Microscope

Adelaide, Savero, & Charissa

(HT : Rizqa A, Putrinata)

P01 Rania apa yg makanan? Rania Tahufiq

 Learning Objectives
❖ To learn parts of a microscope, its functions, and its principle of resolution.
❖ To learn various types of cells and morphology.
❖ To learn the method to differentiate organelles using differential centrifugation.

ATTENTION
The highlighted words represent the keywords, but those are not the only words to
be memorized. Make sure you completely understand the procedure and the
principle used in this lab work. This is not only about memorizing but also
understanding.

1. MICROSCOPE
A. Parts of Microscope and its function
1. Ocular lens: lens closest to our eyes
during observation. (10x
magnification)
2. Tube: connects ocular & objective
lens.
3. Light source: generates light.
4. Stage: table for the specimen.
5. Coarse adjustment/force knob:
largely moves the stage vertically.
Brings the image into focus. Should
only be used when using low power
lens.
6. Fine adjustment/force knob: similar to
coarse adjustment knob but moves
more smoothly. Used to bering sharp
focus on low power lens, used to
bring focus on high power lens.
7. Revolver: holds the objective lens.
8. Objective lens: lens closest to the
object. Magnification and aperture
varies. (4x, 10x, 40x, 100x)
9. Condenser: to focus the light onto the
specimen.
10. Diaphragm: adjust the amount of
light reaching into the specimen.

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 B. Image Formed by Microscope
Total magnification= Objective x Ocular (eyepiece)
magnification
The resolution of the objective lens represents the power of a microscope

𝐶. ʎ
𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛(𝐷) = C : constant (0,61)
𝑛. 𝑠𝑖𝑛ʚ ʎ : wavelength of light
n : refraction index of light between specimen and
NA = 𝑛. 𝑠𝑖𝑛ʚ objective lens.
sinʚ : Aperture = Half the angular width of the cone
of rays collected by objective lens

The numerical aperture (NA) refers to the capacity of a lens in collecting light. The
bigger the number of NA means the higher the resolution. Immersion oil will form
sharper image/image with better resolution.

C. Immersion Oil
When light passes from a material of one refractive index to another (for example: from glass to
air), it bends. In the space between the microscope objective lens and the slide (where air is),
light is refracted, the light scatters and it is lost.

Refractive index:
❖ Air: 1.0
❖ Glass: 1.51
❖ Oil: 1.51

When light passes through both glass and air, it is refracted. Different light bends at different
angles. So, as objects are magnified more, images become less distinct. Why? Because when
using objective lenses with lower power (4x, 10x, 40x) the light refraction is not usually
noticeable. However, once we use the 100x objective lens, the light refraction when using a dry
lens is noticeable (see picture a). If you can reduce the amount of light refraction, more light
passing through the microscope slide will be directed through the very narrow diameter of a
higher power objective lens. In microscopy, more light = 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.

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 D. How to use Immersion Oil
Before using immersion oil, make sure that your 100x objective lens is made for use with
immersion oil.
1. Begin by focusing your sample using the 40x objective lens. Rotate the objective lens part
way between the 40x and 100x lens so you can reach the cover slip on your slide.
2. Place a drop of immersion oil on the top of your cover slip and another drop directly on your
100x oil objective lens.
3. Slowly rotate your 100x oil objective lens into place and adjust the fine focus until you get a
crisp and clear image.
4. When finished viewing with your 100x oil immersion lens, carefully wipe the oil from all
glass surfaces using a piece of lens paper.
5. (NICE TO KNOW) Although the lab manual strictly said to only use lens paper, there are
further methods of cleaning the lens by using a second piece of lens paper moistened with a
small amount of alcohol (ethyl or isopropyl) or lens cleaning solution. Failing to remove
immersion oil from lenses will result in hardened oil on lenses that will affect future clarity.
To remove hardened immersion oil, use xylene or lens cleaning solution.
Reference: https://www.microscopeworld.com/t-using_microscope_immersion_oil.aspx

E. Why Microscopes?
Because human eyes have limited Resolving Power. Resolving Power is the capacity to differentiate
two different points. Human eyes have 100 µm of RP. Animal and bacteria cells are usually sized 10-
20 µm.

F. Types of Microscope
1) Compound Microscope
To observe things with diameter 0,2-0,8 µm (e.g bacteria, mitochondria which sizes 0.5 µm).
The compound microscope heas central (ocular) lens and various objective lens.

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 2) Electron Microscope
To observe things with diameter 2-4 nm (e.g particles, virus, several organelles)

G. Procedure
1. Prepare everything needed.
2. Start by using the smallest magnification of objective
BASIC RULES IN USING
lens (10x) to find the bright field, then insert the
MICROSCOPE:
specimen.
a. Do not insert specimen before we
3. Set the microscope (the coarse and fine adjustment, get the bright field.
tube, etc) and start observing. b. Start observing with the LOWEST
4. To change the objective lens to bigger magnification, magnification.
DO NOT change the focus. Revolve the lens first, and c. If we want to change the
then you may adjust the focus. magnification, DO NOT change
5. For 100x magnification, it is recommended to add oil- the focus before revolving the
immersion especially for observing a very small objective lens.
specimen (mitochondria, etc).
6. Put everything back to the place, clean the table and your hands with streaming water and
soap. To clean oil-immersion, use lens paper.
7. Get the fuck out srsly.

2. CELL FRACTIONATION USING

DIFFERENTIAL CENTRIFUGATION
Differential centrifugation results in the formation of sediments. The sediments are the organelles
of the cell that has been separated on the process. During this lab work, we used liver homogenate.

A. Process of Cell Fractionation

1. Centrifuge the liver homogenate at 5000 RPM for 10 minutes. The result is fraction 1, which
is shown below:

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 Supernatant

Pellet
Supernatant contains organelles that
The pellet is the sedimentation. have not sedimented yet because the
It contains nucleus and cell weight is lighter.
debris that have heavier
weight, so they sediment
faster.

2. In order to get organelles with lighter weight/mass, we need to centrifugate fraction 1

supernatant at 12000 rpm for 15 minutes. Fraction 2 is shown below:

Supernatant

Pellet

This pellet from fraction 2

contains lighter organelles:
mitochondria and lisosome.

3. When fraction 2 supernatant is centrifuged at 100,000 rpm for an hour, it will result in pellet
containing cell membrane, RE, and Golgi apparatus.
4. To observe the nuclei, take pellet from fraction 1 with a micropipette. Put it into a
microtube. Mix the pellet with PBS (phosphate-buffered saline) in a 1:1 ratio. Then, mix
methylene blue in a 1:1 or 1:0.5 (mixture of pellet+PBS:methylene blue). Methylene blue is a
staining agent used to identify nucleus.
5. Take one drop of the mixture and put in onto the specimen glass, cover it, and observe!

There are 2 types of observation preparation using a microscope:

1. Wet preparation
a. This preparation uses a fresh sample and directly given reagent to give color.
b. The sample is easily broken/damaged, usually for about 30 mins to 1 hour.
c. Uses 5x/10x/40x magnification.
d. The staining agent for observing parasites usually is iodine.
2. Dry/permanent preparation
a. This preparation is the most common because the sample lasts longer.
b. The sample takes time to be prepared before being observed, usually 1-2 hours
because it needs to be stored in solution
Can use 100x magnification and add oil-immersion before we observe

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3 & 4. CELL OBSERVATION
Animal cells usually are colorless and translucent. So we have to give color by adding reagent
(staining specimen). Examples of staining agent and the common use are:
❖ Feulgen: DNA
❖ Methylene blue: proteins, nucleus
❖ Comassie: protein
❖ Periodate: carbohydrate
❖ Janus green: mitochondria

How to differentiate parasite cell from others?

❖ Parasites are prokaryotic, must have nucleus and nucleolus. Cell debris, on the other hand,
does not have a nucleus and looks like dust around the nucleus. Cell debris could be a lipid, red
blood cell, etc.

A. Observing Cells
Gram + = purple ; Gram - = pink

1. Escherichia coli
Gram - ; small sized; rod-shaped (bacilli)

Bacterial culture Bacteria – gram stained

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 2. Staphylococcus aureus

3. Candida albicans

budding yeast cells

4. Salmonella sp. Flagella

slender bacilli, petritrichous (projected to all directions) flagella

Todar's Online Textbook of Bacteriology

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5. Aspergillus sp.

6. Cryptococcus neoformans

7. Clostridium tetani

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8. Bacillus subtilis

9. Entamoeba coli (dry preparation)

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 10. Entamoeba coli (wet preparation)

fresh stool --> 30 minutes to 1 hour

Preparing the wet mount:

1. Drop saline solution to the glass slide
2. Take the fecal matters with tusuk sate, then mix it with the saline on the glass slide
3. Drop iodine & mix
4. Put cover slip

B. Prepare ‘dem specimens

1. Immersion : using alcohol or formaldehyde
2. Cutting the cells into thin slices using a Microtome (equipment)
3. Staining : depending on what the specimen is or you want to observe from the specimen
(e.g. methyl blue stain for protein, janus green stain for mitochondria)
4. Centrifugation : seperation of components to obtain the component you want to observe
from the pallete

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