Microbiology

Activity #1
The Compound Microscope

INTRODUCTION:
Often times, the development of science usually parallels the development of instruments that widens
human senses to new limits. The discovery of cells and microorganisms progressed with the invention
and evolution of one of the very significant instrument in Biology, the microscope. The compound
microscope is an optical tool that is used to observe things that are beyond ordinary vision. It is one of the
basic instruments of a microbiologist. Since this course deals with the study of microorganisms, one should
be familiar with the parts of the microscope, comprehend and appreciate the functions and be competent
in using the instrument. Continuous and appropriate use of the microscope will increase one’s expertise.
Parts and Function: The wide base keeps the microscope steady at any position of the stage. The arm
fastened to the base through the inclination joint, permits the adjustment of the stage to the desired angle.
The concave mirror reflects the light into the condenser. The iris diaphragm regulates the amount of light
entering the condenser. Just below the diaphragm is a slot to accommodate different types of light filters.
The condenser concentrates the light rays received from the mirror and sends them to the objective. The
stage is the horizontal platform upon which the specimen to be examined in placed. At the center of the
stage is a circular aperture. The spring clips hold the slide in place on the stage. The objective is that part
of the optical system of the microscope which produces the specimens initial magnified image (real) within
the body tube. The revolving nosepiece, to which three parfocal objectives are attached, allows convenient
exchange of the objectives. The body tube is a hollow cylindrical tube through which the light passes from
the objectives to the eyepiece. The upper position of the body is called the draw tube. The eyepiece or
ocular is that part of the optical system through which the specimen is viewed. 5 The intermediate image
projected by the objective is enlarged by the eyepiece. Hence, the term compound microscope is derived
from the fact that the specimen is magnified twice, first by the objective and second by the eyepiece. The
final image formed is a virtual image. The magnification of the compound microscope is, therefore, the
product of the magnifying power of the objective and the eyepiece. If the magnifying power of the objective
is 100x and the eyepiece is 10x, the total magnification will be 1000x. The coarse focus knob is used to
bring the object into focus. For maximum definition, the fine focus knob is used. The student microscope
has three objectives: a dry low power, a dry medium power and an oil immersion high power objective.
Two important terms in microscopy are magnification and resolving power or resolution. Magnification is
how much larger the object appears compared to its actual size, while, resolving power is a measure of
the clearness of the appearance of the specimen; it is the minimum distance two points can be separated
and still be distinguished as two separate points. For instance, what appears as one star in the sky can
be resolved as twin stars with the use of a telescope. Resolving power of the human eye is limited just as
the resolving power of the telescopes and microscopes is limited as well. Microscopes are designed to
magnify objects as desired but the resolution is limited by the wavelength of light used to illuminate the
specimen.
 Types of Microscopes
Compound Dissection or Confocal Scanning Transmision
stereoscope microscope Electron Electron
Microscope Microscope
(SEM) (TEM)
Description Compound A dissection This SEM use TEM is electron
microscopes are microscope is microscope electron illuminated. This
light illuminated. light uses a laser illumination. gives a 2-D view.
The image seen illuminated. light. This light The image is Thin slices of
with this type of The image is used seen in 3-D. It specimen are
microscope is two that appears because of the has high obtained. The
dimensional. This is three wavelength. magnification electron beams
microscope is the dimensional. It Laser light and high pass through this.
most commonly is used for scan across resolution. The It has high
used. You can dissection to the specimen specimen is magnification and
with the aid of coated in gold high resolution.
view individual get a better
scanning and the
cells, even living look at the
mirrors. Then electrons
ones. It has high larger image is then bounce off to
magnification. specimen. placed on a give you and
However, it has a You cannot digital exterior view of
low resolution see individual computer the specimen.
cells because screen for The pictures
it has a low analyzing. are in black and
magnification. white.
Source of visible light visible light Laser light electrons electrons
Radiation
for Image
Formation
Medium air air air vacuum vacuum
Special glass slides glass slides glass slides Mounted on Thin films of
mounting with dyed aluminum stubs collodion or other
samples and are coated supporting
in gold material on
copper grids
Nature of glass glass glass lenses one one electrostatic
lenses with electrostatic lens and a few
dichromatic lens with a few electromagnetic
mirrors electromagnetic lenses
lenses
Focusing mechanical mechanical digital Electrical Electrical i.e.
computer current of the
motorized objective lens coil
focusing is changed.
mechanism
Magnificatio Changing usually, 1 digitally electrical Electrical i.e.
n objectives objective enhanced changing current
adjustments of the projector
lens coil
Major Light Absorption light scattering laser light with electron electron
means of or light dichromatic scattering scattering
providing reflection mirror
specimen concentrated at
contrast pinhole
I. Objective(s):
a. Demonstrate the correct use of a compound light microscope.
b. Describe how images are formed under the microscope
c. Name the major parts of a compound microscope and give its function
II. Materials:
Microscope prepared slide w/ 3 colored thread Coverslip glass slide
III. Procedure:
A. Operating Procedure:
Place the microscope close to the edge of the table. Select a suitable stool so that when looking into the
eyepiece, your back is straight and your neck is bent at the nape.
1. Lower the body tube by turning the course focus knob until the 10x or 16mm objective reaches the
downward stop.
2. Look through the eyepiece and adjust the mirror to the position which provides the brightest and the
most evenly illuminated field of vision which is the circular area seen in the eyepiece. Raise the condenser
until its top lens at the same level as the stage. Place the slide on the stage and fasten it using the stage
clips.
3. Position the specimen area of the slide over the center of the stage aperture.
4. Looking through the eyepiece, raise the coarse focus knob until the image appears. Focus as sharply
as possible. Low power objective has a much greater depth of focus and is generally used for the initial
focusing and viewing.
5. Adjust the fine focus knob to sharpen the image in the center of the field of vision.
6. 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 higher-powered objective into place and adjust
with the fine focus knob.
7. The eye should be kept at a certain distance from the eyepiece. Be relaxed when looking into the
eyepiece and keep both eyes open. Note: on your worksheet, label the different parts of a compound
microscope. Take note also of the characteristics of your microscope and fill up table 1.
B. Inversion of the Images:
Place under the 10x objective a printed small letter e. The letter should be accommodated in the field of
vision. Sketch the letter e as seen by the unaided eye and through the eyepiece.
C. Movement of the Specimen on the Stage and its Corresponding Movement in the eyepiece:
In referring to direction in the field of vision, use the numerals on the face of a clock. For example, the
specimen is at 12, at 6, between 1 and 2, between 9 and 10. Now with the letter e still under the objective
fill the data in table 2 under observation.
D. Depth of Focus:
Depth of focus is the thickness of the layer within the boundaries of which all appear to be sharply in
focus. As the objective is moved up and down one specimen is in focus at a time. The thickness of this
layer is very nearly inversely proportional to the square of the numerical aperture (N.A.). Hence, an
objective with a large N.A. will have a small depth of focus.
1. Secure a prepared slide containing three colored threads from you instructor.
2. Place the slide on the stage under the 10x or 16mm objective.
3. Focus upward with the coarse focus knob and note which colored thread comes into focus first,
second and third. Write your observation under letter D on your worksheet.
4. Repeat this with the 40x and 4mm objective. Again, make your observation.

\
 WORKSHEET
Activity No. 1

Name/s: Baylon, Gwyneth Mae M. Date Performed: February 04, 2021

Bunyi, Markiely A. Date submitted: February 06, 2021
Encarnacion, Destine John V.
Galera, Samantha Nicole S. j
Group No.: GROUP 2 G

A. Operating Procedure: Label the parts of a compound microscope.

FIGURE 1. The Microscope

Numbered part Part of the Microscope

1 OCULAR LENS (EYEPIECE)
2 DRAW TUBE
3 BODY TUBE
4 REVOLVING NOSE PIECE
5 OBJECTIVES
6 STAGE
7 GLASS SLIDE
8 LIGHT SOURCE/REFLECTOR
9 FOOT
10 DIAPHRAGM
11 STAGE CLIP
12 ARM
13 FINE ADJUSTMENT KNOB
14 COARSE ADJUSTMENT KNOB
Fill in table 1 below for the characteristics of your microscopes.
Table 1 – Characteristics of the microscope
Features Low Power (dry) Medium Power (dry) High Power (immersion
oil)
Focal length (mm) 16mm 4mm 1.8-2.0mm
Working distance (mm) 4-8mm 0.5-0.7mm 0.1mm
Linear Magnification (X) 10x 40-45x 90-100x
Numerical aperture 0.25-0.30 0.55-0.65 1.25-1.4
(N.A.)
Diameter of front lens 2.0mm 0.4mm 0.2mm
(mm)

B. Inversion of images: Sketches of letter e

Fig. 2. Letter “e” as seen by the unaided eye Fig. 3. Letter “e” as seen under the microscope

C. Movement of the Specimen on the Stage and its Corresponding Movement in the eyepiece:
Table 2 – Movement of specimens
If the letter e on stage Is moved to The image in the eyepiece is moved to:
3 o’clock 9 o’clock
6 o’clock 12 o’clock
9 o’clock 3 o’clock
12 o’clock 6 o’clock

E. Other questions for discussion:

What does it mean when a microscope is parfocal?
➢ A microscope is parfocal because it has the ability to remain in focus when the objective lens
is changed. For example, when a microscope's focal point is moved from a low power (10x) to a
higher power (40x or 100x), the item remains in focus
Which objective focuses closest to the slide?
➢ The oil immersion (100x) objective lens is the one that focuses closet to the slide because
more light is equivalent to clear and crisp images. By placing a substance such as immersion oil
with a refractive index equal to that of the glass slide in the space filled with air, more light is directed
through the objective and a clearer image is observed. Also, it has the longest lens among the three
which means that it has a higher power.
What controls the amount of light reaching the ocular lens?
➢ The portion of the microscope that controls how much light enters the ocular lens is known
as the iris diaphragm.

Name two ways in which you can enhance the resolving power.
➢ Using immersion liquids between the objective's front lens and the cover slip is one
approach to improve the microscope's optical resolving power. It improves the resolution of
the image through preventing the refraction of light that travels up to the objective lens.
➢ Use a larger diameter lens of the same magnification. It is known to increase the
brightness and sharpen the focus.

What are the different terms associated with microscopy? Explain each.
Achromatic
➢ It is a term referring to the lens. A lens which brings in light from two parts of the spectrum
(red and blue wavelengths) to the same focus, reducing Chromatic Aberration. This is the most
common lens on a microscope. Color fringes may appear when viewing under bright white light
because not all wave lengths are brought within an acceptable focus range. If you use filtered light
(monochromatic) as in phase contrast the image will be sharper
Barrel Focus
➢ the body tube of the microscope moves to focus the objective lenses and the stage is fixed.
Barrel Focus
➢ Body tube of the microscope moves to focus the objective lenses
Field-of-view
➢ Visible are through the eyepiece when the microscope is in focus
Depth of field
➢ an optical technique where the specimens seen as a bright object against a dark background
Coaxial
➢ Focusing system where the coarse and fine focus are mounted together on a common axis
Abbe Condenser
➢ To see the condenser in the microscope

Describe how images are formed under the microscope

➢ Image creation in a microscope is when light from a condenser illuminates the specimens.
The microscope objective absorbs these diffracted waves and guides them to the focal plane, where
an image of the object is generated by interference between the diffracted waves.

Enumerate the proper way of taking good care of a microscope

1. Handle with care if the microscope must be moved, carry it from the base and support arm, if picked
up by stage or eyepiece holder it can become misaligned.
2. Keep covered Every time you use or clean the microscope be sure to cover the kit to prevent dust
and other contaminants from reaching the apparatus. If you cannot find your cover, order a new one from
the manufacturer or find a suitable substitute, such as a large plastic bag.
3. Store safely Keep the microscope in a draft-free, dry place, where it won’t get bumped, pushed,
wet and where things will not fall on it. Make sure the microscope is level to the table/bench.
4. Keep area clean organized and ready to use for the next person, even if that’s you: throw away
used lens paper/wipes, cover any bottles, wipe the counter clean and place essential items within easy
reach for the user.
5. Take care of the bulbs Turn the microscope off after use. Do not keep the light on all day as this
will shorten the bulb’s life.

Conclusion
The light magnifying instrument is an exceptionally powerful tool for understanding the design and capacity
of tissues, and it is generally utilized in biomedical science courses, as well as in Research and
Diagnostics laboratories. Every part of the microscope has its own function and benefits by using a
microscope. The movement of the specimen can be based on the movement of the eyepiece which result
as inverted view of point of the microscope. There are 3 or 4 objectives can be seen in the microscope
which is the Scanning, LPO (Low Power Objective), HPO (High Power Objective), Oil Immersion (Clearer
view). Taking care of the microscope has many things to follow like we use and unused the microscope,
proper way of holding a microscope and more. Microscope is very important in every medical related field
to understand and taking a clearer view to see to the world of micro-organisms.