Microbiology Lab 1

The Light Microscope

Purpose

1. Identify and describe the functions of the light microscope's parts.

2. Perform brightfield microscopy to view specimens.
3. Use the high-power oil-immersion lens to view prepared specimens.
4. Calculate final magnification.
5. Name three other examples of light microscopy aside from bright-field microscopy.
6. Discuss light microscopy's clinical applications.
7. Describe the basic differences between prokaryotes and eukaryotes
8. Name the three domains and six kingdoms of taxonomy.
9. List the taxonimc hierarchy categories from broadest to most specific.
10. Correctly write a scientific name.
11. List several features of kingdom Fungi and name some representative members.
12. List several features of kingdom Protista and name some representative members.
13. Define the term protozoan and name the four main groups of protozoans.
14. State what feature is primarily used to group protozoans into their four main categories.
15. Descrive the features of select protists viewed on premade and / or wet mount slides.

Introduction

Microbiology is the study of microorganisms too small to see with the naked eye,
however, they can be seen with the aid of a microscope. Antony van Leeuwenhoek, a Dutch
lens maker, was the first to see microbes under a microscope. He identified small organisms
which he called “animalcules”.
The earliest microscopes were simple; a specimen was placed on a pin point and viewed
through a single lens using sunlight. Today’s compound light microscopes use a quartz-halogen
lamp light source and multiple lenses to view a specimen on a slide or other surface. The
microscope you will use has 4 objective lenses (4x, 10x, 40x, and 100x), a binocular 10x lens, a
movable stage, a sub-stage condenser with diaphragm to control light intensity, and a halogen
light source.

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A light microscope allows us to view objects that are as small as 0.2μm (micrometers,
microns). The two properties of the microscope that determine the quality of the image are
magnification and resolution. Magnification is the size of the image we view relative to the
actual size of the object viewed. The magnification is the product of both the objective lens and
the ocular lens. Total magnification on your microscope ranges from 40x (4x objective X 10x
ocular) to 1000x (100x objective X 10x ocular). When viewing an image through the
microscope, the magnification is always specified. Resolution is the ability to separate two
closely spaced objects. Resolution is dependent on the wavelength of light used, the quality of
the lens, refraction and reflection of light as it passes through the object and the air. Resolution
at high magnification (1000x) is enhanced by the use of immersion oil which, when placed
between the slide surface and the lens, prevents refraction of the light. When viewing an
image through the microscope, the magnification is always specified.
Basic Techniques
Carry the microscope with two hands. The lab microscopes have a handle in the arm above the
receptacle for the power supply. Lift using the handle and placing your other hand under the
base. Carry the microscope upright to prevent dislodging loose parts.
Clean the microscope lenses with lens paper only. Other papers or cloths may scratch the lens
or leave fibers behind on the lens. Lens paper is absorbent and will remove oil from the lens.
Parts of the microscopes
Eyepiece (Ocular lens, 10x): The lab microscope is a binocular microscope; objects can
view with both eyes. Adjust the distance between the oculars by opening or closing the gap
between them until you can see equally through both. One of the oculars will have a pointer
which can be moved in a circle to point out particular objects.
Objective lenses: There are 4 objective lenses. The objective lenses are held in place by
the nosepiece. The nose piece allows you to change magnification by rotating an objective into
place. Because the eyepiece and objective lenses are co-focal, an object under the 40x lens will
have a total magnification of 400x. To view objects at 1000x, focus the object at 400x then
rotate the 40x objective to the side, add a drop of immersion oil to the slide over the object,
and carefully rotate the 100x objective into place and into the pool of oil.
Stage: The stage is below the objective lens. The microscope is equipped with a
mechanical stage which can be moved both left -- right and up --down in the field of view. Note
that the direction of stage movement will be opposite of the direction observed in the field.
The knobs used to move the stage are located below the stage on the right side. When placing
a slide on the stage, the stage should be in the lowest position. The slide is placed in the slide
clip which holds it in place. Be certain that the slide is laying flat on the stage and does not
have an edge below the surface of the stage as this might damage the condenser lens.
Condenser: Below the stage is the condenser, which focuses the light on the specimen.
The condenser can be moved up and down using a knob on the left base of the stage. Most
observations of bacteria require the condenser at the highest point. On top of the condenser is
the iris diaphragm, a lever which adjusts the amount of light illuminating an object. The on/off
switch, also, controls the amount of light passing through the specimen. For viewing at low

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magnification, the amount of light should be low while viewing at high magnification requires a
large amount of light.
Focus knobs: Coarse adjustment knob is the large calibrated knob located on the left
side of the microscope. The coarse focus moves the stage up and down. Focus will be close to
the highest position of the stage. Starting with the 4x objective in place, focus on the specimen
by moving the stage to the top position and then adjusting while looking through the eyepiece.
Sharp focus will require the fine focus knob. The Fine focus knob is located inside the coarse
focus on the left and is the large knob on the right side of the arm. Only fine focus should be
used at 400x or 1000x to prevent scratching the lens or breaking the slide.

Materials
Per person:
Microscope
Prepared slides - Spirogyra, Bacteria, various Eukaryotes

Procedure

1. Obtain a microscope from the storage cabinet.

2. Remove the cover and then identify all the parts of the microscope. Move the various
knobs and observe the effect.
3. In the results section of your notebook, record the color and magnification of each of
the objective lenses; calculate the total image magnification for each objective.
4. Plug in and turn on the microscope. Adjust the light to the appropriate amount for the
lens being used.
5. Obtain a slide of the algae Spirogyra from your instructor and place it, right side up on
the stage and in the clip. Record the name of the specimen.
6. Using the 4x objective, focus on the object on the slide. Draw what you see, include
the total magnification. Make your drawing large enough to see all the details visible in
the field of view.
Total magnification = Ocular magnification (10) X Objective magnification
7. Rotate the 10x objective into place and focus. Draw what you see, include the total
magnification. Notice how much larger the specimen appears.
8. Rotate the 40x objective into place and focus with fine focus knob. Draw what you see,
include the total magnification. Make your drawing large enough to see all the details
visible in the field of view. Notice that the size of the object is larger and the field of
view is smaller than the two previous views.
9. Rotate the 40x lens to the side and add a large drop of immersion oil to the slide where
the light is coming through the slide. Now rotate the 100x lens until it clicks into place.

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Focus with fine focus knob only. Draw what you see, include the total magnification.
Note that you can see only a portion of each of the large cells!
10. Calculate the resolution in μm for each of the drawings. Use the equation:
Resolution = λ/2(NA)
Where: λ = the wavelength of light (0.500μm)
NA = the Numerical Aperture (printed on the lens with the magnification:
4x=0.10)
11. Clean the immersion oil off all slides and return to the proper slide tray.
12. Obtain a slide of a bacterial specimen, repeat steps 6 through 9.
13. Clean the immersion oil off all slides and return to the proper slide tray.
14. Clean the three lenses of your microscope, using lens paper, and return the 4x objective
to the down position. Remove any immersion oil from microscope surfaces. Turn off the
microscope, rewind and secure the cord with the Velcro strip, replace the power
adapter in the arm receptacle, cover and place in the microscope cabinet.