INTERNATIONAL UNIVERSITY (IU)

VIETNAM NATIONAL UNIVERSITY – HCMC

SCHOOL OF BIOTECHNOLOGY (BT)
-------------------------------------------

MICROSCOPY
CELL OBSERVATION
OSMOSIS

Instructor: Tong Thi Hang

LABORATORY REPORT 1
TRẦN VĨNH BẢO NGỌC BTBTIU21228

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LÊ HOÀNG GIA HÂN BTFTIU21104
NGUYỄN PHAN TƯỜNG VI BTBTIU21273
LÊ HÀ PHƯƠNG LY BTBTIU21220

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Contents
I. INTRODUCTION:...........................................................................................3
II. MICROSCOPY.................................................................................................4
1. Definition:.......................................................................................................4
2. Structure:........................................................................................................4
3. Usage:..............................................................................................................6
3.1 General instruction:...................................................................................6
3.2 Preparing the specimen:...............................................................................6
3.3 Using the light microscope:.......................................................................8
III. PLANT CELLS AND ANIMAL CELLS OBSERVATION......................9
1. Introduction....................................................................................................9
2. Procedure........................................................................................................9
2.1. Plant cells................................................................................................10
2.2. Animal cells.............................................................................................11
3. Expected results............................................................................................12
3.1. Plant cells................................................................................................12
3.2. Animal cells.............................................................................................12
4. Discussion......................................................................................................13
IV. OSMOSIS.....................................................................................................14
1. Introduction..................................................................................................14
2. Material.........................................................................................................15
3. Procedure......................................................................................................15
4. Expecting results..........................................................................................16
5. Discussion......................................................................................................18
V. Reference:........................................................................................................19
VI. SUMMARY:.................................................................................................19

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 I. INTRODUCTION:

1. Microscopy is the main technique to visualize and study the structure and
function of cells. Different research areas may rely on microscopy in diverse
ways. Since cells are within the micrometer scale, understanding the cellular
basis of human health and disease requires the spatial resolution of
microscopy.
2. When observing cultured cells, it is important to observe cells non-
invasively because the cells are alive. By using the microscope we can
observe more about the structures of each cell.
3. Human blood has a 0.9% salt concentration, is a little less salty than
seawater, which has a salt concentration of about 3.5%. If we take seawater
as an example of a solution, the salt is called the solute (the particles that are
dissolved) and the water is the solvent (the liquid that dissolves the
particles). Osmosis is a biophysical phenomenon occurring commonly in
biologic systems, in which cells of fluid compartments are separated by
semipermeable membranes. Osmosis describes the diffusion of the solvent
through a semipermeable membrane. Diffusion takes place when the
molecules of a substance tend to move from areas of higher concentration to
areas of lower concentration. The net fluid flux ends when the concentration
of osmotic active molecules is equal in the two compartments.
This report will introduce Microscopy, the producer of Cell observation, and
Osmosis and give out the results of each process.

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II. MICROSCOPY
1. Definition:
- Microscopy is the technique of observing and recording images of
samples and objects that cannot be seen with normal eyes (objects that
are not inside the resolution range of normal eyes) by using microscopes.
- Microscopy is divided into 3 well – known branches: optical, electron,
and scanning probe microscopy, along with the emerging field of X-ray
microscopy.
2. Structure:
- All microscopes have a coordinated system of lenses organized to
magnify images of the specimen. The main differences among the
microscopes are the power that the microscopes need to produce images,
nature and to arrange the lens system.

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 Fig. 1: Principle of compound light microscope
and phase–contrast microscopy

Fig 2: Structure of the compound light microscopes

 Ocular lens (eyepiece): Viewers look through to see the specimen. The
eyepiece normally has a magnification power of 10x or 15x.
 Diopter Adjustment: Uses as a means to change focus on one eyepiece to
correct for any difference in vision between your two eyes.
 Head (body tube): Containing a prism bedding the light so that the light
travels through magnifying lenses from objectives to eyepieces.
 Objective lens: Gathering the light of the specimen and helping to magnify
the image of the specimen. The objective lens can magnify the images up to
4x, 10x, 40x, and 100x.
 Mechanical stage: Where to put the specimen and use stage clips of stage
holder to clamp the specimen. In the light microscope, the stage is movable
and it can move horizontal and vertical by using the X-axis knob and Y-axis
knob.
 Condenser: The lens system under the stage is used to gather and focus
light from the light source onto the specimen. There is a condenser
adjustment control used to adjust the height of the condenser.

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  Aperture iris diaphragm: The hole in the middle of the stage which allows
light from the source to reach the specimen.
 Light source (Illumination): Providing light to the specimen. Being
controlled by the light switch (On/Off) or the voltage of the transformer
attached to the illuminator.
 Coarse Focus Knob and Fine Focus Knob: Bring the specimen into the
general focus and fine-tune the focus to increase the detail of the specimen.
Therefore, to calculate the total magnification of an image of the specimen, take
the power magnification of the objective lens multiplied by the power of the
eyepiece.

MAGNIFICATION = EYEPIECE LENS MAGNIFICATION × OBJECTIVE

LENS MAGNIFICATION

3. Usage:

3.1 General instruction:

- Avoid dropping the microscope, hitting it on a lab bench, or having the
eyepieces fall out.
- Use both hands to hold the microscope erect. Keep one hand on the arm
and the other on the microscope's bottom.
- Keep the microscope away from the bench's edge.
- When not in use, turn off the illuminator and disconnect the power cables
from the power supply.
- While focusing, be careful not to break the coverslip, microscope slide, or
even the objective lens.
- Set the stage to the lowest position first.
- Use the lowest power objective lens to locate the ready specimen, then
switch to the higher power objective lenses.
- Never use the coarse adjustment knob to focus the high power lenses, and
never use these lenses to examine thick specimens.
3.2 Preparing the specimen:
Material: a microscope glass slide, a microscope cover glass, observed specimen.[1]

Step 1: Put the specimen in the middle of the glass slide.

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Step 2: Add a drop of water or alcohol or stain onto a specimen to make the
specimen can be seen more clearly (this step is unnecessary if the specimen is
already wet).

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Step 3: Lower slowly the coverslip starting from the edge until the two glasses
slide stick together. Avoiding the forming of air buddle.

3.3Using the light microscope:

- Use lens-cleaning sheets, clean the eyepieces and objective lens (if
necessary)
- Put the prepared microscope glass slide into the clip
- Lower the stage of the microscope and turn the objective lens to the
lowest power objective (4x)
- Turn on the illumination and diagram and adjust the condenser level
- Use the X and Y-axis knobs to move the specimen into the light region
of the stage
- Use the coarse focus (clockwise direction), raise the stage near to the
objective lens while observing through the eyepieces until the specimen
comes into focus.
- When focusing on the microscope, be careful that the objective lens
doesn’t touch the slide, as it could break the slide and destroy the
specimen.
- Adjust the light intensity as needed with the diaphragm, and center the
specimen by sliding the slide with the X and Y-axis knobs.
- Change the scanning objective (4x) to the high-power objective (10x),
(40x), and (100x). Adjust the focus using the fine focus knob solely to
fine-tune it.

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III. PLANT CELLS AND ANIMAL CELLS
OBSERVATION
1. Introduction
- Cells are the basic unit of all living things. Plant and animal cells are typical
of eukaryotic cells, which means that they all have the plasma membrane
enclosing, a membrane-bound nucleus, and organelles. In eukaryotic cells,
DNA is located in the nucleus. Most cells, both animal and plant, range in
size between 1 and 100 micrometers, which is invisible to our eyes. So, to
gain deep knowledge about cell-like structures (shape, size, organelles…)
and functions, we use the technique called “microscope”. Therefore, we can
observe and compare the differences between plant cells and animal cells.

2. Procedure
Materials:
 Onion bulb
 Light Microscopes
 Glass slides
 Coverslips
 Water
 Lugol solution
 Blades
 Forceps
 Tooth-pick

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2.1. Plant cells
- Prepare onion and cut it into small pieces.
- Use the forceps to peel away a very thin outer epidermis layer and
make sure that the layer is not too thick, or else you will not be able to
see the onion cell structure.
- Place the epidermis layer flat in the middle of the slide. After that, add
2 - 3 drops of distilled water and stain the piece of onion with 2-3
drops of Lugol solution.
- Place a coverslip over the skin carefully in order not to get any air
bubbles under it. Excess solution can be removed by touching one
side of the slide with a paper towel or blotting paper.
- Place the microscope slide onto the stage and secure it with the stage
clips.
- Ensure the magnification is at the lowest setting of 4x lens, to begin
with before observing details of cell structure with higher
magnifications up to 100x.

Figure: How to obtain a piece of onion for slide preparation [2]

2.2. Animal cells

- Gently scrape the inside of your cheek with the broad end of a toothpick.
- Add 1-2 drops of water or 0.9% sodium chloride solution which is isotonic
compared to the fluid inside the cells on the slide. Then, smear your cheek
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 scrapings on a slide. After that, your toothpick must be wrapped in a dry
paper towel and immediately disposed of into the waste container provided.
- Stain the check cells with Lugol solution or methylene blue stain.
- Place a coverslip over the skin carefully in order not to get any air bubbles
under it. Excess solution can be removed by touching one side of the slide
with a paper towel or blotting paper.
- Place the microscope slide onto the stage and secure it with the stage clips.
- Ensure the magnification is at the lowest setting of 4x lens, to begin with
before observing details of cell structure with higher magnifications up to
100x.

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 Figure: How to Prepare a Wet Mount of Cheek Cells [3], [4]

3. Expected results
3.1. Plant cells

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 Figure: Onion epidermal cells stained with iodine seen under a light microscope at 40x
magnification
[5]

3.2. Animal cells

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 Figure: Cheek cells stained with methylene blue seen under a light microscope at 40x
magnification [6]

4. Discussion

1. What is the function of Lugol solution in these experiments?

- Lugol solution is a solution of iodine and potassium iodide in water. In this
case, the Lugol solution acts as a staining dye. Since cells under the
microscope are too transparent for us to observe, the addition of brown drops
of Lugol solution makes it easy to identify the cell’s organelles such as the
nucleus, the cytoplasm, or the cell membrane. In addition to the Lugol
solution, we can also use a methylene blue stain for the same purpose.

2. What is the difference between plant cells and animal cells?

- The biggest difference between plant and animal cells is the appearance of
the cell wall. While plant cells such as onion cells have a cell wall, animal
cells like cheek cells do not. The cell wall functions in protecting the cell
membrane and keeping the cell in shape, so that the organelles are arranged
in a certain order. In contrast, due to the absence of cell walls, animal cells
do not share a uniform shape. Consequently, they are cornered disorderly
and overlap each other.

3. Why is the nucleus much deeper than the cytoplasm when stained with
methylene blue?

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- Methylene blue is a strong oxidizer with a high level of biological activity.
Methylene blue may effectively stain DNA and RNA presenting in the cell
nucleus due to its high affinity for them. When it comes into touch with the
two, it produces a deeper stain that can be seen under a microscope. When a
drop of methylene blue is added, the nucleus becomes stained, making it
stand out and be seen clearly under the microscope. Although the entire cell
appears light blue, the nucleus at the cell's center is significantly darker,
allowing it to be identified.

IV. OSMOSIS
1. Introduction
- Osmosis is a special form of diffusion where water molecules flow across a
differentially permeable membrane from a solution with a lower solute
concentration to a solution with a higher solute concentration. Osmosis is an
energetically “downhill” process. However, osmosis does not require
energy.
- Hypertonic solution has water potential outside the cell lower than inside
the cell so the net movement of water will flow out of the cell
- Hypotonic solution has water potential outside the cell greater than inside
the cell so the net movement of water will flow into the cell.
- Isotonic solution has the water potential on each side of a cell membrane is
the same so there is no net movement of water across the membrane.
1.2. Plant cells
- Plant cells have a strong surrounded cell wall. Plant cells begin to swell
when they take up water. Because of the cell wall, they would not burst.
- Plant cells become turgid in dilute solutions

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 - Plant cells become flaccid in concentration NaCl solutions
- Plant cells become plasmolysis in concentrated NaCl solutions when the
cytoplasm of the cells has shrunk and pulled away from the cell wall.
- Plant cells become incipient in the solution which has exactly the same
osmotic strength as in a state between turgidity and flaccidity.
 Objective: Demonstrate and observe the osmosis and osmotic pressure
using epidemic plant cells from Zebrina pendula leaf.

2. Material
 A leaf of Zebrina pendula
 Distilled water
 5% Sodium chloride solutions
 0.85% Sodium chloride solutions
 Glass slides + Coverslips
 Blades + Forceps
 Microscopes
 Paper towel

3. Procedure

Plant cells - Plasmolysis

- Use a scalpel to peel a thin epidermis layer (purple side) of the Zebrina
pendula leaf.
- Put a small drop of 0.85% NaCl on a clean glass slide.

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 - Place the peeled layer to the saline on the slide. Add a coverslip.
- Examine the plant cells with the high-power lens (40x). Locate the region
where the cells are not too dense.
- Add 2-3 drops of 5% NaCl to the edge of the coverslip
- Make another sample by repeating from step 1 to step 4, replace with 2-3
drops of distilled water to the edge of the coverslip

4. Expecting results

Sample 1:

Cells in 0.85% NaCl solution are isotonic solutions. So, there is no net movement
of water across the cells. The color of the purple area is big and not clear. [7]
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Cells in 5% NaCl solution are hypertonic solutions. So there is the net movement
of water out of the cells. The color of the purple area is clearer and bigger. [8]

Sample 2

Cells in 0.85% NaCl

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Cells in water: the purple area is larger and covers the full cell. The water outside
the cell is greater than inside the cell. So there is the net movement of water onto
the cell. [8

5. Discussion

1. Explain the phenomenon:

- 0.85% NaCl solution is an isotonic solution. When cells are added 0.85%
NaCl solution, the water outside and inside are the same, so there is no net
movement of water across the cells
- 5% NaCl solution is the hypertonic solution. When cells are added to 5%
NaCl solution, the water will go out of the cells because the water inside is
greater than outside.
- Cells in distilled water is the hypotonic solution. When cells are in the water,
cells will be bigger because there is the net movement of water onto the
cells.

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 2. What is the concentration of NaCl in the human cell (in isotonic
solution)
- It is 0.9%. The intracellular fluid of erythrocytes is a solution of salts,
glucose, protein, and hemoglobin. A 0.9% NaCl solution is said to be
isotonic: when blood cells reside in such a medium, the intracellular and
extracellular fluids are in osmotic equilibrium across the cell membrane, and
there is no net influx or efflux of water. [*]

3. Difference between plant and animal cells:

- Plant cells have strong surrounded cell walls. When plant cells are in the
water, they will not burst. Animal cells do not have cell walls so they will be
burst in water.

V. REFERENCE:
[1] https://www.youtube.com/watch?v=pjGMxEqzTpk

[2] Rodriguez, J. Preparing Unstained Red Onion Cells for a Microscope Slide.
Studylib. Retrieved from https://studylib.net/doc/25378336/onion-slide

[3] Tami Guy, MS, CPPS (2012). Cheek Epithelial Cells: How to Prepare a Wet
Mount Microscope Slide. Youtube. Retrieved from
https://www.youtube.com/watch?v=i2x3MKSJez4..

[4] Comparing Cells: Plant, Animal, and Bacteria (prokaryote). Integrated Science
2. Retrieved from
https://www.tamdistrict.org/cms/lib/CA01000875/Centricity/Domain/203/
CellLab.pdf

[5] Cell structure and function. Ruzivo Smart Learning. Retrieved from
https://www.ruzivodigitallearning.co.zw/demo/content/1111/2-19/HS/.

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[6]Bardin, M (2015). Cheek to cheek lab-Animal cells. SlidePlayer. Retrieved
from https://slideplayer.com/slide/3846632/.

[7] Blueprint Digital https://www.youtube.com/watch?v=bEAJjzEK8Ko

[8] https://www.youtube.com/watch?v=ICRG5lTBJ0c

Amrita University's CREATE http://www.amrita.edu/create

[*] https://www.medicine.mcgill.ca/physio/vlab/bloodlab/eryfrag1_n.htm

VI. SUMMARY:
To sum up, two processes: Cell observation and Osmosis contribute to much
important research in mostly the Biology field with the modern Microscopy
technique to observe the two mentioned processes. Through the microscope, we
observe specifically the structures of the plant as well as animal cells. Also, we
know the way to measure the osmotic concentration within living cells.

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