Key Words

Microscopy, Dark ground, Bright field, Microscopy, Simple & inexpensive

Key Messages: A simple and inexpensive method of stop patch can convert a conventional light microscope into a Dark ground microscope allowing low contrast specimens to be examined properly.

Introduction

Light microscopy is an important investigative tool for biology that is used regularly in high schools and colleges. The structures of many biological specimens are of low contrast that cannot be revealed by the bright field compound microscopes which are provided in many classrooms. Microscopes that improve the contrast of these specimens through special optics are prohibitively expensive for most teaching budgets.1

This article describes a simple, inexpensive modification that changes a bright-field microscope into a darkfield microscope allowing low contrast samples to be examined. Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Standard bright field microscopy relies upon light from the lamp source being gathered by the sub-stage condenser and shaped into a cone whose apex is focused at the plane of the specimen. Specimens are seen because of their ability to change the speed and the path of the light passing through them. This ability is dependent upon the refractive index and the opacity of the specimen.

Darkfield microscopy relies on a different illumination system. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The entire field appears dark when there is no sample on the microscope stage. (fig 1)

Figure 1—Principle of Dark Ground Microscopy

Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Darkfield illumination uses a carefully aligned light source to minimize the quantity of directlytransmitted (un-scattered) light entering the image plane, collecting only the light scattered by the sample.1

The techniques described here, just requires making filters with opaque light stopper that can be placed under the condenser of the microscope in the filter holder or can be placed in the blue filter.

With a compound microscope, dark field is obtained by placing an occulting disk in the light path between source and condenser. A cheap set of occulting disks can be prepared by cutting circular pieces of black electrical tape. There are several ways to make this ‘patch stop’. You can use a piece of transparent plastic (e.g. OHP transparency) to hold the disc of black cardboard which can be secured in place by a simple sticking tape. Alternatively the central patch can be made by a good quality black marker pen over a piece of transparent sheet or OHP transparency which then can be placed in filter holder. The size of stop depends on the objective lens that is used. A high power lens requires a bigger patch stop. The circle has to be big enough preventing the light to enter the objective lens.2,3,4 (fig 2)

Figure 2 – Central stop patch

Figure 3 – Stop patch from cardboard

Figure 4 – shows epithelial cells in wet mount; in 40X objective lens with light microscopy on left side and same field with in house Dark ground microscope on right showing enhanced contrast.

Figure 5 – Shows Red Blood Cells in wet mount in 40X objective lens with light microscopy on left side and same field with in house Dark ground microscope on right showing enhanced contrast.

Figure 6- shows Fungal hyphae in KOH mount in 40X objective lens, with light microscopy on left side and same field with in house Dark ground microscope on right showing enhanced contrast.

Figure7 – shows Oral spirochetes in wet mount in 40X objective lens with in house Dark ground microscope

Different options over here have been suggested just to ease out the making of stop patch according to the convenience of the user. There is no difference in the visualization of the sample material with any of the stop patch.

The size of patch stop varies with the objective lens used. The higher the magnification used, the diameter of patch stop should decrease.5 With 40X objective a diameter of 19 to 22 mm whereas with 10X a diameter of 16 to 18 mm works very well. For viewing 2,5 .

  1. A specimen is placed on the microscope stage as usual.
  2. Illumination should be made as uniform and as bright as possible. Dark field requires a very bright light source. Remove all neutral density and colored filters from the illuminator.
  3. Aperture diaphragm in the condenser (contrast lever) should be wide open.
  4. Patch Stop placed under the condenser of the microscope in the filter holder or can be placed in the blue filter
  5. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens.2,3,4

  • Motile Treponema
  • Borrelia in blood
  • Motile Leptospira in urine
  • Pathogenic microfilaria in blood. Sheath and nucleus can be easily viewed.
  • Cryptosporidium in CSF
  • Motility of organisms in broth cultures

The advantage of darkfield microscopy also becomes its disadvantage: not only the specimen, but dust and other particles scatter the light and are easily observed. More care in sample preparation needs to be exercised in darkfield application. Glass slides need to be thoroughly cleaned of extraneous dust and dirt. It may be necessary to filter sample media (agar, water, saline) to exclude confusing contaminants. Sample materials need to be spread thinly; too much material on the slide creates many overlapping layers and edges making it difficult to interpret structures. 1,2,3,5

It is a very inexpensive and simple technique and with little trial and error anybody can master the technique. With patience and simple modifications a simple bright-field compound microscope can thus be a useful tool for dark ground microscopy.