Group # 4 January 31, 2017

Dilla, Denise Gamboa, Alissa Jane R.

Duzon, Jhoanna Rein S. Gardaya, Reyzen P.

Elsisura, Rjan Nichole S.

EXERCISE 1

The Microscope: Principle, Parts, Use and Care

I. A. Introduction

The light compound microscope is an optical system consisting of aligned lenses that work
together to magnify or enlarge the image of the specimen under observation. It was the first
microscope to be invented and the most familiar laboratory instrument to microbiologists. The
term light refers to the method by which light transmits the image to your eye. It pertains to the
visible light of the electromagnetic spectrum ranging from 400 nm to 700 nm. Compound deals
with the microscope having more than one lens. Microscope is the combination of two words;
"micro" meaning small and "scope" meaning view. The aligned lenses in a light compound
microscope act like a collection of prisms operating as a unit. It bends and focuses the light to form
images. When a ray of light passes one medium to another, refraction (the ray is bent at the
interface) occurs. There are two sets of lens that is used in a light compound microscope. The first
one is the objective lens that magnifies the image of the specimen and the lens closest to the
specimen that forms a magnified image that is further enlarged by one or more additional lenses.
The image produce is then magnified by the second lens, the ocular resulting in a much enlarged
virtual image.

A microscope can be simple or compound. A simple microscope, which consists of one lens
was invented by Anton van Leeuwenhoek. He was the first one to see bacteria, yeasts, protists,
sperm and blood cells in 1673 using a 300 simple microscope. The compound microscope,
consisting of more than one lens was invented by Zacharias Janssen in 1595. Robert Hooke used
a compound microscope that aid through the discovery of numerous chambers (remnants of plant
cells) in slices of cork or simply cell which was first introduced through scientific literature in
1665. Have you ever wondered why do we need to use a microscope? The reason is simply because
the human eye, an optical system having a resolving power of 0.1 mm has a limitation. It indicates
that objects whose dimension is less than 0.1 mm (ex. 0.04 mm or 0.0001 mm) cannot be seen
anymore by the naked eye and therefore falls into the realm of microscope. A microscope has a resolving
power of 200 nm or 0. 2 micrometer or 0.0002 mm. The resolving power or limit of resolution of a
microscope is measured by its ability to differentiate two lines or points in an object. The greater the
resolving power, the smaller the minimum distance between two lines or points that can still be
distinguished. The highest useful magnification of a light compound microscope is 1000. The Limit of
Resolution is mathematically defined by Ernst Abbes Equation. Ernst Abbe is a German physicist
responsible for much of the optical theory underlying microscope design. The equation is defined as
follows:

Where:

0.61 is Abbes constant derived from light coherence and visibility

(lambda) is the wavelength of light illuminating the microscope can be altered using filters.

Wavelength of visible light= 550 nm Violet Filter= 410 nm

Blue Filter= 450 nm

(eta) is the refractive index of the medium between objective lens and glass slide. Values of

Air= 1.00 Linseed Oil= 1.48 Canada Balsam= 1.52

Water= 1.33 Cedar Wood Oil= 1.50

(theta) is half the value of the angular aperture or the cone angle. It is the cone of light that enters the
objective lens and is directly affected by the moveable sub stage condenser. The best existing value fir
theta in todays lights compound microscopes is 72 o.

What are the values embedded on the barrels of each objective lenses?
1. Magnification of the objective- To get the total magnification take the power of the objective (4X,
10X, 40x) and multiply by the power of the eyepiece, usually 10x. The value varies on the type of
objective lens you will use (LPO, HPO, OIO).
 2. Numerical Aperture- is the ability of the lens to gather light. It is the denominator of Abbes
equation.
3. Tube Length- is the distance from the nosepiece-objective junction to the observation tune where
the ocular is inserted. For most microscopes as set by Royal Microscopical Society (RMS) of
London, the standard value is 160 mm.
4. Thickness of the Coverslip - Coverslips have thickness ranging from 0.11 mm 0.23 mm. A 0. 17
value which is usually engraved on the objective is the standard recommended thickness of the
coverslip in mm.
Types of Light Compound Microscope
1. Bright-field microscope is used to examine both stained and unstained specimens. It is
called bright-field because it forms a dark image against a brighter background.
2. Dark-field microscope- it produces detailed images of living, unstained cells and
organisms by simply changing the way in which they are illuminated.
3. Phase-contrast microscope- converts slight differences in refractive index and cell density
into easily detected variations on light intensity.
4. Stereo Microscope- provides a three-dimensional view of the specimen. It does this with
separate objective lenses and eyepieces for each eye.

B. Objectives

At the end of this exercise, you should be able to:

1. To recognize the different kinds of microscope, their parts, functions and proper care.

2. To compute the microscopic parameters like Total Magnification and Resolving Power of each
objective using Abbes equation and to know their practical application.

3. To develop the proper skills of operating the light compound microscope.

4. To observe the structural detail of microbial cells.

5. To learn the techniques of measuring specimen dimensions under the microscope and the proper
scale presentations using micrometry.

II. Methodology

Proper use and care of the microscope

A. Transport of the microscope

 1. Get your microscopes from the technician counter keeping the microscope in an
upright position.
2. Hold the microscope arm with your right hand and support the base of the
microscope with your left hand. Never hold the microscope like a bag or in any
position other than upright to prevent accidental damage of loose parts.
3. Do not drop the microscope or remove any of its parts. All parts of a microscope
are expensive and difficult to procure.
4. Lost or damage parts of the microscope should be reported immediately to your
teachers.
B. Cleaning of lenses and other parts:
1. Wipe the microscope lenses and its mechanical parts with clean dry lens paper.
2. Gently clean the oil immersion lens (objective marked 100) with clean lens paper.
Xylene is the solvent of choice to clean hardened oil sticking from the Oil
Immersion Objective (OIO). Do not use alcohol for cleaning as some lens adhesives
are soluble in alcohol.
C. Cover slips and working distances
1. Check the positioning of your objectives, the LPO should be initially positioned in
place above the stage aperture.
2. Measure the working distance of the objectives (the distance between the objective
lens and the specimen being focused). The working distance usually decreases with
increasing objective magnification.
3. Avoid breaking your cover slips by carefully checking if your microscope is
parfocal (i.e. objectives can be switched positions for focusing without adjusting
the objectives or the stage up and down). Danger of breaking the cover slips is
increased when the microscope is not parfocal.
4. Use cover slips with approximately 0.17 mm thickness for focusing. Thick cover
slips may introduce chromatic aberration especially under the HPO and the OIO.
D. Light intensity
1. Use light intensity that is most comfortable to your eyes.
 2. Slowly adjust the mirror of the microscope as you see through the ocular to
illuminate the field of vision. Illumination can come from the white light of the
fluorescent lamp, indirect sunlight or built-in lighting system.
3. If using and electric microscope, control light intensity in the operating transformer
or introduce appropriate filters. Avoid too many adjustments in the iris diaphragm
or defocusing the condenser. Built-in light bulbs for microscopes can be tungsten
(incandescent), fluorescent, halogen and arc lamps: mercury vapor, xenon and
zirconium. Incandescent like the common household light bulb is used for less
expensive microscopes; it warns up easily and produces less intense yellowish light.
Fluorescent produces more intense white and cooler light. Halogen provides
brilliant light, uniform illumination and longer life-span.
4. Maintain the proper lens alignment of your microscope by not loosening any part
of the optical system specifically the ocular and the objectives.
E. Care of the eyes
1. With monocular microscope, learn to use the right eye and left eye alternately to
view the specimen.
2. Keep both eyes open while viewing under the microscope, one eye into the oculars
and the other eye may be focused into your illustrations that are simultaneously
done.
3. With binocular microscope, adjust properly the interpupillary distance to obtain
focal balance.
4. Eyeglasses are unnecessary while viewing through the microscope. Compensations
can be done with slight turning of the fine adjustment.
5. Keep a comfortable posture and proper placement of microscope to avoid eye
strain, muscle and joint pain.
II. Results and Discussion

The parts and its function are given as follows:

 The resolving power of the light compound microscope was computed using Abbes equation.

Miceoscopic Parameters LPO HPO OIO

Numerical Aperture (NA) 0.25 0.65 1.24

Half of Angular Aperture 14.48 40. 54 56. 44

Angular Aperture 28.96 81.08 112.88

Resolving power in 1.34 0.52 0.27

IV. Conclusion

V. References

https://www.cas.miamioh.edu/mbiws/microscopes/compoundscope.html