Lab Module 3: Microscope Basics
INTRODUCTION
The microscope is an important tool for any microbiologist. To successfully view bacteria (which
are very small), you must have good technique when using the microscope. The following is an
explanation of the microscopes available in our lab.
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Key:
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Oculars (magnify the image 10x).
Objectives (four lenses of different magnification; 4x, 10x, 40x, 100x).
Stage (the platform on which the slide is placed).
Iris diaphragm control lever on the condenser (controls the size of the opening in the
condenser).
Mechanical stage controllers (move the slide left/right and forward/back).
Coarse focus adjustment (moves the stage up and down rapidly).
Fine focus adjustment (moves the stage up and down incrementally).
Light switch (turns the light source on/off).
Nosepiece Objectives:
Name
Power
Scanning Lens
Low Power
High Power
Oil Immersion
4x
10x
40x
100x
Total
Magnification
40x
100x
400x
1000x
Depth of
Field
Deep
Moderate
Small
Very narrow
Field of View
Comments
Large
Moderate
Small
Very small
Easiest to use
Hardest to use
Note: To figure total magnification, multiply the magnification power of the ocular (10x) by the
magnification power of the selected objective.
Important: The oil immersion lens requires a special procedure involving the submersion of the
lens in a droplet of immersion oil. The oil immersion lens is NOT used with slides that have loose
cover slips (i.e., with wet mounts).
Methods of Light Adjustment:
Light Intensity: For Round-Based Microscopes set at about 4 (on a scale from 0 to 6).
For Rectangular-Based Scopes set at 6 or 7 (on a scale from 0 to 10).
Condenser Height:
Can be adjusted on some microscopes using a knob just under the stage.
Condenser should be set as high as it can go.
Diaphragm Diameter: The diaphragm can be opened up or closed down allowing more or less
light through the stage. The diaphragm is controlled by a small swing-arm
located just below the stage on the front of the condenser. In general, the
diaphragm should be in a fairly closed down position when you first
look at a slide. THE DIAPHRAGM IS THE MOST IMPORTANT
TOOL IN LIGHT ADJUSTMENT.
Tips for Focusing:
1. Always start with the scanning lens in place, and with the diaphragm closed down.
2. Start with the stage up as close to the scanning lens as possible.
3. Start with a macroscopic object (e.g., the edge of the cover slip or an air bubble) in the
middle of the field of view.
4. Jiggle the slide back and forth using the mechanical stage control knobs.
5. Focus on the macroscopic object (e.g., the edge of the cover slip or an air bubble) which
should be moving back and forth in the same way as you are jiggling the slide.
6. Using the mechanical stage controls, move the stage so that the specimen is in the middle of
the field of view.
7. Jiggle the slide back and forth and focus (gently) until objects are visible that are moving
back and forth in the same way as you are jiggling the slide.
8. Switch to the low power objective. These microscopes are parfocal, which means the
image stays in rough focus when you switch from one objective to another. Jiggle the slide
and focus on the moving object.
9. Repeat step 8 whenever you switch from one objective to the next higher powered objective.
ACTIVITIES
Activity 1: Size of the field of view.
1. Obtain a clear plastic ruler from your drawer.
2. Place the ruler on the stage by laying it over the stage clips, and then bring it into focus
using the scanning (4x) objective.
3. Move the ruler so that a millimeter mark is touching one edge of the field of view.
4. Estimate the diameter of the field of view of the scanning objective (in millimeters). Write
this measurement in the box for the scanning objective’s diameter (mm) in the chart
below.
a. The fields of view for the longer objectives are inversely proportional to the diameter
of the field of view for the scanning objective.
5. Complete the rest of the column (diameter in mm) by using the following formula:
& Magnification of Scanning Objective #
!!
Diameter of Objective N (mm) = Diameter of 4x Objective (mm) X $$
Magnification of Objective N
%
"
6. Convert the diameters from millimeters to micrometers ( m) by multiplying by 1000.
Objective
Scanning
Low Power
High Power
Oil Immersion
Magnification
4x
10x
40x
100x
Diameter (mm)
Diameter ( m)
7. Knowing the diameter of the fields of view for the different objectives will allow you to
estimate the sizes of specimens that you observe.
Activity 2: Observing prepared slides.
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Obtain a prepared slide from the table at the front of the room.
Work through the tips for focusing given above.
View the specimen with the scanning, low power, and high power stage objectives.
When in focus at high power, move the 40x objective out of the way, but do not rotate the
100x objective into place yet.
5. Place a drop of immersion oil on the slide in the field of view, over the center of the hole in
the stage where the light is passing through it.
6. Rotate the 100x (oil immersion) objective into place.
a. CAUTION: The 100x objective is built to be put into oil. The 40x objective is
NOT. Oil will leak into the barrel of the 40x objective and ruin it. NEVER
USE ANY OBJECTIVE EXCEPT THE 100x OBJECTIVE WHEN OIL IS ON
THE SLIDE.
7. Use the fine focus adjustment to focus the view. Be very subtle with your focusing. Even
the slightest slip can cause you to overshoot the depth of focus for the specimen.
Sometimes, focusing with the oil immersion objective takes a great deal of patience.
Remember to jiggle the slide back and forth.
8. When finished viewing the specimen with the oil objective, directly rotate the scanning (4x)
objective into position.
9. Remove the slide from the stage. Using lens paper (not bibulous paper) daub the oil from
the oil objective, and wipe the oil off the slide. Put the slide back in the proper tray.
10. Repeat with another prepared slide if you desire.
Activity 3: Cleaning a slide for making a wet mount.
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Obtain a slide from your drawer. Make sure the slide does not have broken edges.
Go to one of the two main lab sinks.
Wet the slide and your fingers.
Shake some Bon Ami cleanser onto your fingers.
Scrub the slide with your fingers and cleanser to get rid of any grit on the slide.
Remove the cleanser by rinsing with tap water.
Soak the slide in a Coplin jar full of isopropyl alcohol (one minute). This will remove any
oil that may remain on the slide.
8. Remove the slide (holding it by the edges). You may allow the slide to air dry or you may
dry it with paper towel.
Activity 4: Making wet mounts.
1. Obtain a glass cover slip from the front of the lab room.
2. Put a dollop of Vaseline in the palm of your hand.
3. Run all four edges of the cover slip through Vaseline to create a border.
Cover Slip
Vaseline
4. Put a drop of specimen (pond water, hay infusion, or other liquid specimen) on the cover
slip.
5. Place a clean slide on the cover slip. The slide should stick to the Vaseline. The Vaseline
acts as a barrier against evaporation.
6. View the slide using the scanning, low power, and high power stage objectives. DO NOT
USE THE OIL OBJECTIVE ON WET MOUNTS!
7. Make more wet mounts as time and your interests allow.
QUESTIONS
1. Of the methods for light adjustment, which is most important?
2. Roughly how large is the field of view when using the high power lens?
3. Suppose a typical bacterial cell is 2 m in diameter. In the circle below (which represents the
field of view), draw what a group of five such a cells would look like when viewed with the oil
immersion objective.