Cell biology and Genetics - course module Cell biology 1

Short handbook for using lab microscopes

The parts of a simple light microscope:

A. The eyepiece can be adjusted to focus the image. Sometimes there are two.
B. The lenses can be rotated to low or high power. Always start with the low power lens (the short one). Move it as close to the slide as possible (as you watch from the side), using the coarse focusing knob F.
C. The stage holds the glass slide containing the object to be viewed.
D. The aperture allows you to control the amount of light entering the lens.
E. The light source (the brightness can be adjusted).
F. The coarse focus adjustment knob. Use this one first.
G. The fine focus adjustment knob.
H. The handle. Use one hand here and the other under the base when carrying the microscope.

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1. How to prepare and view a FRESH MOUNT slide

1. Get a clean slide and cover slip.
2. Place the specimen in the center of the slide (or put a drop if the sample is a liquid).
3. Remember that the specimen should be THIN and SMALL. Light should be able to shine through it.
4. Use a dropper (a pipette) to add 1 SMALL drop of water to cover the specimen.
5. Place the edge of the cover slip to the side of the water drop and specimen.
6. Carefully lower the cover slip over the specimen letting any air bubbles escape.
7. If you end up with air bubbles, tap the top of the cover slip gently with the bulb of the dropper.
8. Dry off any extra water with a piece of paper towel. If the sample contains bacteria, remember to throw away the paper in the proper place.
9. Remember DO NOT GET ANY WATER ON THE MICROSCOPE OR OBJECTIVE!
10. If the lens is dirty or gets wet, ask you teacher for help.

2. Moving and transporting the microscope

Grasp the arm of the microscope with one hand and support the base of the microscope with the other. Handle the microscope gently, they cost a lot.

3. Switching on the microscope

Before you plug in the microscope, turn the voltage control dial on the microscope to minimum. Now plug in the microscope and use the on/off switch to turn it on. Make sure the entire cord is on the bench top and not hanging down where it could be caught by a leg.

4. Adjusting the eyepieces

These microscopes are binocular, that is, they have 2 ocular lenses (eyepieces). To adjust them, first find the proper distance between your eyes and the eyepieces by closing one eye and slowly moving your head toward that eyepiece until you see the complete field of view - about 1 inch away. Keep your head steady and both eyes in the same plane. Now open the other eye and gradually increase the distance between the eyepieces until it matches the distance between your eyes. At the correct distance you will see one circular field of view with both eyes.

5. Positioning the slide (objektglas på svenska)

Place the slide specimen-side-up on the stage so that the specimen lies over the opening for the light in the middle of the stage. Secure the slide between - not under- the slide holder arms of the mechanical stage. The slide can now be moved from place to place using the control knobs.

6. Adjusting the illumination

Adjust the voltage by turning the voltage control dial. For oil immersion microscopy (1000X), more light will be needed. At lower magnifications less light will be needed.

Adjust the amount of light coming through the condenser using the iris diaphragm lever. Light adjustment using the iris diaphragm lever is critical to obtaining proper contrast. For oil immersion microscopy (1000X), the iris diaphragm lever should be set almost all the way open. For low powers such as 100X the iris diaphragm lever should be set mostly closed.

The condenser height control should be set so the condenser is all the way up.

7. Obtaining different magnifications

The final magnification is a product of the 2 lenses being used. The eyepiece or ocular lens magnifies 10X. The objective lenses are mounted on a turret near the stage. The least powerful magnifies 10X; the middle lens magnifies 40X, and the oil immersion lens (with the black circle) magnifies 100X. Final magnifications are as follows:

• ocular lens X objective lens = total magnification
• 10X X 10X (yellow) = 100X
• 10X X 40X (blue) = 400X
• 10X X 100X (white) = 1000X

8. Focusing from lower power to higher power

Rotate the 10X objective until it locks into place (total magnification of 100X).

Looking at the microscope from the side, use the coarse focus until the stage and objective (shortest one) are as close as possible. DO NOT TOUCH THE OBJECTIVE TO THE SLIDE!

Look through the eyepieces and turn the coarse focus control (larger knob) slowly increasing the distance between the objective and slide until the specimen comes into focus.

Get the specimen into sharp focus using the fine focus control and adjust the light for optimum contrast using the iris diaphragm lever.

If higher magnification is desired, simply rotate the blue-striped 40X objective into place (total magnification of 400X) and the specimen should still be in focus. (Minor adjustments in fine focus and light contrast may be needed.)

For maximum magnification (1000X or oil immersion), rotate the 40X objective slightly out of position and place a drop of immersion oil on the slide. Now rotate the white-striped 100X oil immersion objective into place. Again, the specimen should remain in focus, although minor adjustments in fine focus and light contrast may be needed. Make sure that you do not crash the objective into the cover slip!

9. Cleaning the microscope

Clean the exterior lenses of the eyepiece and objective before and after each lab using lens paper only. (Paper towels may scratch the lens.) Remove any immersion oil from the oil immersion lens before putting the microscope away.

10. Reason for using immersion oil

Normally, when light waves travel from one medium into another, they bend. Therefore, as the light travels from the glass slide to the air, the light waves bend and are scattered similar to the "bent pencil" effect when a pencil is placed in a glass of water. The microscope magnifies this distortion effect. Also, if high magnification is to be used, more light is needed.

Immersion oil has the same refractive index as glass and, therefore, provides an optically homogeneous path between the slide and the lens of the objective. Light waves thus travel from the glass slide, into glass-like oil, into the glass lens without being scattered or distorting the image. In other words, the immersion oil "traps" the light and prevents the distortion effect that is seen as a result of the bending of the light waves.

11. Fluorescence microscopes

The epi-fluorescence microscopes in the laboratory are only to be operated under the supervision of an instructor.

12. When you are done for the day

a. Follow all instructions to clean up your slides and station when you are finished.
b. Turn off the light.
c. Make sure the microscope is clean and dry.
d. Make sure the LOWEST power objective (the shortest) is in place.
e. Move the stage as far away from the objective as possible.
f. Put the cover on (if there is one) and unplug.