Laboratory Activity 2.

Magnification and Parts of Microscope

Objectives
1. Review the principles of light microscopy and identify the major parts of the microscope.
2. Learn how to use the microscope to view slides of several different cell types, including the use of
the oil immersion lens to view bacterial cells.

Introduction

The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen.
In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe
the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells
under the microscope in 1675—he described many types of cells, including bacteria. Since then more
sophisticated and powerful scopes have been developed that allow for higher magnification and clearer
images. Microscopy is used by scientists and health care professionals for many purposes, including
diagnosis of infectious diseases, identification of microorganisms (microscopic organisms) in
environmental samples (including food and water), and determination of the effect of pathogenic
(disease-causing) microbes on human cells. This exercise will familiarize you with the microscopes
we will be using to look at various types of microorganisms throughout the semester.

Light Compound Microscope

The compound light microscope is an important tool in the study of cells, tissues and
organs. It is designed to enlarge the image of these structures that may be too small to be seen by
the naked eye. It is important to know the parts and functions of the microscope, to understand its
proper use and care. There are several designs of the compound microscope, but all of them consist
of magnifying (lens system), illuminating (illumination system) and mechanical (geared focusing
system) parts.

The Lens System

The compound microscope has a lens system, described as magnifying parts, which function
to enlarge objects. It consists of two kinds of lenses: the ocular lens or eyepiece located at the
upper end of the body tube and the objective lenses located just below the body tube. The ocular
lens, inscribed with a 10x at its top side, magnifies the image of the specimen by 10 times.
Generally, there are two objective lenses attached to the revolving nosepiece that allows them to
be rotated in or out of position with reference to the slide on the stage. When rotating the revolving
nosepiece, look from the side and make sure that the lenses do not hit the stage. The shorter
objective, referred to as the low power objective (LPO), with an inscribed 10x at its side, has a
magnification of 10X. The longer objective lens, referred to as the high power objective (HPO)
may be inscribed with a 40x or 60x at its side which also indicates its magnification. If the
microscope has 3 or more objectives, these may include a scanner with a magnifying power of 4x
and/or an oil immersion lens with a magnifying power of up to 100x.

Figure 1. This shows the main parts of two types of commonly used compound microscopes.
The Illumination System
The illumination system consists of the following:
a) An adjustable mirror that reflects the light from a source into the condenser.
b) The condenser beneath the stage that concentrates light to provide proper illumination to
the field of view. The condenser may be raised or lowered using the substage knob.
c) The iris diaphragm is a thin structure with an adjustable aperture located at the mouth of
the condenser. It regulates the amount of light that enters the condenser. The size of the
aperture is regulated by the iris diaphragm lever located at the side of the condenser

Mechanical Parts
Mechanical parts function to support, hold or help in the adjustment of parts of the microscope.
The drawtube holds the eyepiece. The body tube provides appropriate distance and ensures
proper alignment between the eyepiece and the objectives. The revolving nosepiece supports the
objectives and facilitates shifting from one objective to another to achieve desired magnification
of the object under observation. The dust shield is set above the revolving nosepiece and provides
a protective covering for unused objectives, preventing dust from settling and collecting on their
surfaces.

The stage is the platform where the mounted specimen is placed for observation. It is generally
provided with a pair of clips to hold the slide in place. In some models, a mechanical stage may
take the place of the clips.

The substage supports the condenser and the mirror. It may have a substage knob that can be
used to adjust the position of the condenser between the stage and the mirror.

The arm connects the body tube to the pillar. The pillar is connected to the base and provides
major support for the microscope. An inclination joint may be found between the arm and the
pillar, and functions to allow the tilting of the microscope for comfortable viewing. When
transporting a microscope from one place to another, make sure to hold the arm with one hand and
support the base with the other hand.
Proper focusing requires the attainment of the correct distance between the objective lens and
the slide. This distance is called the working distance. Adjusting the distance between the
objective lens and the slide is carried out by means of the geared focusing mechanism consisting
of two rounded knobs located on each side of the microscope. The larger knob, referred to as the
coarse adjustment knob, is used for raising or lowering the objective lens to a considerable
distance. The smaller knob, referred to as the fine adjustment knob, is used for critical focusing.
Note that turning the right knobs clockwise will shorten the distance between the objective lens
and the stage. On the other hand, turning the right knobs in a counter clockwise direction will move
the lens away from the stage. In some models, it is the stage that moves up or down with the
objective lens remaining stationary.

Figure 2. Resolving Power of Microscope

Proper use of the microscope

a) Place the microscope in front of you, no less than an inch and no more than two inches
from the edge of the table.
b) Turn the nosepiece so that the LPO is in line with the tube; then lower the tube as far as it
will go.
c) Adjust the iris diaphragm to give maximum opening.
d) While, looking through the eyepiece, adjust the mirror until the field of view is properly
lit. Proper illumination means the field of view has uniform lighting. If the microscope has
a condenser, the plane mirror should be used with the HPO; the concave mirror may prove
more satisfactory with the LPO, especially when a desk lamp is used as a light source. If
the microscope does not have a condenser, the concave mirror should be used with all
objectives.
e) Place the prepared slide on the stage. While looking through the eyepiece, move the slide
until the desired part of the specimen is positioned in the center of the circular field of view.
Use the stage clips to keep the slide in place. Slowly turn the right coarse adjustment knob
 clockwise until the image comes into focus. For sharp and clear image, use the fine
adjustment knob to get the proper working distance.
f) For viewing under higher magnification, use the revolving nosepiece to shift from LPO to
HPO. Note that the HPO is longer than the LPO. To prevent damage to the microscope or
breakage of prepared slides, look from the side and make sure the HPO fits nicely into the
space above the stage.
g) Most well-maintained microscopes are parfocal, which means that when the object is in
clear focus under LPO, it will also be in clear focus under HPO. Thus shifting from one
objective to another will not require any other focusing adjustment.
h) If the microscope is not parfocal, make sure to lower the HPO very carefully until it touches
the upper surface of the mounted specimen. Then, using the adjustment knob, very slowly
increase the distance between the HPO and the slide until the specimen comes into clear
focus.

Care of the Microscope

a) Examine the microscope and report any damage immediately.
b) When transporting the microscope, it should be carried in an upright position with one
hand holding the arm and the other hand supporting the base.
c) Before putting away the microscope, adjust the LPO so that it is in line with the body tube
and lower it until it is as close to the stage as possible. Adjust the stage clips parallel to
each other, close the iris diaphragm and put the mirror in a vertical position.
d) The eyepiece should not be removed from the tube. If the unexposed lens of the eyepiece
requires cleaning, inform the instructor.
e) Lens are cleaned only with lens paper.
f) If any liquid is spilled on the stage, or comes in contact with the lenses, wipe it off
immediately. Make certain that any liquid that may have dripped down below the stage is
also removed.
g) The microscope is an expensive precision instrument, it is your responsibility to use it
properly.
Guide Questions

Direction: Fill out the table below

Name of lens Objective magnification Total magnification

Scanning 4x 40x
Low 10x 100x
High 40x 400x
Oil immersion 100x 1000x

Direction: Label the parts of the microscope.

 Ocular lens (eye
piece)

Nose piece

Arm
Objective lens

Stage control
Mechanical stage
Stage clips
Diaphragm
Course focus

Illuminator
Fine focus

Base

1. Why is it important that health care professionals know about microorganisms?

Knowledge about microorganisms is essential for healthcare professionals to know since they deal with the diseases that may
arise from the microorganisms. They need to know how to properly detect infections as well how to prevent their transmission and
spread as well as how to treat them.
2. What are the major parts of microscope?
• Mechanical part - base, c-shaped arm and stage.
• Magnifying part - objective lens and ocular lens.
• Illuminating part - sub stage condenser, iris diaphragm, light source.
3. Why is it important to use immersion oil when using the 100X objective?
When examining specimens with 100x objectives, immersion oil is utilized to assist generate a light pathway. The way light moves through glass and air is not the same.
This distortion is more apparent the higher the magnification. The immersion oil acts as a kind of conduit between the objective and the microscope slide.
4. Are there things that are too small to be seen with a light microscope? Explain.
According to. Alberts et al (2002) , Practically speaking, the smallest items whose form can be readily observed using a light microscope are
usually bacteria and mitochondria, which have a width of approximately 500 nm (0.5 μm). Details smaller than this are usually obscured by
effects coming from the wave structure of light. Light does not travel in precisely the idealized straight ray routes that geometrical optics
predicts due to its wave nature. Rather, light waves take several, marginally different paths through an optical system, interfering with one
another and producing optical diffraction effects.

Reference:

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Looking at the Structure of
Cells in the Microscope. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26880/#
5. Fill in the blanks: As magnification increases, the area of the field of view
Decreases
_________________, Decreases
the depth of the field of view _______________________, the
Decreases
working distance __________________, and the amount of light required
Increases
____________________.

6. What do you think would happen if you tried to view a slide using the oil immersion lens but
forgot to add the oil?
The immersion liquid's function is to lessen the specimen's light refraction and reflection while improving
the objective's capacity to catch this otherwise aberrant light. Due to this, using an oil immersion lens
without the oil will just result to a poor resolution view.