Dark-field Microscopy: Principle and Uses

Dark-field microscopy is a technique that can be used for the observation of living, unstained cells and microorganisms. In this microscopy, the specimen is brightly illuminated while the background is dark. It is one type of light microscope, others being bright-field, phase-contrast, differential interface contrast, and fluorescence.

Principle

Dark-field microscopy uses a light microscope with an extra opaque disc underneath the condenser lens, or a special condenser having a central blacked-out area, due to which the light coming from the source cannot directly enter into the objective.

The path of the light is directed in such a way that it can pass through the outer edge of the condenser at a wide-angle and strike the sample at an oblique angle. Only the light scattered by the sample reaches the objective lens for visualization. All other light that passes through the specimen will miss the objective, thus the specimen is brightly illuminated on a dark background.

Uses of Dark-Field Microscopy

Dark-field microscopes are used in the microbiology laboratory for the following purposes;

• Visualization of spirochetes such as Treponema pallidum (syphilis), Borrelia burgdorferi (lyme borreliosis) and Leptospira interrogans (leptospirosis) in clinical samples.

Spirochetes can not be seen by light microscopy because of their thin dimensions.

• Observation of microbial motility; tufts of bacterial flagella can often be seen in unstained cells by dark-field or phase-contrast microscopy

• Observation of internal structure in larger eukaryotic microorganisms such as algae, yeasts, etc.

Advantages of Dark-Field Microscopy

1. Resolution by dark-field microscopy is somewhat better than bright-field microscopy.
2. It improves image contrast without the use of stain, and thus do not kill cells.
3. Direct detection of non-culturable bacteria present in patient samples.
4. No sample preparation is required.
5. It requires no special setup, even a light microscope can be converted to dark field.

Limitations of Dark-Field Microscopy

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

References and further readings

• Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
• Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
• Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.
• Willey, Joanne M, Sherwood, Linda M, & Woolverton, Christopher J. (2016). Prescott’s Microbiology (10 edition). McGraw-Hill Education.