Introduction to the Microscope
Copyright ©2009by Gary Fromert
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1. Demonstrate an understanding of the various parts and usage of a compound
microscope.
2. Demonstrate an understanding for the handling and care of a compound microscope.
3. Observe specimens on permanently mounted slides with proficiency.
Since an unaided eye cannot detect anything smaller than 0.1 mm in diameter such things
as dust particles, animal and plant cells, and many small organisms are beyond our visual
capability, so we need equipment to magnify objects which are too small to be seen with
the unaided eye. Magnification allows us to see details that we would not see with the
unaided eye. Magnification is the enlarged view of an object by use of an optical
instrument. A microscope is an optical instrument that allows us to see these objects by
magnification.
A simple microscope is one that uses only one lens to magnify, such as a magnifying
glass. In microbiology, the organisms are too small to be magnified by a simple
microscope containing one lens. By combining two or more simple lenses in series, a
compound of lenses, we are able to observe microorganism with detail. The
compounding of these lenses along with appropriate housings and mechanical
movements for focusing creates a compound microscope. Thus the compound
microscope sometimes called the student microscope or light microscope is a microscope
that contains at least two magnifying lenses in series. One magnifying lens is in the
ocular or eyepiece, which when combined with a second magnifying lens, the objective,
compounds the magnification of the image being observed. Each lens contributes to the
magnification of the object on the stage of the microscope. The total magnification of any
set of lenses is determined by multiplying the magnification of the objective lens by the
magnification of the ocular lens. Generally a compound microscope magnifies from 40
times (40X) to 1000 times (1000X) that of the actual specimen.
The compound light microscope is a delicate instrument composed of many parts that are
accurately fitted together (Figure 1). Some common parts and features of a compound
light microscope are:
1. Ocular Lens
The ocular lens or eyepiece lens is the lens you look through, it is inserted at the top of
the body tube. If your microscope has one ocular, it is a monocular microscope, if it has
two, it is binocular microscope. The most common magnification of the ocular eyepiece
is 10X and is usually printed on the eyepiece. Binocular microscopes also have a diopter
adjustment. When you look through a microscope with two eyepiece lenses, you must be
able to change the focus on one eyepiece to compensate for the difference in vision
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between your two eyes. The diopter adjustment does this. The way to correctly adjust this
is to first close the eye over the eyepiece with the diopter adjustment and normally focus
the microscope so that the open eye sees the image in focus. Next, switch eyes (close the
open eye, open the closed eye) and without changing the main focus knobs, focus on the
image by turning the diopter adjustment only. Now with both eyes open, the image
should be clear with both eyes.
Figure 1. The compound light microscope. (http://www.saskschools.ca)
2. Body Tube
The body tube is the optical housing for the objective lenses.
3. Objective Lenses
The objective lenses are a set of three to four lenses mounted on a rotating turret call the
revolving nosepiece at the bottom of the body tube. The magnification of the objective
lens is printed on the lens. The four objective lenses of your microscope and their
magnifications are:
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Magnification
Objective Lenses
Ocular Lens
4X - Scanning
10X
10X - Low Power
10X
40X - High Power
10X
100X - Oil Immersion
10X
Total Magnification
Objective multiplied by ocular
40X
100X
400X
1000X
Compound microscopes consist of two lens system: the objective lens, which magnifies,
and projects a “virtual image” into the body tube and the ocular lens, which magnifies the
image further and projects the enlarged image into the eye. The total magnification of a
microscope is the product of the magnification of the objective lens and the ocular lens. If
the objective lens has a magnification of 40X and the ocular lens 10X, then the image
produced by these two lenses is 400 times larger than the specimen being observed.
Note: With the exception of the oil immersion lens all the objective lenses are used dry.
The magnification of oil immersion lens requires using the lens with special immersion
oil for proper resolution. A drop is placed upon the cover slip and the objective is
lowered until it just touches the drop. Once brought into focus, the oil acts as a "light"
bridge between the glass slide and the glass in the lens. This concentrates the light path
and increasing the resolution of the image.
4. Stage
The horizontal surface on which the slide is placed is called the stage. It may be equipped
with simple clips for holding the slide in place or with a mechanical stage, a geared
device for precisely moving the slide. Two knobs, either on top of or under the stage,
move the mechanical stage in the x-axis and the y-axis directions.
5. Condenser Lens
The condenser lens system (not show in Figure 1), located immediately under the stage,
contains a system of lenses that focuses light on your specimen. The condenser may be
raised or lowered using the condenser knob.
6. Iris Diaphragm
The iris diaphragm is located below the condenser or immediately below the stage in
microscopes without a condenser. It functions in regulating the light intensity passing
through to the stage. More light is required at higher magnification.
7. Projection Lens
Most microscopes have a built in light source which has an ON/OFF switch and may
have adjustable lamp intensities and color filters. The projection lens concentrates and
projects the light into the iris diaphragm.
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8. Base
The base is the bottom support on the microscope and is used along with the body arm
when carrying the microscope.
9. Arm (Body Arm)
The body arm connects the body tube to the base and is used when carrying the
microscope in conjunction with the base.
10. Nosepiece
The nosepiece is the mounting for the objective lenses which rotates to bring the desired
objective into position.
11. Coarse Focus
The coarse focus adjustment knob is a large knob located at either side of the microscope
which functions in controlling the distance between the objectives and the stage. Use the
coarse adjustment only with the scanning (4X) and low power (10X) objectives. The
coarse adjustment is used for rapid focusing of the specimen until the specimen is
roughly in focus and then left alone, in which the fine adjustment knob controls precise
focusing of the specimen being observed.
12. Fine Focus
The fine focus adjustment is a small knob located at either side of the microscope. This is
used for the control of the stage, for precise focusing you should use just the fine
adjustment knob with the higher magnification objective lenses. Because using the coarse
adjustment knob with the higher objective lenses may damage the lens and/or the slide
you are observing.
Microscope safety precautions:
 Always carry the microscope in an upright position using both hands, one under the
base with the other hand grasping the arm of the microscope.
 Keep the microscope away from the edge of the lab bench.
 Always examine a slide first with the scanning or low power objective, never use the
high power or immersion oil objective to view thick specimens.
 Remove the slide only after the scanning objective has been rotated into viewing
position, never when a higher power objective is in position.
 Keep the stage dry at all times. A wet stage will prevent the slide from being accurately
positioned.
 When returning your microscope to its proper place in the cabinet always:
 Remove the slide from mechanical stage.
 Clean all lens surfaces and the stage.
 Rotate the nosepiece so that the scanning lens is in the viewing position.
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Procedure for cleaning and storing a microscope:
 Turn off the light.
 Using the coarse adjustment knob, lower the stage to obtain maximum working
distance and remove the slide from the stage.
 Using only lens paper, clean all the ocular and objective lenses. Do not use anything
other than lens paper to clean the lenses on the microscope, as any other type of paper
may scratch the optics.
 Clean any oil off of the stage using paper towels.
 Rotate the scanning objective into place. Use the coarse adjustment knob to obtain
minimum working distance.
 Return the microscope to the appropriate storage area.
Procedure for cleaning a microscope slide:
 Before placing a specimen on a slide, it must be clean, as any small foreign body might
mislead the observation. If your slide is not clean, do the following:
 Hold the slide from its ends the fingers of one hand.
 Apply a detergent liquid to the slide; rub the surface of both sides of the slide with the
detergent with one finger of the other hand.
 Wash the slide under running tap water; rub again, until no trace of the detergent is left.
 Rinse the slide with distilled water to remove the tap water.
 Blot dry the slide by placing it between two pieces of paper towel.
 Never touch the slide in the middle, always holds it from its ends.
Preparation of microscope slide:
Two types of slides are commonly used in microbiology labs: permanent slides and
temporary wet mounts.
Permanent slides
Permanent slides have been professionally prepared. They have often been stained to
show better contrast of structure. A permanent slide may be a whole mount of the entire
specimen or section of the material.
Wet mounts
 Wet mounts are temporary slides that you prepare yourself. When doing a wet mount
follow the procedure outlined below:
 Place the specimen (mixed culture, tissue, etc.) on the center of a clean slide.
 Add a drop of water or designated stain if required. (Note: liquid cultures do not require
adding water).
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 Place one edge of the cover slip on the slide near the specimen. (This is done by
holding the cover slide at a 45ºangle). Gently lower the cover slip on top of the specimen.
 Try to avoid trapping air bubbles.
 Blot any excess fluid with bibulous paper or paper towel before you place the slide on
the stage of the microscope.
 After you have made your observations the slide and cover slip should be washed and
dried.
Materials (per student)
1 Compound Light Microscope
1 Newspaper "e" Mounted Slide
1 Thread Mounted Slide - contains three different colored threads: red, yellow and blue
Lens Paper
Procedure
Viewing a slide:
 Obtain a permanent mounted slide.
 Check that the ocular and all objective lenses as well as the slide clean.
 Use the coarse adjustment knob to obtain maximum working distance.
 Place the slide on the stage, the slide should fit into the slide holder. Use the stage
adjustment knob to move the slide over the hole in the stage.
 Rotate the lower objective in place.
 Use the coarse adjustment knob to obtain the minimum working distance.
 Look through the ocular. Adjust the light with the iris diaphragm lever if necessary.
Slowly turn the coarse adjustment knob until something comes into focus. Use the fine
adjustment knob to sharpen the focus.
 Using the stage adjustment knob move the slide around until you find an area you wish
to examine more closely. Move the slide until the object you wish to examine is in the
center of the field.
 Rotate the high power objective in to place. Use the fine adjustment knob to sharpen the
focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm
lever if necessary.
 Rotate the high power object halfway to the next position, place a drop of immersion oil
on the slide, and then rotate the oil immersion objective into place. The objective should
be immersed in the oil on the slide. Use the fine adjustment knob to sharpen the focus.
Adjust the light using the iris diaphragm lever if necessary.
 When finished observing the slide use the coarse adjustment knob to maximize the
working distance and remove the slide from the stage.
 When you are finished with the microscope clean it and return it to storage.
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Observations and Interpretations
Using the Newspaper "e" mounted slide, draw the "e" as represented in the field at the
specified magnification as observed in the ocular.
"e" at 40X
"e" at 400X
Laboratory Review
1. Based on your observations, what does each of the two different magnifications
provide in terms of detail for the "e" as observed at 40X and 400X?
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2. How do you determine the total magnification of a set of lenses?
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3. What objective should always be in place, both when beginning to focus and when
preparing the microscope for storage?
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4. What part of a compound microscope houses the objective lenses?
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