BASIC TOOLS IN MICROBIOLOGY

Activity No. 01
Name: Kapunan, F.A.C. Date Performed: Feb. 23, 2021
Members: Barraquias, F.S. Date Submitted: Feb. 23, 2021
Narag, S.C.
Tawasil, A.N.
Group No: Group 3

Introduction
Microbiology is the study of microscopic life forms and is used by scientists studying viruses, plants,
fungi, protozoans, cells, and parasites. Microbiology equipment is a large category covering all kinds of items
used in laboratories. The type of microbiological equipment a laboratory requires depends on the type of
organisms it is studying. A basic instrument and the most commonly used of a microbiologist are compound
microscope, an optical tool that is used to observe things that are not seen by our naked eye. “Micro” refers to
tiny, “scope” refers to view or look at. Compound microscopes are light illuminated and the image seen with this
type of microscope is two dimensional. You can view individual cells and it has a high magnification but low in
resolution. Equipment for microbiology also comprises slides; test tubes; petri dishes; inoculation loops; pipettes
and tips; incubators; autoclaves; and laminar flow hoods.

Objectives
At the end of the activity, the students should be able to:
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
d. Identify and describe the functions of the different basic tools used in the laboratory

Procedure/Methods
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 16m m objective reaches the
downward stop.
2. Look through eye piece and adjust the mirror to the position which provides the brightest vision seen
through 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 dips.
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 final focus knob to sharpen the image in the center of the field of vision.
6. If the specimen is examined at a higher magnification, move the slide towards the center of the field of
vision. The higher the objective, the lesser the surface arear included in the field of vision. Shift the higher-
powered objective into place and adjust with the first focus knob.
7. The eye should be kept at a certain distance from the eyepiece. Relax when looking into the eyepiece and
keep both eyes open.
8. For the laboratory tools that are commonly used in microbiology, the following tools are:
Identification Function
Transparent material used as a growth medium of cells being cultured, and it
a. Petri dish
can be used as a platform for other liquids that needs to be experimented upon.
A measuring apparatus for volumes of liquid components, and is rated to be
precise but not as accurate as the graduated cylinder. It is also used for mixing
b. Erlenmeyer flask
chemical liquids due to its form, and a container in preparation of the chemical
liquids to be used.
Used to contain chemical liquids in preparation to the experiment or to hold the
c. Test tubes
results of the experiments, either in liquid of solid state.
d. Hotplates Generally, for heating purposes that can be easily controlled.
It is used to operate the temperature and for decontamination/sterilization of the
e. Autoclave
biological wastes.
f. The Laminar Flow It is a closed chamber used to employ aseptic technique that prevents the risk of
Hood bacterial contamination during transfer or contamination of bacterial culture.
 g. Glass slide Used as a platform for the specimens being examined for the experiment.
h. Inoculating A simple tool used by microbiologists to transfer small specimens from culture
loop/needle of microorganisms such as culturing plates.
Used to cause a reaction to happen in a faster rate in spreading liquid
i. Stirring rods substances on solid surfaces, and glass rods are preferable due to the material
that it is not a good conductor of heat.
A measuring apparatus for volume of liquid components which comes in
j. Graduated cylinder
different sizes, and are more accurate than flasks and beakers.
Used for heating/combusting, and sterilization in the laboratory for experiments
k. Alcohol lamp
in need of heat to further analyze other observable changes.

Results and Discussions

Ocular Lens/Eyepiece

Body
Tube

Head
Tube
Course and Fine
Adjustment Knob Revolving Nosepiece

Arm

Stage Clips Objectives

Stage
Aperture Diaphram

Illuminator

Base

The Microscope
Figure 1
These are the parts of a compound microscope. A compound microscope is used to also referred to as a
light microscope, an optical instrument for forming magnified images of small objects, consisting of an objective
lens with a very short focal length and an eyepiece with a longer focal length, both lenses mounted in the same
tube using a visible light through the illuminator.
Table 1 – Characteristics of the microscope
High Power
Features Low Power (Dry) Medium Power (Dry)
(Immersion Oil)
Focal length (mm) 16 mm 4 mm 1.8 – 2.0 mm
Working Distance (mm) 4 – 8 mm 0.5 – 0.7 mm 0.1 mm
Linear Magnification (x) 10x 40x 100x
Numerical Aperture (N.A.) 0.25 0.55 – 0.65 1.25 – 1.4
Diameter of front Lens (mm) 2.0 mm 0.4 mm 0.2 mm

This table represents the characteristics a microscope consists where the distance to the focal point from
the center of the lens is called the focal length, wherein 16mm for low power, 4mm for medium power and 1.8-
2.0mm for high power. Working distance refers to the distance from the front lens element of the objective to the
closest surface of the coverslip when the specimen is in sharp focus, for high power it is 0.1mm, medium power
is between 0.5 and 0.7mm, while in low power it is 4-8mm. The ratio of image length to object length measured
in planes perpendicular to the optical axis refers to linear magnification, whereas 10x for low power, 40-45x for
medium power, and 90-100x for high power. A microscope objective's numerical aperture is a measure of its
ability to absorb light and resolve fine specimen information at a distance from a fixed object, where for high
power it is between 1.25 and 1.4, for medium power 0.55-0.65 while 0.25 for low power. The diameter of front
lens for low power is 2.0mm, 0.4mm for medium power, and for high power it is 0.2mm.
 Table 2 – View of Letter “e” through the unaided eye and under the microscope

Letter “e” as seen by the unaided eye Letter “e” as seen under the
Figure 2 microscope
Figure 3
Sketches of letter “e”

This table contains figure 2 where the letter of “e” is seen by the naked eye of its actual position and the
compact color it persists. Figure 3 on the other hand is seen under the microscope, and different from how the
naked eye view the letter “e”, it is inverted and flipped view under the microscope. Images under the microscope
are inverted and flipped because it lies in the focal length of the objective lens. The image focused by the lens
crosses before the eyepiece further magnifies what the observer sees, and the objective lens inverts
the image because of the lens' curvature. This real image is inverted at the focal length.
Table 3 – 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
9 o’clock 3 o’clock
12 o’clock 6 o’clock
6 o’clock 12 o’clock

This table is the representation of the movements of the specimen through the unaided eye, and the
movement of the specimen as seen through the eyepiece as explained in Table 2.

Observation

Linear Magnification: 10x Linear Magnification: 40x Linear Magnification: 100x

The virtual experiment conducted included the materials of red, blue, and green colored threads to examine
the depth of focus of the objective lenses. For the objective lens 10x magnification, there was no colored thread
that come to focus at first, second or third, however it focused on all colored thread simultaneously as observed
under the 10x objective. Under the objective lens 40x magnification, the colored thread that came into focus first
was the yellow thread then the red thread, and last to focus was the blue thread which was under the yellow and
the red threads. Lastly, for the highest-powered magnification of 100x, the threads could be focused alternatively
once the fine course adjustment know is focused but the order remained to be yellow first since it is on top, then
red and then the blue colored thread.

Question & Answer

1. What does it mean when a microscope is parfocal?
It is a state of the microscope where the condition of the specimen is in clear focus in the
field of view under one objective, and the specimen will be in at least partial focus after switching
to the next objective.
2. Which objective focuses closes to the slide?
The objective focuses that can be closest to the slide or the specimen in the 100x oil
immersion objective.

3. What controls the amount of light reaching the ocular lens?

The diaphragm is responsible on the amount of light that reaches the ocular lens usually
aided with condenser that gathers the wavefronts from the light source and concentrate it to the
cone of light in order to properly view the specimen with uniform intensity.

4. Name 2 ways in which you can enhance the resolving power?

Two ways that can enhance the resolving power is switching to a higher aperture lens and
decreasing the amount of light being used to less the wavelengths.

5. What are the different terms associated with microscopy? Explain each.
Different terms associated with microscopy are light (optical) microscope, electron
microscope, and scanning probe microscope. The light microscope is used for visualizing fine
details, and is used for examining living cells for regular use when low magnification and
resolution is enough. Electron microscope provides higher magnifications and higher resolution
images. It has the capability to magnify more than 500,000 times that allows us to see and visualize
tiny microbes. Lastly, scanning probe microscope is used for atomic surfaces at nanoscale
resolution. This type of microscope does not have lenses, but it uses needles to interact with the
surface of the sample examined.

6. Describe how images are formed under the microscope.

When you look at your microscope, the simulated image you see is not exactly the same as
the true image you can see through your eye. The orientation of the image is different, the bottom
is really what you think of as the "top" of your picture, and what you think of the "right" is really
left. As the light rays actually move through the place where the image resides, it forms an upside-
down and magnified picture called a real image. For this real image, the ocular lens, or ocular lens,
acts as a magnifying glass.

7. Enumerate the proper way of taking good care of a microscope.

- Carry the microscope by holding the C-shaped arm with one hand and other hand under
the base. Never swing the microscope while carrying.
- Never allow direct light to fall on the microscope. Cover the microscope with a plastic
cover when not in use.
- While using oil immersion objective, do not adjust the coarse screw.
- After using the oil immersion objective, the revolving nosepiece should be adjusted to 10x,
then to 40x before putting the stage to its original height.
- Oil immersion objective should be cleaned after use by wiping with soft cotton cloth or
lens paper.
- Dry objective should never come in contact with oil.
- At the end of every experiment, clean the lenses with lens paper.
- The scanning objective or the 4x objective should be locked in place in the revolving nose
piece, the stage should be centered and objectives should be rolled up away from the stage,
when the microscope is replaced after use.
- When the microscope is replaced in the cabin the microscope's arm/pillar must face the
opening of the cabin.

8. Why is there a need to familiarize yourself with the different laboratory tools?
Students should familiarize how to use and how to handle the laboratory apparatuses to
avoid/prevent accidents because it could lead to dangerous effects when doing experiments. Also,
familiarizing how to use the laboratory equipment/tools will help us to do laboratory experiments
correctly, and avoid false results but secure precise and accurate results.
Conclusion/Generalization
In this virtual experiment, the instruments used in the microbiology laboratory include a variety of tools
needed, namely microscope, coverslip, letter e, prepared slide with 3 colored thread, glass slide, alcohol lamp,
graduated cylinder, inoculating loop, stirring rods, petri dish and Erlenmeyer flask for several processes use in
performing the laboratory activities and the basic instrument and most commonly the use of a compound light
microscope. The students were able to demonstrate the correct use of a compound light microscope and name the
major parts and give its function through the sketches of letter “e” and depth focus using different colors of thread
as observed under 10x where in there was no colored thread that come to focus at first, second or third, however
it focused on all colored thread simultaneously but when observed under 40x and 4mm objectives, the colored
thread that came into focus first was the yellow thread then the red thread and last to focus was the blue thread
which was the yellow and the red threads. In sketches of letter “e”, images under the microscope are inverted and
flipped because it lies in the focal length of the objective lens. The image focused by the lens crosses before the
eyepiece further magnifies what the observer sees, and the objective lens inverts the image because of the lens'
curvature. This real image is inverted at the focal length. In conclusion, the images are formed under the
microscope is inverted because light from a condenser illuminates specimens that’s why when you look at your
microscope, the simulated image you see is not exactly the same as the true image you can see through your eye
because it forms an upside-down and magnified picture called a real image.

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