G1B1 Group no.

_____ Date ________________________

Leader: __________________________________________
Members: _______________________________________ _________________________________________
_______________________________________ _________________________________________
PRELAB ACTIVITY
Basic Microscopy
An Important Skill for Laboratory
OBJECTIVES:
To further equip the students the following objectives as set forth. Primarily, this activity aims the students to become
familiar with the components of microscopes commonly used by Biologists. These likewise aim that student become
familiar with the:
1. proper care and maintenance of microscopes.
2. steps for proper focusing on material and observation with a microscope.
3. determination of the magnification of materials being observed using a microscope.

INTRODUCTION:
You are about to proceed on the adventure of a lifetime. A microscope is one of the most useful tools a
biologist has when trying to identify the causal agents of diseases. Sometimes they will provide you with information
that will allow you to know exactly what is causing a disease. They may be important in other cases in determining
that certain pathogens are not causing a problem.
Proper microscope use is one of the most important skills that a beginning laboratory student can learn.
Microscopes were developed during the late 17th century and continue to be important in identifying microscopic
specimen and other causal agents of diseases. Although microscopes often are introduced to students early in their
science classes, students frequently do not learn how to use and care for microscopes. In fact, in many classes
microscopes are set up by someone else and students are told to not change anything other than possibly the focus.
This teaching aid provides basic information and instructions on how to set up, use, and care for microscopes,
essential skills for biologists.
Compound microscopes are used for the observation of smaller specimens which are placed on microscope
slides and topped with a cover slip. Such specimens should be transparent or translucent because light must be
transmitted through the specimen to reach the lens of the microscope. Magnifications commonly found on compound
microscopes are between 10x and 1000x.

Components of this exercise include: MATERIALS:

Parts of microscopes Compound light microscope
How to move and transport microscopes properly Microscope slides and cover slips
Determination of magnification Scissors
Adjustment of eyepieces Dropper bottle with water
Placement and focusing of specimen slide Immersion oil
Orientation of materials on a slide

PROCEDURES:
Parts of Microscopes. You should familiarize yourself with the different parts of the microscopes before using them
because you will need to know where the different parts can be found on your microscopes. Some of these
components may be located in slightly different positions on different models of microscopes. Take a few minutes to
label the Figure 1 before proceeding. The major components of the compound microscopes and their purposes are
outlined in Table 1.
 Fig.1

GUIDE QUESTIONS:

1. What components are objective lenses present on the compound microscope? Identify the total magnification for each.
objective lenses total magnification
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2. What is the maximum magnification which you can obtain with your compound microscope?

With use of oil? ____________________________

Without use of oil? ___________________________

Table 1. Major components of microscopes and their general description and purpose.

PARTS OF THE MICROSCOPE

I. Illuminating Parts- parts that need light for illumination, regulate/control the light entering the microscope.
1. Mirror - located below the stage used to catch the light and direct it to the object being studied. The mirror has one flat or plane
surface (used under natural light) and one concave mirror (used under artificial light).
2. Iris Diaphragm - with a small lever at one side of the condenser which opens and closes a screen of metal plates. It is used to
regulate the amount of light entering the microscope.
3. Condenser – located immediately below the stage. Used to concentrate or focus the light coming from the diaphragm for the object
found on the stage.
II. Magnifying Parts- for enlargement of objects.
4. Ocular or Eyepiece – contains lenses (eye lens and field lens). Fits into the top of the draw tube. The eyepiece of most microscopes
has a magnification of 10. the eyepiece may contain a short hair which can be used as a pointer, as it appears as a black line.
5. Objectives – small tubes containing lenses screwed into the nosepiece which magnifies the object being examined. A number
followed by an x, stamped on the objective, indicates the magnifying power.
a. Lower Power Objective (LPO) – is the shortest tube stamped 10x affording the lowest ocular magnification.
b. High Power Objective (HPO) – is the longer tube marked either 43x or 45x, sometimes 40x.
c. Oil Immersion Objective – same length as the high power objective but affording a much higher magnification being
stamped 97 or 100x. Needs a special oil to be placed on object studied.
III. Mechanical Parts – the framework
6. Draw tube – the tubular, upright portion attached at the top of the arm; this is where the ordinary eyepiece or ocular is inserted.
7. Body tube – found immediately below the draw tube, thus, serving as connection between the ocular inside the draw tube and the
objectives found at the other end of the body tube. The body tube is the carrier of optical parts of the microscope.
8. Coarse Adjustment Knob (Pinion Head) – the large wheel with milled edges used with the LPO for locating the outline of the
specimen. Rotation moves the tubes upward or downward.
9. Fine Adjustment Knob – the smaller wheel with milled edges used to see the finer detail of the object. Rotation provides a delicate
control of the tube movement
10. Dust Shield – dark metallic disc located just above the revolving nosepiece used to prevent dust from entering the objectives.
11. Revolving nosepiece – located at the lower end of the body tube serving for attachment of objectives and to facilitate the shift from
one objective to another. When an objective is in proper alignment below the body tube there is a faint click.
12. Arm or Handle – the C-shaped pillar rising from the stage or base used for carrying the microscope.
13. Stage –the platform attached to the arm where the glass slide containing the specimen is placed
14. Stage clip/ spring clip – found on the stage used for securing the slide in position.
15. Inclination joint – located between the pillar and the arm and enables one to tilt the upper part of the microscope to a desired position
for convenience.
16. Pillar – a very short connection between the base and the rest of the microscope.
17. Base – the U-shaped or horse-shoe shaped iron portion on which the microscope rests. It supports the entire apparatus.

How to Move and Transport Microscopes Properly

Microscopes are often stored in cabinets when they are not in use and must be removed in
order to use them. To remove microscopes from the storage area, place one hand completely
around the arm, and then place your other hand underneath the microscope base. Always
carry the microscope in an upright position. Carrying in any other way may allow parts to
fall from the microscope. Use care to ensure that electrical cords are not entangled with
those of other microscopes. Place microscope on a clean area of the desk or laboratory
bench.

Light sources are required for most microscopic observations. The cord for the light should be plugged into a suitable
outlet. Dissecting microscope lights are often a unit separate from the microscope. Such lights may be attached to the
arm to illuminate the stage from above the sample. Alternatively, light sources may be placed next to the stage at the
level of the sample, or below the stage.

Determination of Magnification

Magnification is a measure of the ability of the microscope to enlarge an image. Resolution is a measure of the ability
of the microscope to separate different points of the image. Determining magnification is vital when comparing the
sizes of different objects being viewed with a microscope. Note the magnification of the eyepieces and the different
objectives on your microscope. The magnification is printed or etched on the side of the objective and on the side or
top of the eyepiece. This magnification is shown as a number followed by an x (e.g. 15x).

Compound microscopes often have objectives which are designed strictly for use with immersion oil. These
objectives are identified by having the word "oil" engraved on the side, near the number stating the magnification of
the objective. Oil objectives cannot be used without immersion oil. Other objectives cannot be used with oil and can
be damaged if inadvertently immersed in oil. The use of an oil immersion lens is essential when viewing structures
less than 10 µm in size. For example, magnification requiring an oil immersion lens is necessary to determine the
shape of an individual bacterium. Oil immersion does not increase the magnification of the lens, but it improves the
resolution or sharpness of the image produced by the objective. When light passes through any material to another,
such as from glass to air, the light is refracted or bent. Light of different wavelengths bend at different angles. Such
refraction results in distortion which can be significant at higher magnifications. By putting a drop of immersion oil,
which has the same refractive index as glass, between the 100x objective and your slide you significantly reduce the
light scattering that would otherwise occur, thereby increasing the resolution of the image.

To determine the magnification of any image, you will need to multiply the eyepiece magnification by the objective
magnification. If an auxiliary lens is present on a dissecting microscope, its magnification must be multiplied times
the objective and eyepiece magnifications. It is important to note the magnification on all drawings so that you can
compare the relative sizes of images that you observe at various times. In many cases you will need to use two or
more different magnifications to view all the details of a sample.

Adjustment of Eyepieces

Several adjustments are necessary before looking at specimens. On binocular microscopes (microscopes with two
eyepieces) the distance between eyepieces should be adjusted so that you can look through both eyepieces easily at
the same time and see a single image. There are several different methods of adjusting this distance depending on the
microscope being used. In some microscopes there is a knob at the top of the microscope between the eyepieces that
adjusts the eyepiece distance (Figure 5A). Other microscopes are adjusted by pushing on the eyepieces (Figure 5B).
This adjustment is specific to each person so minor adjustments may be required when one person looks through a
microscope set up by another person. Make this adjustment before continuing. Adjusting for differences between a
person's eyes using the focusing sleeve on left or both eyepieces is another important adjustment to prevent
discomfort and fatigue of the microscope user. The procedure for making this adjustment follows the initial focusing
since a specimen has to be observable for making the adjustments.

Orientation of Materials on a Slide

Making accurate drawings of your observations can be extremely important. These drawings will be especially
important when you are making comparisons of drawings made at different times. It may be advantageous to draw a
circle representing your field of view and then add a grid. The circle and grid will assist you in drawing details in the
correct place and size. Make sure to note the magnification at the bottom of your circle.

3. If you need to look at the material on the left side of your image which way do you need to move the slide?
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4. If you need to look at the material at the top of your image which way do you need to move the slide?
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5. Why is this important?

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 6. What do you mean by parfocal?

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ILLUSTRATION:

Provide illustration(s) on the specimen used for this microscopy activity.

________________________________ ________________________________

To increase the magnification you should proceed as follows: Make sure that your sample is in focus with
lower power objective.

While observing the objective to make sure it clears the slide holder and slide safely, turn the turret containing
objectives to next higher power objective and make sure it is in proper position over the sample. (Most turrets have
positions which the objectives will snap into indicating that they are in the correct position. If the light is not
observable or the circle of light is not centered make sure the turret is in the correct position.)

Use the fine focus to sharpen the image. You should not have to use the coarse adjustment.

Special Note: Be sure not to get oil on other objectives because it can damage them.

When you finish with an oil immersion objective raise the tube and wipe oil off the objective immediately and
remove the slide with oil. Clean oil off slides if they are to be used further or are permanent laboratory slides.
Objectives may be cleaned with lens paper and distilled water. Use only lens paper for cleaning objective because
other materials (Kimwipes, paper towels, etc.) will damage them.

Any time you are ready to remove a slide, always raise the tube prior to removing the slide to ensure that you don't
inadvertently damage an objective by contacting it with the slide.

After you have finished using your microscope for the day, care should be taken in properly storing it. Make sure that
objectives are clean and slides are removed. Turn off all lights. Unplug electrical cords and loosely wrap around the
base. Remove lights that are associated with dissecting microscopes. Place all microscopes and lights carefully back
into their proper storage area. Instructors may provide further instructions as appropriate.

References:
Riley, M. 2003. Basic Microscopy - An Important Skill for Plant Pathologists.The Plant Health Instructor. DOI: 10.1094/PHI-I-2003-0130-02
Melissa B. Riley, Department of Plant Pathology and Physiology, Clemson University