DARK FIELD MICROSCOPY

1. DARK FIELD
MICROSCOPY

2. Contents
• Introduction
• History
• Principle
• Path of Light in Dark - Field Microscopy
• Uses of Dark – Field Microscopy
• Pros & Cons of Dark – Field Microscopy

3. Introduction
• Darkfield microscopy is a special form of microscopy in
which the light beam is split in such a way that the edges
of objects in the samples are illuminated so that they
appear as silhouettes against a dark background.
• Darkfield can be used to view wet samples, including live
blood and other liquids or apparently liquid substances.
• This is similar to the ordinary light microscope; however,
the condenser system is modified so that the specimen is
not illuminated directly.
• The condenser directs the light obliquely so that the light is

4. History
In 1830, J.J. Lister
(the father of Joseph
Lister) invented the
darkfield microscope,
in which the standard
brightfield (Abbe)
condenser is
replaced with a single
or double-reflecting
dark field condenser.
In 1906 in Vienna,
Karl Landsteiner and
Treponema pallidum – Dark Field
Microscopy
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5. Principle
Light enters the microscope for
illumination of the sample.
A specially sized disc, the patch stop
blocks some light from the light
source, leaving an outer ring of
illumination. A wide phase annulus
can also be reasonably substituted at
low magnification.
The condenser lens focuses the light
towards the sample.
The light enters the sample. Most is
directly transmitted, while some is
scattered from the sample.
The scattered light enters the
Microbes

6. Path of Light
Light enters the microscope to
illuminate the specimen.
A special disc, called patch stop
blocks some light from the light
source, leaving an outer ring of
illumination.
A wide phase annulus can also be
reasonably substituted at low
magnification.
Now the condenser lens focuses the
light towards the specimen.
The light enters the specimen.
Most of the light rays are directly
transmitted, while some of them are
scattered from the sample.
Only Scattered light started to enter
the objective lens and creates an
image of the specimen.
While the directly transmitted light
Microbiolo
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7. Centering
Dark Field
Condensers
the condenser, a point will
be reached where a bright
spot will appear in the field
of view.
b) As the condenser is
slightly raised or lowered, a
dark spot will appear, if the
condenser is properly
centered.
c) In cases where the
condenser is not properly
aligned and centered.
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8. Method of using Darkfield Condenser
• Make a very thin preparation of the field to be examined by using a
clean dry glass slide & cover it wit a thin cover slip. Air bubbles &
any excess of objects (blood cells, pus cells, etc.) should be avoided.
All these objects tend to brighten the background & reduce the
contrast.
• Remove the regular substage condenser & Introduce the Dark – field
condenser.
• To facilitate centering a series of concentric circles are ruled on the
top of the condenser. Bring these circles to the centre of the field of a
low power objective by means of centering screws.
• Place a drop of immersion oil on the apex of slide & focus a low
power objective.

9. Uses of Dark Field Microscope
• It is useful for the demonstration of very thin bacteria not
visible under ordinary illumination since the reflection of
the light makes them appear larger.
• This is a frequently used method for rapid demonstration
of Treponema pallidum in clinical specimens.
• It is also useful for the demonstration of the motility of
flagellated bacteria and protozoa.
• Darkfield is used to study marine organisms such as algae,
plankton, diatoms, insects,

10. Types of Specimens
• Very thin histological sections can be used if unstained or
if only certain components are stained, as in silver stains.
• Biological fluids from animals and plants, cell cultures,
microbes, foods, fibers, crystals, colloids, and sub-
microscopic particles are all suitable for dark field
microscopy.

11. Advantages
• Dark-field microscopy is a very simple yet effective
technique.
• It is well suited for uses involving live
and unstained biological samples, such as a smear from a
tissue culture or individual, water-borne, single-celled
organisms.
• Considering the simplicity of the setup, the quality of
images obtained from this technique is impressive.
• Dark-field microscopy techniques are almost entirely free

12. Disadvantages
• Not only the specimen, but dust and other particles scatter
the light and are easily observed.
• Glass slides need to be thoroughly cleaned of extraneous
dust and dirt.
• It may be necessary to filter sample media (agar, water,
saline) to exclude confusing contaminants.
• The sample has to be adequately illuminated. sometimes
strong illumination may become necessary and care must
be exercised , so that the specimen is not damaged by

13. Conclusion
• Dark field microscopy is a simple yet useful and effective
type of microscopy technique that is especially useful for
very minute organisms such as Treponema pallidum &
other spirochaetes. There are many undeniable advantages
to using dark field microscopy.
• Normally the bacterial cell are stained to observe under
microscope which requires killing them. But in Dark – field
microscopy the living bacterial cell can be observed in
unstained preparation by hanging drop techniques.
• The entire field appears dark when there is no sample on

14. References
• www.darkfieldstudies.com
• www.olympus-lifescience.com
• www.microbenotes.com
• www.microbeonline.com