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How to use a microscope

For the Koi Keeper, a microscope is an essential piece of equipment that can assist in various ways, especially the identification of parasites. Microscopic parasites are almost always present on fish but when they are there in large numbers, the fish will be unable to cope and intervention is necessary. The identification of these microscopic parasites is not easy and the shape and movement of the various parasites should be studied, so that  you can know what you are looking for. An excellent web-site to visit is Koi Quest where videos appear on what to expect when searching for parasites.

A normal microscope that is used in schools is adequate but it remains an expensive piece of equipment and should be handled carefully. Handle a microscope properly and carefully and it will last for many years!

When you purchase a microscope, make sure the magnification is 40x, 100x and 400x. Normally the objective lenses are marked 4x, 10x, and 40x. Remember the eyepiece also has a lens of 10x, so the actual magnification is ten times what the objective lens indicates.


What you need before you start

This is very simple. A microscope slide and cover strip. Also a stable surface in the shade to place the microscope. If you need a power point, make sure it is close by.


Setting up the microscope


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Before moving the microscope make sure all skate-boards are out of the passage! When moving your microscope, always carry it with both hands. Grasp the arm with one hand and place the other hand under the base for support.
Remove the cover, plug the microscope in, and place the excess cord on the table! If you let the excess cord dangle over the edge, your knee could get caught on it, and the next sound you hear will be a very expensive crash.
Turn the revolving nosepiece so that the lowest power objective lens is "clicked" into position (This is also the shortest objective lens).

Your microscope slide should be prepared with a cover strip or cover glass over the mucus sample.  This will help protect the objective lenses if they touch the slide.  Place the microscope slide on the stage and fasten it with the stage clips.  You can push down on the back end of the stage clip to open it.

Place the slide on the microscope stage, with the sample directly over the centre of the glass circle on the stage (directly over the light). Then you have a 9 out of 10 chance of finding the specimen as soon as you look through the eyepiece! If you wear glasses, take them off. If you see only your eyelashes, move closer. If you see a dark line that goes part way across the field of view, try turning the eyepiece.  That dark line is a pointer that will be very valuable when you want to point out something to someone else.

Look at the objective lens and the stage from the side and turn the coarse focus knob so that the objective lens moves downward (or the stage, if it moves, goes upward).  Move it as far as it will go without touching the slide! 
Now, look through the eyepiece and adjust the illuminator (a light or mirror) and diaphragm for the greatest amount of light. You will adjust it again later.

Slowly turn the coarse adjustment so that the objective lens goes up (away from the slide). Continue until the image comes into focus. Use the fine adjustment, if available, for fine focusing.  If you have a microscope with a moving stage, then turn the coarse knob so the stage moves downward or away from the objective lens.

Move the microscope slide around so that the image is in the centre of the field of view and readjust the mirror, illuminator or diaphragm for the clearest image.

Adjust the Diaphragm as you look through the Eyepiece, and you will see that more detail is visible when you allow in less light!  Too much light will give the sample a washed-out appearance. Once you have found the specimen/parasite on Low Power (40x or 100x), centre the specimen in your field of view, then, switch it to High Power (400x). If you don’t centre the specimen you will lose it when you switch to High Power.

Now, you should be able to change to the next objective lenses with only minimal use of the focusing adjustment. Use the fine adjustment, if available. Do not allow the objective lens to touch the slide! Once you have it on High Power remember that you only use the fine focus knob! Also, do not remove the slide when it is on High Power.” -- The High Power Objective (400x) is very close to the slide. Use of the coarse focus knob will scratch the lens, and crack the slide.

The proper way to use a monocular microscope is to look through the eyepiece with one eye and keep the other eye open (this helps avoid eye strain). If you have to close one eye when looking into the microscope, it's ok.  Remember, everything is upside down and backwards. When you move the slide to the right, the image goes to the left!

Do not touch the glass part of the lenses with your fingers. Use only special lens paper to clean the lenses.

When finished, raise the tube (or lower the stage), click the low power lens into position and remove the slide.

Always keep your microscope covered when not in use.  Dust is the number 1 enemy!


Taking a scrape

Take a mucus sample from the fish that you have identified. You can use a blunt instrument like a stainless steel teaspoon or if you have some experience, you can use the slide. Move the teaspoon along the edges of the gill covers, behind the pectoral fins or the sides of the dorsal fin. All these places are the preferred hiding spots of parasites. Please do the scrape from front to back of the fish, otherwise scales will come off. Just gather a small sample of mucus and transfer it onto the slide.

Place ONE drop of water directly over the mucus sample. If you put too much water over the specimen, then the cover strip will float on top of the water, making it harder to see and identify the parasites as they float past the field of view! Place the cover strip at a 45 degree angle (approximately), with one edge touching the water drop, and let go. If you just put the cover-slip on top of the slide, it will trap small air bubbles underneath the cover.

As described above, view the mucus sample under minimum magnification and get the focus and amount of light correct. Once this is ok, rotate the objective lens to higher magnification, and look for anything that moves first. Some parasites are so small that it will be necessary to rotate to 400x to properly identify them.


How to stain a slide

Staining may not be necessary, and the staining may/will kill the parasites. However to stain a slide, place one drop of Methylene Blue stain on one edge of the cover strip, and the flat edge of a piece of paper towel on the other edge of the cover strip. The paper towel will draw the water out from under the cover strip, and the cohesion of the water will draw the stain under the cover strip. As soon as the stain has covered the area containing the sample you are finished. If the stain does not cover the area needed, get a new piece of paper towel and add more stain until it does.

Be sure to wipe off the excess stain with a paper towel, so you don’t end up staining the objective lenses. You are now ready to place the slide on the microscope stage.

Be sure to follow all the instructions above as to how to use the microscope.

 

Last Updated on Thursday, 06 August 2009 15:44