 Direct smears: Prepared from the specimen.
 Indirect smears: Prepared from the growth of
Microorganisms on culture plate.

3. Applications of Microscopy in
Microbiology
 Rapid
 Inexpensive
 Preliminary identification of organisms.
 Final identification of organisms in some cases. (Ex:
Identification of M tb, M leprae by ZN staining,
Identification of C diphtheria by Albert staining)
 Detection of different organisms present in the
specimen.
 Detection of organisms that are difficult to grow.
 Detection of late growing organisms.
 Detection of anaerobic bacteria where facilities for
anaerobic culture is not available.
 Providing information on what organisms to be expected

4. Applications of Microscopy in
Microbiology
 Helps in selection of Culture media for growing
the organisms.
 Helps in initiation of empirical treatment with
antibiotics.
 Helps in assessment of quality of specimen.
 Helps in deciding clinical significance of
organisms.
 Helps in detailed study of organisms (Electron
Microscope).

5. Types of Microscopes
 Bright field Microscope (Compound/Light
Microscope): Field of view is brightly illuminated.
 Dark field Microscope: Filed of view is dark. But
organisms are illuminated using a special condenser.
 Phase contrast Microscope: Here the organisms
are observed by enhancing their contrast. Mainly
used for examination of live unstained organisms.
 Fluorescence Microscope: Fluorochromes are used
here for staining the organisms.
 Electron Microscope: Beam of electrons are used
for illuminating the organisms.

6. Bright field
Microscop
y
Fluorescen
ce
Microscopy
Dark field
Microscop
y
Electron
Microscop
y
Bacteri
a
+ + +
_
Fungi + +
_ _
Parasit
es
+ +
_ _
Viruses
_
+
_
+

7. Phase contrast Microscope
 Principle:
 Instead of using stains to enhance the contrast, here
the differences in refractive index (density) of
different cell components are used to create contrast.
 When beam of light pass through cells, the
differences between refractive index of different
components of cell result in difference in light
intensity thus creating contrast.
 Applications:
 Used for examination of live unstained organisms, for
detecting microbial motility and for detection of
Intracellular Inclusion bodies.

8. Dark field Microscope
 Filed of view is dark. But organisms are illuminated
using a special condenser.
 Principle:
 Special condenser with a central opaque disc is used.
This condenser doesn’t allow light to directly pass
through the specimen, but directs the light to hit the
specimen at an oblique angle.
 Only the light rays that are deflected by the specimen
enter the objective lens. As a result specimen is
illuminated and the background appears dark.
 Applications:
 Used for detecting thin bacteria like Spirochetes:
Treponema pallidum

10. Spirochetes: Treponema pallidum in Dark field
Microscope

11. Electron Microscope
 Principle:
 Here electrons are used instead of light and
electromagnetic fields are used to focus the electrons.
 Because of increased resolution, the final magnification
of the image will be >100000 times.
 Scanning electron microscope (SEM): It uses electron
beam to scan the surface of an object and provide 3D
view of surface structures.
 Transmission electron microscope (TEM): Here electron
beam passes through the object and hence internal
structures of an object can be studied.
 Applications: Used for detection of viruses, studying
intracellular components of cell, studying thin structures

12. Fluorescence Microscope
 Certain dyes (Fluorochromes) can be raised to high
energy level after absorbing UV rays. When they
return to their original, low energy level, they
release excess energy in the form of visible light.
This process is called as Fluorescence.
 2 types:
 Fluorochroming: Here Fluorochromes are used
directly to stain the smear.
 Immunofluorescence: Here Fluorochromes are
tagged with specific antibodies.

13. Principle:

14.  Applications:
 Acridine orange: Used for detection of Malarial
parasites and detection of cell wall deficient bacteria
(Ex: Mycoplasma)
 Auramine-Rhodamine: Used for detection of
Mycobacterium tuberculosis.
 Calcofluor white: Used for detection of Fungi.
 Immunofluorescence: Here dyes are tagged with
antibodies that will bind with antigens present on the
surface of cells. Mainly used for detection of bacteria
that are difficult to grow or slow growing (Legionella,
Bordatella, Chlamydia). Ex: Fluorescein
isothiocyanate (FITC) is the dye commonly used for
conjugation.

15. Bright field Microscope
Compound/ light Microscope
 Here visible light is used
 Total magnification is product of magnification of
Ocular and Objective lenses.
 Magnification:
 Ocular lens: 10x
 Total magnification is product of Ocular and
Objective lenses
 Objective lenses:
 Scanning lens: 4x
 Low power lens: 10x
 High power lens: 40x

16.  Resolution:
 It is ability of Microscope to produce separate images
of closely spaced objects.
 Resolving power of human eye: 0.2 mm
 Resolving power of light Microscope is 0.2 𝜇𝑚.
 Oil used in Oil immersion lens gives very good
resolution as its refractive index is almost same as
glass slide
 Contrast:
 Because Microorganisms are almost transparent
because of high water content, they can not be easily
visible among the specimen.
 Hence to enhance their contrast, Staining
techniques are used.

17.  Numerical aperture (NA): It is a measure of light
gathering power of a lens. High NA of a lens
indicates high resolving power.
Objective lenses Numerical aperture
(NA)
10x 0.25
40x 0.65
100x 1.25

19. Parts of Compound Microscope
 Base: It supports the Microscope and holds various
parts like source of light, brightness adjustment knob,
light switch.
 Arm: Backbone of the Microscope. Bears Adjusting
knobs and Mechanical stage.
 Source of light: Halogen lamps are preferred over
Tungsten lamps as the former emits white light, have
higher luminosity and have long life.
 Coarse adjusting knob: To raise the stage and bring
the slide near the objective lens.
 Fine adjusting knob: To focus the smear present on
the slide.

20.  Mechanical stage: Stage with stage clips hold the
slide in place. Stage control knobs can be moved to
move the slide during observation.
 Condenser: It focuses light on the slide.
 Filter: Filters may be used below the condenser. Ex:
Blue/Green filters. Blue filter may be used to change
the light from ordinary electric bulb into a natural white
light.
 Iris diaphragm: Used to control the amount of light
that enters condenser.
 Nose piece with Objective lenses:
 Scanning lens: 4x
 Low power lens: 10x
 High power lens: 40x
 Oil immersion lens: 100x

21.  Microscope tube: It contains Prisms that bend light
thus providing comfortable viewing angle.
 Eye piece with Ocular lens: Magnification of
Ocular lens is 10x. Eye piece can be Mono ocular or
Binocular. It can have a movable pointer inside to
focus an area on the smear.

22. Care of the
Microscope
 Keep the Microscope on a table away from
chemicals/water source to avoid any kind of
splashes.
 The table should not be shared by vibrating
devices like centrifuge/ refrigerator.
 Turn off the light of the Microscope after use.
 Dust should be cleaned with air brush/artist’s
brush.
 Clean/ wipe the lens after use.
 Cover the Microscope with polythene/plastic bag
when not in use.

23.  Cleaning of the lens:
 Clean the lens with lens cleaning fluid
recommended by the manufacturer.
 Usually Ethyl ether/Xylene are used.
 Alcohol/Acetone are not used unless specified by
the manufacturer.
 Use lens paper for cleaning. If it is not available,
use Muslin cloth/thin silk cloth.

24.  Storing of the Microscope:
 Microscope should generally be closed with cover
after use and stored in well lit and ventilated area.
 If it is stored in a closed space, keep drying
agents like Silica gel, rice grains, salt along with it
which will absorb moisture.
 In humid areas store the Microscope every night
in a cabinet fitted with an electric bulb (5W to
40W). Light is switched on at night to reduce the
humidity.

25.  Preventing fungal growth on the Microscope:
 Fungal growth on the Microscope is common in
hot & humid areas. They generally grow on
objective lenses, eye piece & prisms.
 To prevent fungal growth, store the Microscope in
a cabinet fitted with light. Use hygroscopic
materials like silica gel, regularly clean the
Microscope and store in a well lit and ventilated
areas.
 Film of fungus can be removed from the
Microscope by using fungus cleaner/lens cleaner
fluid.

26. Micrometry
 It is a process where the size of the
Microorganisms are measured on the
Microscope.
 Micrometer is used for this purpose.
 Ocular Micrometer is a circular disc with
graduations that fits in the eye piece.
 Stage micrometer is clipped on the stage of the
Microscope.

27. Thank you