Dark-field microscopy is a technique used to illuminate unstained samples, causing them to appear brightly lit against a dark background, utilizing a special condenser that forms a hollow cone of light. It offers better resolution and contrast than bright-field microscopy without the need for staining, making it suitable for observing live bacteria and other small organisms. However, it has limitations such as the need for strong illumination, potential damage to samples, and challenges with sample density affecting image quality.

1. SIVASANGARI SHANMUGAM
DARK FIELD MICROSCOPY

2. CONTENTS
1. Introduction
2. Principles
3. Parts of Microscope
4. How does it works?
5. Applications
6. Advantages
7. Disadvantages

3. INTRODUCTION
 Dark-field microscopy is ideally used to illuminate unstained samples causing them to
appear brightly lit against a dark background.
 This type of microscope contains a special condenser that scatters light and causes it to
reflect off the specimen at an angle.
 Rather than illuminating the sample with a filled cone of light, the condenser is
designed to form a hollow cone of light.
 This microscope uses reflected light instead of transmitted light used in the ordinary
light microscope.
 It prevents light from falling directly on the objective lens.
 Light rays falling on the object are reflected or scattered onto the objective lens with
the result that the microorganisms appear brightly stained against a dark background.

4.  In dark-field microscopy the condenser is designed to form a hollow cone of
light (see illustration below), as opposed to bright-field microscopy that
illuminates the sample with a full cone of light.
 In dark-field microscopy, the objective lens sits in the dark hollow of this cone
and light travels around the objective lens, but does not enter the cone shaped
area.
 The entire field of view appears dark when there is no sample on the
microscope stage. However, when a sample is placed on the stage it appears
bright against a dark background.
 It is similar to back-lighting an object that may be the same color as the
background it sits against - in order to make it stand out.

5. PRINCIPLES
 A dark field microscope is arranged so that the light source
is blocked off, causing light to scatter as it hits the
specimen.
 This is ideal for making objects with refractive values
similar to the background appear bright against a dark
background.
 When light hits an object, rays are scattered in all
directions. The design of the dark field microscope is such
that it removes the dispersed light, so that only the scattered
beams hit the sample.
 The introduction of a condenser and/or stop below the stage
ensures that these light rays will hit the specimen at
different angles, rather than as a direct light source
above/below the object.
 The result is a “cone of light” where rays are diffracted,
reflected and/or refracted off the object, ultimately,
allowing the individual to view a specimen in dark field.

6. PARTS OF DARK FIELD MICROSCOPE
1. Eye piece
2. Arm
3. Rotating nosepiece
4. Objective lens
5. Condenser
6. Dark disc
7. Light source
8. Course and Fine adjustment

7. HOW DOES IT WORKS?

8. HOW DOES IT WORK?
 Light reaches the object mounted on the stage through the condenser.
 The condenser lens focuses the light towards the sample.
 The patch stop provided in the condenser lens blocks light around the central
region allowing light to pass only the periphery of the lens.
 The light that enters the specimen, most is directly transmitted, while some is
scattered from the sample.
 The scattered light alone enters the objective lens and produce the image.
 The directly transmitted light is not collected and is omitted.
 Thus the field of vision is rendered dark and the image created is by the scattered
light and therefore, cell components that reflect light are clear in the image.

9. APPLICATIONS
 It is useful for the demonstration of very thin bacteria not visible under
ordinary illumination.
 Used for rapid demonstration of Treponema pallidum in specimens.
 Useful for the demon clinical of motility of flagellated bacteria and
protozoa.
 Used to study marine organisms such as algae, plankton, diatoms, insects,
fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin
polymers and some ceramics.
 Used to study mounted cells and tissues.
 It is more useful in examining external details, such as outlines, edges, grain
boundaries and surface defects than internal structure.

10. ADVANTAGES
 Resolution by dark-field microscopy is better than bright-field microscopy.
 Improves image contrast without the use of stain, and thus do not kill cells.
 Direct detection of non-culturable bacteria present in patient samples.
 No sample preparation is required.
 Requires no special set up, even a light microscope can be converted to dark
field.
 Dark-field microscopy is a very simple yet effective technique.

11.  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 of artifacts, due to
the nature of the process.
 A researcher can achieve a dark field by making modifications to his/her
microscope.

12. DISADVANTAGES
 The low light levels seen in the final image.
 The sample must be very strongly illuminated, which can cause damage to the
sample.
 Necessity to examine wet, moist specimens containing living organisms very
quickly, because visualization of the moving bacteria is essential to detection.
 Besides the sample, dust particles also scatter the light and appear bright.
 Sample material needs to be spread thinly, dense preparations can grossly
affect the contrast and accuracy of the dark field’s image.

13. REFERENCE
1. https://microbenotes.com/darkfield-microscopy/
2. https://www.slideshare.net/abhishekindurkar/dark-field-microscopy-81857005
3. https://microbeonline.com/dark-field-microscopy-principles-use-advantages-and-
limitations/
4. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boun
dless)/3%3A_Microscopy/3.3%3A_Other_Types_of_Microscopy/3.3A%3A_Dark-
Field_Microscopy
5. https://www.microscopeworld.com/t-darkfield_microscopy.aspx
6. Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier
India.
7. Cappuccino, J. and Welsh, C. (2014). Microbiology: A Laboratory Manual, Global
Edition. 1st ed. Pearson Education.

14. THANK YOU