Introduction
Dark field microscopy is a simple and popular method for rendering unstained and transparent specimens clearly visible.
Good candidates for dark field observation often have refractive indices very close in value to that of their surroundings and are difficult to image with conventional bright field techniques.
As an example, small aquatic organisms, oocytes, and cells in tissue culture have a refractive index ranging from 1.2 to 1.4, resulting in a negligible optical difference from the surrounding aqueous medium (refractive index of 1.3).
Principle
The dark-field microscope produces detailed images of living, unstained cells and organisms by simply changing the way in which they are illuminated.
A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective.
In dark-field microscopy, a dark-field stop (inset) is placed underneath the condenser lens system. The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen.
Only light that has been reflected or refracted by the specimen forms an image. The field surrounding a specimen appears black, while the object itself is brightly illuminated.
The dark-field microscope can reveal considerable internal structure in larger eukaryotic microorganisms. It also is used to identify certain bacteria such as the thin and distinctively shaped Treponema pallidum, the causative agent of syphilis.
Procedure
- Take a drop of pond water /hay infusion/Yeast/Fungi onto the clean glass slide.
- Place a glass cover slip on the specimen and press it down to remove any air bubble.
- Examine the slide immediately under Phase Contrast as well as Dark Field Microscope.
