Microbiology

How to use a Microscope



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When using a compound light microscope (a scope with two sets of lenses) students frequently become discouraged after the object they are trying to view can't be found at all or repeatedly disappears from view every time the scope is switch to a higher objective magnification. The following is a troubleshooting guide for diagnosing and correcting problems commonly associated with finding and focusing on microscopic objects.

* Not Enough Contrast *

Contrast is the difference in brightness between the object being viewed and the background. A light microscope employs visible light, which is shone upward through a window in the stage of the microscope and then through the specimen itself. The object being viewed will appear darker than the area surrounding it. If there is not enough contrast, the specimen is difficult to see. Difficulty seeing the object can result from too much or too little light.

Solution: Most compound scopes with electric light illumination have two mechanisms for adjusting light:

1. a dial, often in the base, that will increase and decrease the light level

2. an iris diaphragm, like that associated with the lens of a camera, that opens and closes the aperture, to allow more or less light in. This is adjusted with dial or lever directly below the stage.

Make sure to check the adjustment in both places: light source and iris diaphragm. Often the light level is turned up all the way, but the student is unaware that the iris diaphragm is closed.

Depending on the type of object being viewed, staining the specimen prior to viewing may also make it easier to find, especially when trying to view very transparent, colorless objects such as animal cells.

* Dirty Objective Lenses *

There is no guarantee that the person who last used the microscope properly cleaned it after use. The longer objective lenses (400xTM and 1000xTM) can easily become covered with stain or other liquid that dries and obscures the view.

Solution: To avoid this frustration, always use lens paper (and only lens paper) to clean the ocular and all objective lenses prior to using the microscope.

Tip: There is a simple test for determining if dirty lenses are obscuring the view. While looking through the oculars at the specimen, move the slide. If the image that you are seeing doesn't move, this may mean that the lens is dirty.

* Specimen Is Not Centered *

When magnification is increased by switching to a higher power objective lens, the field of view (the area of the specimen in view) gets smaller.

Solution: It is important to make sure that what you are trying to view is centered prior to switching to a higher magnification; otherwise the area of interest may end up outside of your field of view.

* Starting at Too High a Level of Magnification *

Compound microscopes are parfocal. This means that once the specimen is in focus at a lower objective power (for example 40xTM or 100xTM), when the objective is switched to a higher magnification, the specimen should still be pretty much in focus, with only fine focus adjustment required to get a crisp, clear image.

Solution: Depending on the size of the specimen, start off with the 40xTM scanning power objective (reads 4x right on the objective lens itself) or with the 100xTM low power objective (reads 10x on the objective lens itself). When viewing multicellular objects or large cells, scanning power is the best starting point. When viewing small cells, such as cheek cells, blood cells and bacteria, start with the low power objective.

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