Processing Specimens for Microscopy

Not all microscopic specimens can be observed directly with a compound microscope, many of them need to be brought into a form which is suitable for observation. Different specimens have to be processed differently. This article gives an overview of different preparation methods.

A specimen for compound microscopy must fulfill several criteria:

• It must be sufficiently thin.
• It should not be too dark (too heavily pigmented).
• If it is not pigmented at all, then it should possess a different refractive index compared to its surrounding medium, otherwise the structure is invisible.
• It should possess sufficient color contrast.

What should one do if the specimens do not fulfill the above criteria? It depends on the type of specimen.

• Thin and strongly pigmented specimen: bleaching. Depending on the type of specimen, different bleaching methods can be used. It is also possible to remove some pigments (such as chlorophyll of plants) by immersing the specimen in alcohol.
• Thin specimen with low contrast: staining. Selective stains react differently with different parts of the specimens. Certain DNA stains (careful, potentially carcinogenic!) interact with the DNA and make nuclei visible. Other stains interact with other substances. Here it is necessary to consult a catalog to determine the right stain for the task.
• Thin specimen with low contrast: observing in phase contrast. Phase contrast microscopy is an optical method in increase contrast. A prerequisite is, that the specimen possesses a different refractive index than the surrounding medium, which is the case most of the time.
• Thick and soft specimen: squeezing. The specimen can be squeezed between the slide and the cover glass. One example of this method is the observation of various fruits, such as a soft kiwi.
• Thick and soft specimen: hardening followed by microtoming. Soft specimens (ripe fruits, soft leaves etc.) are often difficult to cut into thin sections. They have to be hardened first. Plant materials can be hardened by placing them into alcohol for a few days. This removes water and makes the object easier to cut into small slices. Be careful again, this method is not suitable for children, due to the sharp tools involved. Also note, that the removal of water by the alcohol may cause the specimen to shrink.
• Thick and hard specimen: softening. Certain specimens can be softened by boiling them. Alternatively, certain chemicals also achieve the same effect. The soft specimen can then be squeezed between the slide and cover glass before microscopic observation.
• Thick and hard specimen: grinding them thin. This method is sometimes used when observing rocks and other hard substances which can not be softened. Specialized tools are required.
• Thick and hard specimen: use stereo-microscopes. The easiest way is to observe them with a stereo microscope using epi-illumination (light from the top).

[box]Not all of these methods are suitable for children and beginners. The purpose of this page is to give the reader an overview of possible methods.[/box]