Name:
BIOL 191 Introductory Microbiology
Microscopy PreLab
All Prelab reports are due when students initially come into the lab, before lab instructions.
The purpose of the PreLab is to encourage students to read the exercise before coming to lab,
thereby increasing students’ understanding and facilitating the flow of lab activities. Therefore,
PreLabs are not accepted if a student is absent or tardy (meaning arrival after the lab instruction
has begun).
1. (0.5pt) Each student is assigned a numbered microscope. That specific microscope must be
used every time by that student.
a. True
b. False
2. (0.5pt) Each individual student must read and complete a Student Microscope Checklist each
time a microscope is used.
a. True
b. False
3. (4pt.) Label the microscope parts below.
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12/1/14
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LAB EXERCISE: MICROSCOPY
Only objects 0.1mm and larger can be visualized by the human eye. Because most
microorganisms are much smaller than 0.1mm, a microscope must be utilized in order to
directly observe them. In general, the diameter of microorganisms ranges from 0.2 - 2.0
microns.
A light microscope, which uses light as a source of illumination, will be employed in
this lab. There are several types of light microscopes. The type used in this course is a
brightfield microscope, where the specimen appears darker against a bright background.
Resolving power
The resolving power (resolution) is a very important characteristic of a microscope and
different from magnification. Resolving power is the ability of the lenses to differentiate
between two adjacent objects. Objects that are 0.2 microns (or farther) apart will be seen as
two separate objects. Therefore, the resolution determines the amount of detail that can be
visualized.
Read ALL of the following BEFORE obtaining a microscope.
1. Students must be assigned a specific microscope he/she will use throughout the
semester. The scopes are labeled by number.
2. EVERY time a microscope is used, each student must record their scope number on the
‘Student Microscope Checklist’.
3. Scopes are found in main microscope cabinet at the front of the lab and in the far right
cabinet under the lab supplies bench at the rear of the lab.
4. These microscopes are large, heavy, and expensive. Always use both hands when
carrying the microscope. One hand holds the arm while the other hand supports the
base.
5. Always carry the scope in an upright position. Do not bump the scope while removing
it from and returning it to the cabinet. Always remove the specimen slide before moving
the microscope.
6. Always take care when removing the dust cover. After removal, check the inside of the
dust cover for parts that may have come off.
7. Follow cleaning instructions carefully (See Preparation for viewing).
8. If using, do not let the electric cord dangle off of the table or become entangled when
moving the scope.
9. Always be sure the stage is at its lowest level (by rotating the course adjustment knob
counterclockwise until it is at its lowest position) before placing a slide on the stage and
while cleaning the objectives after use.
10. Always start with the lowest magnifying objective (10X with bacteria).
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* The observation head with the eyepieces may be oriented toward the front of the scope
or toward the back of the microscope (the opposite orientation from the above picture).
**Any mention of ‘right and left’ sides of the scope refers to the scope when viewing from
the back of the scope.
Parts of the light microscope
1. Stand: Arm and base.
All other parts of the microscope are attached to the arm or base.
2. Mechanical stage: The clamping device that secures and moves the slide while it rests on
the stage.
3. X-Y mechanical stage control knobs: These knobs, under the left side of the stage, move
the slide horizontally (lower X knob) and vertically (upper Y knob). Never force any knobs.
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4. Lamp.
The lamp on/off switch is located in the rear on the base. The light intensity is adjusted with
the light intensity regulator on the right side of the base. The lamp should be adjusted to a
medium level at the start of viewing.
5. Lens systems:
There are three lens systems: the eyepieces (ocular), the objectives (four), and the condenser.
a. Eyepieces (ocular): Magnification of 10X. The eyepieces are held rather
loosely in the eyepiece tubes. Never remove the eyepieces from the eyepiece
tubes. A rubber eye shield should be on the top of each eyepiece. The width
between the eyepieces should be moved until a full circle (the viewing field) is
visible with both eyes simultaneously. Students can record their proper setting
by reading the scale between the eyepieces.
b. Objectives: There are four objectives: 4X, 10X, 40X, and the 100X (oil
immersion objective). The objectives are attached to a rotatable nosepiece.
The total magnification is calculated by multiplying the
ocular magnification and the magnification of the objective in use.
c. Condenser: The condenser is located directly beneath the stage. It gathers and
conducts the light to the specimen. The condenser height adjustment knob (on
the right side of the scope arm below the stage) should be moved clockwise until
the condenser is in the highest position. If the field of view is not uniform
enough, it may be improved by lowering the condenser slightly.
The iris diaphragm controls the amount of light from the condenser and is
adjusted with a thin lever under the stage.
6. Focusing knobs: located on the sides of the microscope.
The larger coarse adjustment knob moves the stage up and down much faster and farther than
the smaller fine adjustment knob. The coarse adjustment knob is used ONLY with the lower
power (4X, 10X) objectives. When focusing under the 40X or 100X objective, ONLY use the
fine adjustment, never the coarse adjustment.
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Preparation for viewing- Refer to each of the following procedures every time the
microscope is used. Read FIRST, then perform the step.
1. If you have not done so, carefully remove a scope from the cabinet and set it on your lab
bench. Remove the dust cover. Check that no parts are loose or missing). They might be
in the dust cover. Immediately contact the instructor if parts are missing or anything is
wrong with the scope. Write down the number of your scope (found on the front of each
scope) on the microscope checklist at the end of the lab exercise instructions.
2. Plug in the microscope. Be sure the electrical cord is not dangling off of the lab bench and
cannot become entangled.
3. Use the coarse adjustment knob to move the stage to its lowest level.
4. Cleaning the microscope. Clean all objective lenses with swabs and liquid lens cleaner.
Place a small amount of liquid lens cleaner on the swab. Use a fair amount of pressure to
clean the lens in a circular pattern. Dry the lens with the dry, unused end different swab.
Use different swabs for each lens.
Do not use liquid lens cleaner on the eyepieces. Dust on the eyepieces, or elsewhere, may
be removed using the blower brush. NEVER remove any parts.
5. Before use, clean all slides, top and bottom, with Kimwipes. Place a coverslip over the
specimen on the slide. Do not use a coverslip if viewing prepared slides.
6. Rotate the nosepiece until the 4X (10X with bacteria) objective clicks into place.
Turn on the lamp. Adjust the on/off switch to a medium intensity.
7. Gently place the slide on the stage so that it is held within the mechanical stage clamping
device. The bow-shaped lever of the mechanical stage clamping device should be pulled
outward and released gently after the slide is placed on the stage within the clamping device.
The lever should not be released in the middle or the slide could be broken. The slide must lie
flat on the stage.
8. Using the mechanical stage X-Y movement control knobs, position the slide so that the
specimen is in the exact center of the light coming through the condenser. Do not use your
hands to move the slide; only use the X-Y control knobs.
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Viewing and focusing the specimen under the 4X, 10X, and 40X objectives
1. Always start with a low power objective (4X or 10X when viewing bacteria) clicked into
place.
The lower power objectives have the largest field of view (a larger portion of the slide can be
seen), making it easier to initially find the specimen.
2. Only now, with a lower power objective in place, move the stage to its highest point by
rotating the course adjustment know clockwise. In other words, the stage should be moved as
close to the objective as possible.
3. Optimal viewing by adjusting the eyepieces while focusing (steps
a. Inter-pupillary distance: While looking through the eyepieces, adjust the width between the
eyepieces (inter-pupillary distance) until a single image is seen simultaneously with both eyes.
The left and right fields of view should coincide completely. The position of the dot indicates
the inter-pupillary value. Note your inter-pupillary value.
b. 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
between your two eyes. The diopter adjustment does this.
1) Rotate the right eyepiece to match your inter-pupillary distance value.
2) Close the eye over the eyepiece with the diopter adjustment (left eye), and normally focus
the microscope so that the open right eye sees the image in focus.
3) Next, switch eyes (close the right eye, open the left eye) and without changing the main
focus knobs, focus on the image by turning the diopter adjustment ring on the left eyepiece
only. Now with both eyes open, the image should be clear with both eyes.
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c. Eye Shades
When wearing eyeglasses: Use the eye shades in the normal,
folded-down position. This will prevent the eyeglasses from
being scratched.
When not wearing eyeglasses: Extend the folded eye shades
outwards (direction of the arrow) to prevent extraneous light
from entering into your line of vision.
4. Light control
VERY IMPORTANT!!!!!
Light intensity is a essential aspect of microscopy. Perform the following steps to adjust the
light. For optimal viewing, the light must be adjusted several times while focusing. Always
adjust the light while looking through the eyepieces.
a. Adjust the light intensity switch that turns the lamp on. Begin at a medium level.
Adjust again while focusing.
b. The entire viewing area (field) must be filled with light. If this is not the case,
contact the instructor.
c. Locate the thin, iris diaphragm lever under the stage condenser. Adjust this lever to
a medium/low light level. The iris diaphragm will need to be adjusted as magnifications
increase and focusing continues.
5. Under low power, SLOWLY focus with the coarse adjustment knob counterclockwise,
moving the stage down, until the specimen comes into view. Adjust the light as instructed in
step 3 above.
Many specimens, especially bacteria, are very small and may look like specks at this
magnification. If the stage moves too quickly, you may go past the specimen without seeing it.
Look for ‘color’ since the specimens are stained.
6. Refine the image with the fine adjustment knob and by adjusting the light.
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7. Important! Before switching to the next objective, move the slide so that the desired
specimen is located in the center of the field (circular viewing area).
8. SIGNIFICANT POINTS WHEN FOCUSING UNDER THE 4X, 10X, AND 40X
OBJECTIVES!
a. Always start with a low power objective (4X or 10X). After focusing and making
appropriate observations, rotate the nosepiece until the next objective clicks into
place. Do not skip objectives.
b. Do not move the stage up or down before rotating the nosepiece to the next
objective. When properly focused, there is no need to adjust the objective's distance
from the stage before increasing the magnification.
c. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING
UNDER THE 40X OBJECTIVE! ONLY USE THE FINE ADJUSTMENT
KNOB!
d. Before increasing the magnification, always use the X-Y control knobs to move the
slide so that the desired specimen is located in the center of the field.
e. Remember to adjust the light each time the magnification is changed and while
focusing.
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100X Oil Immersion Procedure
Immersion oil is used with the 100X objective because it increases the resolution. The oil
should come in contact with both the lens of the 100X objective and the slide/coverslip. This
prevents light rays from escaping.
1. Be sure that the specimen has been optimally focused using the 40X objective and is in the
EXACT CENTER of the viewing field under the 40X objective.
2. NOTE! Again, do not move the stage up or down before rotating the nosepiece to the 100X
objective.
3. Rotate the 40X objective away from the slide but do not yet click the 100X objective
into place.
4. Put a small drop of immersion oil on the slide/coverslip directly over the light.
5. Rotate the nosepiece until the 100X oil immersion objective is clicked into place. If there are
oil bubbles, revolve the nosepiece slightly to remove the bubbles.
6. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING UNDER
THE 100X OBJECTIVE! ONLY USE THE FINE ADJUSTMENT KNOB!
6. Adjust the light for optimal viewing.
7. If problems are encountered during viewing, re-clean the objectives and repeat the
procedure. If problems persist, review the Common Problems section at the end of this
document.
9. In order to determine the size of your specimen, you will need to estimate utilizing the size
of the field (circular viewing area). When you observe your specimen, estimate its size by
comparing it with the size of the field.
Objective
Total Magnification Size of field of view
40X
400X
~0.45 mm
100X
1000X
~0.18 mm
When finished viewing and drawing your specimens, complete the Student Microscope
Checklist. Turn it in with your lab report.
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Common Problems
1. The field is dark.
Is the light on?
Is the objective securely clicked into place?
Is the diaphragm open?
Is the slide lying flat on the stage?
2. You are not sure if you are looking at dirt on the objective lens or the specimen.
Use the mechanical stage knobs to move the slide slightly while looking through the eyepieces.
If what you are looking at does not move, it is probably dust or dirt on the objective. If it does
move, it is on the slide.
Rotate the ocular gently between your fingers. If what you are looking at rotates, it is probably
dirt on the ocular.
3. You cannot find the specimen.
The stage may be too far from the objective. Did you start with the stage as close as possible to
the low power objective?
Is the specimen directly over the light?
Is the slide secure and flat in the mechanical stage?
Did you start with a low power objective and focus on the lower objectives first?
Have you adjusted the light?
Are you moving the adjustment knobs too quickly? Work slowly so you do not miss the
specimen. Remember, bacteria look like specks at low magnifications.
4. You are having trouble focusing.
Always start on a low power objective, and focus here first.
Focus SLOWLY! It is very easy to move past the specimen if the adjustment knobs are moved
too quickly.
Be sure to look in the ocular while you are focusing with the adjustment knobs or changing the
light intensity.
Adjust the light.
Press down on the stage with your fingertips. Does the stage move? If so, tighten using the
knob next to the coarse adjustment.
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5. You lose the specimen when switching from the 40X objective to the oil immersion
objective.
Was the specimen in the exact center of the field before switching to the 100X objective?
Is the 100X objective lens clean?
Have you adjusted the light?
Have you refined the image with the fine adjustment?
6. If double vision persists after adjusting the width of the eyepieces…
a. Open the iris diaphragm all the way.
b. Using your left eye only, use the 10X objective and focus on the specimen by
adjusting the coarse adjustment knob.
c. When the image is in view, refine the image to its sharpest focus by turning the fine
adjustment knob.
d. Using both eyes, rotate the right eyetube collar below the eyepiece (dioptric
adjustment) until the sharpest image appears.
e. Repeat several times to check.
8. One of the most common problems when switching objectives several times, especially
after using oil, is that the objectives need RE-CLEANING, and the student must refocus
beginning with the 10X objective again.
Assignment (see attached Lab Report):
Each student must view and draw two prepared slides.
When viewing bacteria, start with the 10X objective, and work your way up to the 100X
objective.
Call the instructor after you have focused at 100X on each slide.
Label and draw the specimens on the Lab Report.
Remember to complete the Student Microscope Checklist. Be sure to write the microscope
number of the scope you used on the Checklist. The Checklist and the Lab Report will be
returned to the instructor.
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Name:
Section:
Introductory Microbiology
Lab Report
Microscopy
1. (0.5pt.) Draw and label the two specimens after focusing with the oil immersion objective.
2. (0.5pt.) Define resolution.
3. (0.5pt.) What is the total magnification of the following?
Ocular magnification
Objective magnification
10X
40X
10X
100X
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Total magnification
4. (0.5pt.) What is the function of the iris diaphragm?
5. (0.5pt.) List two instances when the coarse adjustment knob is never used.
6. (0.5pt.) Why is immersion oil used with the 100X objective?
7. (1pt.) When should the lenses be cleaned? What is the correct way to clean them?
10. (1pt.) List two common problems associated with using the microscope and how you would go about solving
it.
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Student Microscope Checklist
When finished using the microscope, complete the following.
Check off each step when completed.
Name:
Date:
Lab Section:
Microscope Number: HCC
Bring the stage to its lowest level.
Click the 4X objective into place.
Remove the slide and dispose of appropriately. Wipe prepared slides until ALL oil/dirt is
removed (top and bottom).
Clean objective lenses with swabs and liquid lens cleaner. Dry each objective after using the
liquid.
Clean eyepieces with dry swabs.
Clean condenser lens with dry swabs.
If necessary, clean the stage with a damp Kimwipe.
Turn the light switch OFF. Do not move the microscope for a few minutes
before putting it away.
Rotate the head so that the eyepieces are facing forward (away from the arm).
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet in the appropriate postion.
Check to be sure all waste is removed from the sink and floor.
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