Darkfield Microscopy

In standard brightfield microscopy, specimens are visible because of they change the speed and the path of the light passing through them (refraction). To see a specimen clearly in a brightfield microscope, the light rays passing through it must be refracted enough to cause interference and produce sufficient contrast (differences in light intensities) to make the specimen visible. Most biological specimens consist primarily of water and hence have inherently low contrast unless they have been artificially stained. Fine detail is very difficult to see in unstained specimens, and stained specimens are usually dead.

Darkfield microscopy is a specialized technique which enhances the contrast of specimens, forming a bright image of the specimen superimposed onto a dark background. Small details such as flagella which cannot be seen under brightfield illumination become visible. An analogy which is often used is the bright appearance of dust particles in a dark room illuminated by strong light coming in through a side window. The dust particles are very small, but are easily seen because they scatter the light rays.

In the first example, Arthrobacter cells are seen - compare this with the same specimen viewed under phase contrast illumination.

The following example shows a highly motile culture of Bacillus halodenitrificans. In the video you can see the long cells moving in a corkscrew motion as they are driven along by their flagellae. The very bright dots are endospores which are clearly visible using this technique:

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