Dark-Field Microscopy (A Type of microscope )

Dark-Field Microscopy
 
This method is valuable in the examination of unstained living organisms. For example the causative agent of syphilis, Treponema pallidum, can be identified in the specimen under dark-field microscopy by its characteristic spiral shape and motility. The dark field microscopy uses a light microscope equipped with a special condenser and objective which brightly illuminate the objects against a dark background. The object can be compared with the moon shining brightly in the dark night sky because it reflects the sun rays to the earth and appears as a self-illuminated star. The same principle is also observed when the dust particles floating in the air in a dark room appear bright in a stream of sunlight. 

Principle 

Dark-field microscopy, also known as dark-ground illumination, makes use of a special condenser which has a central blacked-out area. Therefore, the light coming from the source cannot directly enter into the objective and the microscope field appears dark. The path of light is directed in such a way that it can pass through the outer edge of the condenser at a wide angle (Fig. 3.24). 

In its path, the light ray may strike the object in the object plane which will reflect the light. This reflected light then passes through the objective, making the object appear to be luminiscent against a dark background. Setting up dark-field microscope The special dark-field condenser produces the best form of dark-ground illumination. 

It is very useful when a high magnification is required. Ordinarily, adequate dark-ground can be obtained with 10X and 40X objectives using a simple opaque disc which is inserted into the filter holder of the substage to prevent light passing through the condenser to the objective.

Requirements

1. An oil immersion or dry dark-ground condenser with centering screws.

2. A funnel stop for insertion in the 100 X objective to reduce its numerical aperture to less than one.

3. A 6X or 10X eyepiece. The lower the power of the eyepiece, the brighter will be the image.

4. A high intensity microscope lamp. if the microscope is not provided with a built-in source.

5. Good quality slides, not more than one mm thick, free from scratches and absolutely clean.

Setting up-

1. Replace the ordinary condenser with the dark-ground condenser and raise to almost stage level.

2. Using a thoroughly clean slide and cover slip, make a thin wet preparation of the object.

3. Add a drop of oil on top of the condenser and place the slide on the stage so that the oil is between the slide and the condenser.

4. Place a high intensity lamp about 18 inchesaway. Adjust the mirror so that light passesupwards into the condenser.

5. With the 10x objective, focus on the specimen until a small ring of light is seen illuminating a part of the specimen.

Some examples of application of dark-ground microscopy

1. Detection of Treponema pallidum in chancre fluid, the motility of which cannot be seen in ordinary light microscope.

2. Detection of Leptospira in urine.

3. Detection of Borrelia in blood.

4. Examination of stool for the presen

6. Focus the condenser up or down until the small ring of light becomes just a spot of light.

7. using the centering screws, bring the spot of light to the centre of the field.

8. Focus with the 40X objective and examine. Further adjustments of the centering screws and focusing of the condenser can be made.

9. The oil immersion objective can be used after inserting a funnel stop of Vibrio cholera and Campylobacter species.

Problems in the use of dark-ground microscope Problems are frequently encountered in the use of dark-ground microscopes. These include:

1. Difficulty in focusing or centering of the dark-ground condenser.

2. Using slides and cover slips that are not completely clean.

3. Examining too dense a preparation.

4. The presence of air bubble in the immersion oil.

5. Insufficient oil contact between the slide and the condenser.