Exercise 1.

Microscopy

The development of instruments that extend the human senses allowed the
discovery and early study of cells. Cells vary in size, but they are generally quite small.
Instruments like microscopes magnify the objects otherwise too small to be seen, such as
microbial cells, producing a larger image of the object. There are two general types of
microscopes: the light microscope which uses light waves and lenses, and the
electron microscope which employs electron beams and magnetic fields to visualize
the object.

A compound light microscope is an instrument that considers three parameters:

magnification, resolution, and contrast. Magnification extends our ability to observe
the details 1000 times, so that we can see objects as small as
0.1 micrometer in diameter. The image of an object is magnified through at least one
lens, but magnification is of little value to the observer if the resolution is not increased.
Resolution or resolving power is the ability to distinguish two adjacent objects as
distinct and separate, which also depends on the wavelength of light used and on the
numerical aperture (NA), a characteristic of microscopes that determines how much light
enters the lens. In addition, improving contrast typically improves the final image, as
this will accentuates differences in parts of the specimen. These parameters are useful to
clearly observe the microscopic specimens like bacteria, fungi, protozoa, and some
helminths. Without microscopes, our understanding of the characteristics and
morphological features of cells and tissues would be severely limited.

This exercise is designed to familiarize students with the parts, functions, use and
care of a compound light microscope.

By the end of this exercise, the students should be able to:

● identify the parts of a compound light microscope and elucidate each

function;
● learn the proper handling and care of a microscope; and
● calculate the total magnification and measure the size of the sample specimen.

MICR 21: Microbiology and Parasitology AY 2022-2023

Cavite State University
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A. Parts of a Compound Light Microscope and their Functions

1. Eyepiece/Ocular Lens: This is the lens where you look through and has different
magnifications (e.g., 10X, 16.5X, or 20X), which are stamped on the side of the
eyepiece.
2. Body Tube: Contains a prism that bends the light rays so that they will pass
through the oculars. Sometimes the body tube is straight, whereas in others, the
oculars are held at an angle.
3. Objective Lenses: These are a group of 3 or 4 objective lenses that are attached to
a revolving nosepiece at the base of the body tube. Locate the nosepiece and notice a
click as each objective snaps into position. The names of the objective lenses based
on their magnifying power are as follows: Scanning power (4X); low power (10X);
high power (40X); and oil immersion (100X).
Note: The total magnification for each objective is calculated by multiplying the
magnification of the eyepiece and objective lens on your microscope.

Important features of the objectives:

a. Focal length (mm), an optical constant of the lens system, is the distance from the
center of the lens to the point where parallel rays entering the lens are brought to a
focus.
b. Resolving power of an objective is the property to recognize features of a
specimen that are close to each other as separate and distinct. The greater the
resolving power, the greater the definition of an object. This property is dependent
on the wavelength of light used and an optical property of the objective lens known
as numerical aperture.
c. Numerical aperture (N.A.) (indicated on the side of the lens) is a measure of
the resolving power of an objective. An objective with a 0.25 N.A. allows the viewer
to distinguish as separate 25,000 lines per inch. If a specimen is known to be of the
order of 26,000 lines per inch, the observer can never see the lines as separate no
matter how much magnification is employed. Lenses with higher magnification
usually have higher N.A. but the medium through which the light passes also affects
N.A.
d. Parfocal means that the objectives are optically and mechanically designed so that
the distance between the specimen and the aerial image is always constant. Slight
refocusing with the aid of the fine knob is sufficient to restore critical sharpness of
the image after changing from one objective to another, thus, the coarse focus knob
need not be operated.

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4. Stage: The surface or platform on which you place the prepared slide. It has an
opening (stage aperture) in the center and with clips to hold the slide in place. On
some microscopes, stage is stationary, whereas some have movable stage called
as mechanical stage. The movement of the stage is controlled by 2 knobs, which
move the slide in horizontal and vertical scales.
5. Substage: The area under the stage, which contains a diaphragm and condenser.
a. Diaphragm: Regulates the amount of light passing through the specimen. Proper
adjustments provide better contrast between the surrounding medium and your
specimen, resulting in a clear image.
b. Condenser: Consists of a series of lenses that concentrates the light rays onto
the specimen; improve the clarity of the specimen.
6. Light Source: An illuminator is built into the base of the microscope and is
controlled by an on/off switch. The amount of light entering the specimen is
controlled by adjusting the diaphragm, whereas the light intensity is controlled by
adjusting the voltage of a transformer attached to the illuminator. Higher setting
of voltage is used when using the OIO lens. On the other hand, some compound
microscopes use mirror instead of a built-in illuminator to capture light, which is
coming from different light sources (lamp and/or natural light).
7. Focusing knobs: Coarse focus knob is commonly used to draw the entire body
tube up and down to bring the object into approximate focus, whereas the fine
focus knob is used for maximum definition.
8. Base: Keeps the microscope steady at any position of the stage.
9. Arm: Fastened to the base through the inclination joint, permits the adjustment of
the stage to a desired angle.

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B. Proper Handling of a Compound Microscope

The following are the considerations in handling a microscope.

1. Place the microscope close to the edge of the table.

2. Lower The body tube by turning the coarse focus knob until the 10X or 16 mm
objective reaches the downward stop.
3. Look through the eyepiece and adjust the mirror to the position which provides the
brightest and most evenly illuminated field of vision (the circular area seen in the
eyepiece).
4. Place the slide on the stage and fasten it using the stage clip.
5. Position the specimen area of the slide over the center of the stage aperture.
6. Looking through the eyepiece, raise the coarse focus knob until the image appears.
Focus as sharply as possible. The LPO has a much greater depth of focus and is
generally used for initial focusing and viewing.
The free space between the specimen surface and the objective is the working
distance (mm). The higher the magnification of the objective, the smaller is the
working distance.
7. Adjust the fine focus knob to sharpen the image in the center of the field of vision.
8. When a feature on the specimen is to be examined at a higher magnification, move
the slide so that the feature is centered in the field of vision. The higher the power of
the objective, the lesser is the area of the specimen surface included in the field of
vision. Shift the HPO into place and adjust with the fine focus knob.
9. Keep your eye at a certain distance from the eyepiece. Relax and keep both eyes open
when looking into the eyepiece.
If a sharp image is not obtained despite application of the above procedure, it is
possible that:
a. the fine focus knob has reached a stop.
b. focusing attempts are too rapid; adjust fine focus slowly.
c. the objective is covered with dried oil from previous use (dried oil can be
removed using xylene or 95% ethanol).
d. the cover slip is too thick (optimum thickness is 0.17 mm)
e. the slide is inverted.
10. Use of the 1.8 mm or Oil Immersion Objective
a. Examine the specimen under the 16 mm objective. Locate first the smear. Focus and
select an area where the yeast or bacteria are clearly seen and move this to the center
of the field.
b. Shift to the 4 mm or 40X objective and focus again using the fine adjustment knob to
get a clearer image of the specimen.

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c. Shift into place the 1.8 mm or OIO. Use the fine focus knob for final focusing.
Write down your observations and draw the stained smear as seen under the 1.8
mm objective without oil.
d. Slightly rotate the revolving nosepiece to allow application of a small drop of oil on
that part of the specimen at the center of the stage aperture.
e. Return the 1.8 mm objective into place. The front lens is now immersed in oil
and almost touches the slide.
f. Look into the eyepiece and use the fine focus knob for sensitive focusing. After
using the oil immersion objective:

g. blot out the oil with lens paper.

h. clean the lens with lens paper wet with xylol (with instructor’s supervision)
i. blot-dry the lens with fresh lens paper.

.
C. Care of a Compound Microscope
The following rules, cautions and maintenance hints will help keep your microscope in
good operating condition.

1. Use both hands when carrying the microscope: one firmly grasping the arm of
the microscope; the other beneath the base. Avoid jarring your microscope.
2. Never touch the lenses. If the lenses become dirty, wipe them gently with lens
tissue in a circular motion to avoid scratching.
3. If blurred specks appear in the field of view this may be due to lint or smears on
the eyepiece. If the specks move while rotating the eyepiece, the dust is on the
eyepiece and cleaning the outer lens of the eyepiece is in order. If the quality of
the image is improved by changing objective lenses, clean the objective lens with
lens paper.
4. Never leave a slide on the microscope when it is not in use.
5. Always remove oil from the oil-immersion objective lens after its use. If by
accident oil should get on either of the lower-power objective lenses, wipe it off
immediately with lens tissue.
6. Keep the stage of the microscope clean and dry. If any liquids are spilled, dry the
stage with a piece of cheesecloth. If oil should get on the stage moisten a piece of
cheesecloth with xylol and clean the stage, then wipe it dry.
7. When not in use, store your microscope in its cabinet. Put the low power objective
lens into position at its lowest point above the stage. Be sure that the mechanical
stage does not extend beyond the edge of the microscope stage. Wrap the
electrical cord around the base.

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8. Never force the adjustments. All adjustments should work freely and easily. If
anything does not work correctly, do not attempt to fix it yourself, immediately
notify your instructor.
9. Never allow an objective lens to jam into or even to touch the slide or coverslip.
10. Never focus downward with the coarse adjustment while you are looking through
the microscope. Always incline your head to the side with eyes parallel to the
slide and watch the objective as you move it closer to the slide. This will prevent
you from smashing the objective into the slide.
11. Never exchange the objective or eyepiece lenses of different microscopes, and
never under any circumstances remove the front lenses from objective lenses.
12. Never attempt to carry two microscopes at one time
If you follow these rules, you will never have trouble with your microscope.

Materials
Compound microscope Prepared
slides (Paramecium) Ruler
Glass slide
Cover slip
Lowercase letter ”e”
Dropper

Lab Kit (per group): Alcohol, Tissue, rag, dishwashing liquid, sponge First
aid kit (per group/class): betadine, band aid, gauze pad Individual: Lab
gown, gloves

A. Actual Focusing
1. Cut a lowercase letter “e”. Select the smallest size possible.
2. Place the letter “e” on the center of a slide, a put a drop of water. Put a
coverslip.
3. Place the slide at the center of stage. Be sure that the top of the letter is away
from you.
4. Turn the nosepiece until the LPO clicks into position.

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5. Look into the ocular lens and focus slowly upward using the coarse adjustment
knob.
6. Slowly rotate the nosepiece so that the HPO clicks into viewing position.
7. Make a drawing of these views (as seen by the unaided eye and as an image
under the microscope – LPO and HPO) in the lab report sheet.

B. Measuring Microscope Field Diameter

a. Lower Power Field Diameter
1. Measure the diameter of the LPO by focusing a plastic metric ruler under the
microscope.
2. Focus the ruler (mm) directly without mounting it on a slide.
3. Place the vertical line at the left of the microscope field. The distance
between two vertical lines is a millimeter.
4. Calculate the length of the remaining segment of the next millimeter.
b. High Power Field Diameter
Since the HPO magnifies greater then the LPO, its diameter is also reduced in
size. Thus, it is smaller than a millimeter. The method used in measuring LPF
does not apply in this case.
1. Determine the magnification of both the LPO and the HPO.
2. Divide the magnification of LPO by the magnification of HPO.
3. Multiply the quotient with the LPF diameter obtained in B.a.

1 mm = 1000 µm To

determine HPF, the formula below is used:

𝐿𝑜𝑤 𝑃𝑜𝑤𝑒𝑟 𝑂𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛
𝐻𝑃𝐹 = 𝐻𝑖𝑔ℎ 𝑃𝑜𝑤𝑒𝑟 𝑂𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑥 𝐿𝑃𝐹

C. Calculating Specimen Size

1. Focus the prepared slide (Paramecium) under LPO.
2. Estimate the number of cells that would fit end to end in the lower power field
of view.
3. Using the calculated LPF diameter, determine the size of the specimen by
dividing the diameter field of view by the number of cells.
4. Do the same using HPO.

𝐹𝑖𝑒𝑙𝑑 𝑜𝑓 𝑣𝑖𝑠𝑖𝑜𝑛 (µ𝑚)

𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑐𝑒𝑙𝑙 (µ𝑚) 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 (𝑛)

MICR 21: Microbiology and Parasitology AY 2022-

Cavite State University
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Writing discussions and answering study questions

1. There are discussion sections and study questions listed at the last part of your
laboratory worksheet. Discuss with your groupmates on how you will answer
each of the questions. Every member is expected to contribute.
2. All of your answers are expected to have appropriate citations from references.
When constructing your answers, always paraphrase and do not forget to cite your
references using APA format. Use references which are accepted by the academic
and scientific community. These references include, scientific/journal
articles, .edu websites, libretext website, books, and e-books. Using Wikipedia as
reference is strictly not allowed.
3. When writing your references list, always follow APA format. Provide all the
necessary information (authors, date, title of the reference, publisher, URL or DOI
etc.)

Grading of your laboratory report

Your laboratory instructor will thoroughly check the contents of your laboratory report,
namely, its scientific soundness, proper formatting, credibility and timeliness. The rubrics
for grading are as follows:

A. Completeness, scientific relevance, conciseness and proper

presentation (50%)- the output presents sound scientific information and
synthesis of important points in connection to the topic, concisely written and
properly arranged. (points:50% if all points above were met; 45% if few
points are missing; 30% if most points are not met; 20% for the effort)
B. Credibility and References (30%)- the output follows proper
paraphrasing and the standard APA format in citations and list of references.
References used are credible/accepted for use by the scientific and teaching
community. No copy-pasted entries are observed or at the minimum.
(points:30% if all points above were met; 25% if few points are missing; 15%
if most points are not met; 10% for the effort)
C. Punctuality (10%)-the output was submitted on/before the set deadline.
(points:10% if the activity was submitted on time; 8% if the activity was
submitted a day after the deadline; 6% if the activity was submitted 2-7 days
after the deadline; 4% if the activity was submitted more than 8 days after the
deadline.)
D. Matrix of work assignments (10%)- each group must fill-out the matrix
of work assignments. The matrix contains the name of all the members of the
group who contributed to the output and their

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respective contributions/assignments leading to the completion of the output.

Each member will be given a rate by your instructor based on the quality of
their work. The group must specify each member’s contribution and ensure
that the distribution of tasks is equally divided among members. (Points: 10pts
for excellent; 8pts for good; 6pts for average; 5pts for needs improvement.

Fernandez, W. L., Dalmacio, I. F., Raymundo, A. K., Zamora, A. F., Mendoza, B.

C. (2008). Laboratory Manual in General Microbiology. Seven Lakes Printing
Press.

Eisenback, J. D. (2003). Nematology: Laboratory Investigation: Morphology and

Taxonomy. Mactode Publications.

Madigan, M. T., Bender, K. S., Buckley, B., Sattley, W. M., & D. Stahl, A. (2019).
Brock biology of microorganism. 15th ed. Boston, MA: Pearson Publisher.

Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V. and Jackson, R.B.
(2014). Campbell Biology. 10th Edition. Pearson Education, Inc.
Virtual Microbiology. Proper care of the microscope.
https://instr.bact.wisc.edu/book/displayarticle/85 Accessed on September 14, 2021

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Exercise 1.
Microscopy
Group
Course and Section:
No.

Group Leader:

Members:

ACTIVITY PROPER

A. Actual Focusing

Unaided eye LPO HPO

a. When you move the slide upwards, the image moves .

b. When you move the slide downwards, the image moves .
c. When you move the slide to the left, the image moves .
d. When you move the slide to the right, the image moves .

B. Measuring Microscope Field Diameter

LPF Computation

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HPF Computation

C. Calculating Specimen Size

Computation:

STUDY QUESTIONS

1. Explain why an inverted image is seen under a compound microscope.

2. How can the location of the dust particles in the optical system be determined?

3. What is the purpose of the oil when using the oil immersion objective?

4. Why should the microscope be calibrated for each objective and prior to each use?

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Cavite State University
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Cited References in Answers to Questions

Matrix of work assignments

Group member Contribution Rating (to be filled by

your instructor)

MICR 21: Microbiology and Parasitology AY 2022-