Experiments in the use of dark-field optical microscopes
Experiments in the use of dark-field optical microscopes
Living bacteria are transparent and not easy to see when viewed under a bright-field microscope. The principle of dark-field microscopy is the same as that of a beam of strong light from a slit passing through a dark room, in which fine dust can be clearly seen, i.e., the light illuminating the sample does not pass directly through the objective lens, but the light reflected or refracted on the sample enters the objective lens, and is therefore often used in the observation of living bacteria and bacterial motility.
Operation method
dark-field optical microscope
Principle
Living bacteria are transparent and not easy to see when viewed under a bright-field microscope. The principle of dark-field microscopy is the same as the phenomenon that when a beam of strong light from a slit passes through a dark room, the fine dust in it can be clearly seen, i.e., the light that illuminates the sample does not pass through the objective lens directly, but the light reflected or refracted on the sample enters into the objective lens, and then, in the dark field of view, the sample is able to be seen more clearly due to the increase of the contrast. Because the dark-field method allows you to see the bright organisms in a dark field of view, it is often used when observing living bacteria and bacterial motility. Darkfield microscope construction is mainly the use of a special condenser, in the condenser below the center of the round black disk for the cover, the light only from the periphery into the light will converge on the slide, and oblique illumination of the object, the object by the oblique illumination, issued by the reflective light can be entered into the objective lens, which results in a dark field of view of the microscope, and the object in which the bright. If there is no dark field microscope, just remove the bright field microscope on the concentrator, replace the dark field concentrator can be; can also be in the bright field microscope concentrator below the filter bracket put a piece of star-shaped baffle (star diaphragm) constitute the dark field of view, this method is suitable for low magnification observation. In the dark field, because some living cells are brighter than dead cells, the dark field is also used to distinguish between dead and living cells, and this technique is now used to identify dead and living yeast cells. In addition, dark-field microscopy is particularly useful for observing delicate microorganisms such as Treponema pallidum.
Materials and Instruments
Brewer's yeast Move 1. Select a clean slide of 1.0-1.2 mm thickness, apply a drop of brewer's yeast suspension and cover the slide (be careful not to have air bubbles). Caveat 1. dark field observation, the spotting mirror and the slide between the drop of cedar oil should be filled, otherwise the illumination light in the spotting mirror above the full reflection, can not reach the object being examined, so can not get dark field illumination. 2. before darkfield observation of the specimen, be sure to carry out the center of the spotting scope adjustment and focusing, so that the focus and the object being examined. 3. As the numerical aperture of the dark-field spotting scope is larger (NA=1.2~1.4), the focus is shallower, therefore, the over-thickness of the examined object can not be adjusted to the focus of the spotting scope, the general thickness of the slide is about 1.0 mm, and the thickness of the coverslip is suitable to be 0.16 mm or less, and at the same time, the slide and the coverslip should be very clean, free of grease and scratches, or else it will seriously disrupt the final image.Otherwise, the final image will be seriously disturbed. For more product details, please visit Aladdin Scientific website.
Cedar oil
Darkfield Optical Microscope Slides
2. Set the spotting scope aperture to 1.4.
3. The aperture hole of the light source is set to maximum.
4. Place a large drop of cedar oil on the condenser, place the specimen on the carrier, and screw the condenser up so that the oil is in contact with the slide (no air bubbles can occur).
5. Use a low-power objective lens and 7 × eyepiece to match the light to the object. Adjust the height of the condenser, first on the slide there is a black dot in the middle of the circle of light, and finally a bright point of light, the smaller the point of light, the better, the point of light will be moved up and down the condenser are to make the point of light increased.
6. Replace the required eyepieces and high magnification lenses, and slowly raise the objective lens for focusing until a luminous sample appears in the center of the field of view.
7. Place a drop of cedar oil on the coverslip and turn the oil mirror to the position it should be in to adjust the light distribution and make observations.