Bacteriology 102:
Review of Microscope Technique

This page concerns the microscope we use to observe stained smears – i.e., the Olympus Model CH30 which is found in the cabinets along the wall. The handout provided in lab on the first day for the Olympus microscope is expanded below, covering the important points for adjusting illumination, focusing and magnification and emphasizing the absolute need for clean slides and lenses.

Clean Slides and Lenses: Clean slides must be used initially. It should be obvious that we will need to focus only on the top surface of the slide, where the smear is. Any residual stain or other material adhering to the bottom of the slide could be observed with the 10X objective, but then the 100X objective will hit the slide when it is swung into place. With the 10X objective, one can observe material on the condenser lens as well. Before observing the slide, it is best to clean off the accumulated debris, etc. on the bottom surface of the slide (the side not specifically stained) and also make sure the condenser lens is clean. Dirt on the ocular lens can be recognized when the lens is turned. To clean the bottom surface of a slide, a paper towel soaked in alcohol-acetone can be used, but only lens paper (with no solvents!) should be used on the lenses. (A diagram pointing out the three levels that can be focused on is shown here.)

Note the various parts of the microscope as labeled on Figure 1. Figure 2 (Parts A, B and C) highlight the parts concerning slide placement and illumination. (Clicking on these links opens the images in separate windows.)

Place slide on stage. Make sure the slide is secured on the stage as shown on Figure 2B. The slide can be positioned by moving the X- and Y-axis knobs (see Figure 1).

Illumination: (Figures 2A and 2C)

• The condenser is set all the way up such that the light is focused properly on the smear.
  
  
• The light setting should be at 4. ("4.1" is actally optimal. High settings tend to distort the shape of the cell.)
  
  
• Move the aperture iris diaphragm to the right for the 10X objective and to the left for the 100X objective.

Focusing and Magnification:

• Use the 10X (yellow) lens for initial focusing of the smear. Remember that there must not be anything on the bottom surface of the slide which can be focused on!
  
  
• Without raising or lowering the stage, move the 10X lens to the side and apply a large drop of immersion oil to the smear. (We will skip the 40X (blue) lens which is probably a waste of time.)
  
  
• You will always use the 100X (black) lens for your definitive observations of stained smears. Swing the lens into place, making sure that it gets immersed in the oil. You may find you need to turn the fine adjustment up to a quarter turn one way or the other to get the image in focus.

An Explanation for the Use of Immersion Oil:  As the 100X objective lens is very small, it tends to miss much of the light that is projected upward from the condenser lens system. Note on the diagram below that light coming up from the sides will be bent when it enters the glass slide due to the slide's "refractive index" which is actually a measure of the ability of a substance to refract (bend) light. When the light again is travelling through air – above the slide – it goes off in the same direction in which it entered the slide. The immersion oil has the same refractive index as the slide and will thus prevent scattering of much of the light, directing the light into the objective lens and resulting in better illumination of the subject.

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Page last modified on 2/23/07 at 6:15 PM CST.

John Lindquist, Department of Bacteriology

University of Wisconsin – Madison