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Answering Reader Questions

Found in: Microscopy Basics, Microscopy FAQ, Theory

Why is refractive index of mounting media important?

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The refractive index is important for several reasons. First, it influences the resolution of the image. Second, if the refractive index of the specimen is too similar to the refractive index of the mounting medium, then it may be difficult to see the specimen if it is not stained. Phase contrast microscopy relies on a different refractive index between medium and specimen. If the refractive index is too different (eg. mounting the specimen in air), then the specimen may appear to be too dark.

What are some differences electron microscope and light microscope concerning cost and skills required? Generally, electron microscopes require substantially more sample preparation time than light microscopes. However, this is only a generalization. Some staining techniques in light microscopy are also highly elaborate and time consuming. It depends much on the actual preparation technique used. These two types of microscopes can hardly be compared. Due to the elaborate sample preparation techniques which are required by electron microscopy, the chances are much higher to introduce artifacts. It requires much skill in identifying these. Compound light microscopes are sufficiently simple to be used in schools and allow for a fast observation of specimens.

What are some disadvantages of permanent slides? Permanent slides contain specimens that are fixed, dehydrated and possibly also microtomed (sliced into thin sections). The organisms are therefore not moving. Over time the specimen may also start to lose color. Generally, permanent slides require much more elaborate preparation. The advantage is, however, that once prepared the slide can be used over and over again and can be stored for longer time periods.

Why is a mounting medium important? The mounting medium physically supports the specimen, conserves it, and provides the correct refractive index in order to see the relevant details.

Why is it important to apply a coverslip (cover glass) at a 45 degree angle when making a wet mount? This reduces the possibility of air-bubble formation. It does not have to be exactly 45 degrees. The point is, that one should not drop the cover glass horizontally on the water droplet on the slide. By lowering the cover glass at an angle, the water slowly replaces the air from one side. Read the following post (and video) on how to correctly make a wet mount: Making a wet mount microscope slide

Why are unstained bacteria difficult to see? They are difficult to see in bright-field microscopes, because they are small, transparent and lack color. Beginners also have problems distinguishing bacteria from dust and debris. Phase contrast microscopes are much better for viewing of unstained bacteria.

Why is water added when mounting tissue onto a microscope slide? The water is the mounting medium. It supports the specimen, and most importantly, improves the resolution of the image by providing the correct refractive index. Try it out yourself. Look at some dry specimens (insect wings, pollen) with and without water. Read the following post for more information and pictures: The effect of the mounting medium on specimen and image quality

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