DEPARTMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING FACULTY

OF ENGINEERING
UNIVERSITI PUTRA MALAYSIA

ECH3903
MATERIAL ENGINEERING LABORATORY

GROUP 6

EXPERIMENT WEEK 8: ANALYSE MATERIAL USING

MICROSCOPE TECHNIQUE

LECTURER: DR. MUHAMMAD HEIKAL BIN ISMAIL

EXPERIMENT DATE: 5/11/2019

Authors
Matric Number

DIVAGAR A/L SIVASELVAM

194892

MIIRAA A/P MURUGA

192606

TASNIM ASYIQIN BT TARMIZI

192178

NORHALISA HUSNA BINTI MOHD HALIM

193471
 ABSTRACT

In this experiment, cells are analyzed using the light microscope. For the first part,
onion cell was analyzed by using the outer skin of the onion. The sample was analyzed with
different magnifications. The shape, size, and features are obtained through the analysis using
the light microscope. For the next part, yeast suspension is prepared. From the yeast suspension
prepared, one concentrated yeast suspension and diluted yeast suspension was prepared using
the dilution method. This specimen was also analyzed using the light microscope and
hemocytometer gridlines. The number of cells on the hemocytometer gridlines was counted
between the concentrated and diluted yeast suspension. The hemocytometer gridlines help in
counting the number of yeast cells in a small square area. The number of yeast cells in the
concentrated yeast suspension is higher than the diluted yeast suspension.
 TABLE OF CONTENT

No. Items Pages

1. Introduction 1

2. Objective 2

3. Theory and working equations 2

3-4
4. Materials and method

5. Results 5-8

5. Discussion 9

6. Conclusion and Recommendation 9

7. Acknowledgment 10

8. References 11

9. Appendices 11
 LIST OF FIGURE AND TABLE

No List of figure and table Pages

1. Figure 1.0: Anatomy of a Microscope 6

2. Table 2.0: Image if the cells from each of the mechanical stage 6-7

3. Table 3.0: Cell counted in each image of each set 8

4. Table 4.0: Concentration of the cell for each set 8

 INTRODUCTION

A light microscope uses focused light and lenses to magnify a specimen, usually a cell.
In this way, a light microscope is much like a telescope, except that instead of the object is very
large and very far away, it is very small and very close to the lens.

Light microscopes send light through a path that first focuses the light into a tight beam
and then passes that light through a sample, which creates an image. That image then passes
through one or more lenses to magnify it until it reaches the user's eye or a camera. Because
light needs to pass through the sample, it must be either very small or very thin. Most cells are
both small and transparent, and so light can easily pass through them. This instrument has
several lenses, namely optical lens and objective lens, that produce an enlarged image of a
specimen placed in the focal plane of the lenses.
The ocular lens at the eyepiece has a 10x magnification. Three common lenses are 4x,
low 10x and high 100x. Biological samples for the light microscope particularly compound
microscopes often need to be ‘stained’ colored in some way to make it easier for users to
understand what they’re seeing. This is important because these samples often lack contrast,
which makes it hard to distinguish between parts of the sample. In this experiment methylene
blue solution was used as a staining agent to stain the cells. The onion cell is used as a sample
for this part in the experiment.
Cell counting is rather straightforward and requires a counting chamber called a
hemocytometer. A hemocytometer consists of a thick glass microscope slide with a grid of
perpendicular lines etched in the middle. The grid has specified dimensions so that the area
covered by the lines is known, which makes it possible to count the number of cells in a specific
volume of solution. hemocytometer has an “H” shape engraved in the middle that encloses two
separate mirror-like polished grid surfaces and provides the coverslip mounting area. The
difference between the number of cells in concentrated yeast suspension and diluted yeast
suspension is studied.

1
 OBJECTIVES

From this experiment, the correct method of using a light microscope to examine specimens
is learned. Besides, image processing software is used to extract detailed information on the
specimen.

THEORY AND WORKING EQUATION

The concentration of the corresponding yeast cell suspension for each set could be estimated
from the following large cell formula:

DxN

SxK

where D is the dilution factor,

N is the average number of cell counter,

S is the number of squares counted,

K is the volume of each square.

2
 EQUIPMENT AND MATERIALS

Equipment: Light Microscope equipped with an image acquisition system

Material/chemical: 1. Specimen slide- Onion cell

2. Specimen slide-Yeast suspension (concentrated)

3. Specimen slide-Yeast suspension(diluted)

METHODS

Preparation of specimen for viewing under a microscope

Part A -Specimen: Onion Cell

The top of the onion is cut off and the outer skin is removed. Each onion layer is separated
by a thin layer of skin. A section of this thin layer is peeled off using tweezers. Then, this
section of the skin is placed in a suitable staining agent for about 10 minutes before transferring
onto a clean glass slide. Diluted staining is used to avoid overstaining the specimen. The
specimen is covered using a coverslip.

Part B-Microbial culture suspension-Specimen: Yeast cell

Enough dry yeast is weighed to prepare the specified concentration of culture suspension,
(1000 mg dry yeast in 50ml water). Warm distilled water is added to the volume. The
suspension is centrifuged at 4000 rpm for 10 minutes and the supernatant (liquid part) is
removed carefully without disturbing the accumulated solid at the bottom of the container.
Then, the staining agent is added and the sample is top up with distilled water to volume.

3
Vortex mixer is used to re-suspend the cells for 10 minutes. The suspension is then
centrifuged again at 4000 rpm for 10 minutes and the supernatant is removed. Distilled water
is added to the volume. Vortex mixer is used to re-suspend the cells again. The three steps
above are repeated until a clear supernatant is obtained. The final supernatant is discarded
and distilled water is added to the volume and the vortex mirror is used to re-suspend the
cells. The final suspension is denoted as the stock solution (Suspension A). Another
suspension is prepared with a concentration of choice (Suspension B) by diluting the stock
solution. Soon, a hemocytometer slide and coverslips are prepared is used.

Firstly, both the hemocytometer and its coverslip are ensured to be clean by removing
any dust particles with lens paper. The coverslip is made sure to be placed over the counting
surface before loading the cell suspension. Then, the pipette tip is placed wit the sample into
one of the V-shaped wells and the sample was gently expelled. The area under the coverslip
will be filled by capillary action. Enough liquid should be introduced so that the mirrored
surface is just covered, usually around 10 - 11 µl, but the surface is not overfilled. Two
samples can be load on one hemocytometer, one into each of the two grids. The loaded
hemocytometer is then placed on the microscope stage and the counting grid is brought into
focus at lower power. The sample is allowed to settle for a couple of minutes and the
coverslip is avoided from moving as it might introduce the air bubble and make the counting
difficult.

4
 RESULT

Part 2:

Main part of a microscope:

Main Part Function

Eye-Piece The lens the viewer looks through to see the specimen. The eyepiece
usually contains a 10X or 15X power lens.

Microscope The microscope tube is attached on top of the arm. It can be of the
Tube monocular or binocular type. It supports the eye-piece on the upper end

Nose-Piece A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses.

Objective
One of the most important parts of a compound microscope, as they are
the lenses closest to the specimen. A standard microscope has three,
four, or five objective lenses that range in power from 4X to 100X.

Mechanical
Stage The mechanical stage holds the slide and allows it to be moved to the
left, right, forward or backward by rotating the knobs. It is fitted with
fine vernier graduations as on a ruler. This helps in relocating a specific
field of examination.

Condenser
Gathers and focuses light from the illuminator onto the specimen being
viewed.

Coarse and fine

focusing knob The coarse and fine focusing knobs are used to change the distance
between the specimen slide and the objective.

Illuminator
The light source for a microscope

Table 1.0: The main parts of a microscope and its function

5
 Figure 1.0: Anatomy of a Microscope

Mechanical Stage Image of the Cell

5x10

10x10

6
20x10

40x10

50x10

Table 2.0: Image if the cells from each of the mechanical stage

7
Part 3c:

Set A Set B

Image 1 25408 6208

Image 2 24820 6392

Image 3 20090 5541

Image 4 26001 6673

Image 5 25025 5809

Average 24268.80 6124.6

Table 3.0: Cell counted in each image of each set.

Dilution factor of the yeast cell suspension used in Set B: 11

Set Concentration (mol/m3)

Set A 5.339x10^-6

Set B 1.347x10^-6

Table 4.0: Concentration of the cell for each set

8
 DISCUSSION

From the second part of this experiment, images of cell are obtained when observed
under a light microscope with different magnifications. When the lowest magnification is
used, the cell can be seen as a whole and many features of the cell are observed. The shape of
cell walls can be seen when an intermediate magnification is used while structures inside the
cell wall boundary can be seen as the highest magnification is used. There are some
precaution steps when running this experiment. Safeguarding clothing needs to be worn
before using the light microscope. It is a principal safety to protect the observer's body as the
slides that are going to be looked into might contain dangerous biological materials or
chemicals. Precautions are required when preparing samples to seize volatilized materials or
to steer clear of the formation of volatile materials. When examining samples, gases released
from the reaction should be notified whether they are toxic or not.

CONCLUSION

As a conclusion, there are correct process of examining specimen using light

microscope such as observing the samples starting with the lowest magnification first and
ending with the highest magnification. The sample must be brought into its central point to
look over interested region and enhanced properly.

9
 ACKNOWLEDGMENT

Assalamualaikum and Salam Sejahtera, first of all, we are hereby would like to express
our gratitude to all the parties who are involved in this experiment and to those who are
participating to make this report successful. We would like to thanks our dear lecturer, Dr.
Heikal bin Ismail for giving us the opportunity to have the experience to conduct an experiment
on how to use the light microscope that is used for the analysis of the specimen. At the same
time, we also would like to thank our lab assistant, Mr. Termizi for spending his time to assist
us on how to use the machine correctly and efficiently, without her assistant, this experiment
might not have done successfully. It was very hard for us to identify the grid lines in the
hemocytometer, in which En Termizi helped us to identify it with ease.

Our thanks and appreciation go to our group members because of the effort and
commitment given while making this report and also not forgotten the people who have
willingly given their best to help us out with their abilities. It would not have been possible
without the help and support from the people involved itself.

10
 REFERENCES

Grigoryev, Y. (2019, September 2). Cell Counting with a Hemocytometer: Easy as 1,

2, 3. Retrieved from https://bitesizebio.com/13687/cell-counting-with-a-
hemocytometer-easy-as-1-2-3/.

MicroscopeMaster. (2019). Parts of a Compound Microscope with Diagram and Functions.

[online] Available at: https://www.microscopemaster.com/parts-of-a-compound-
microscope.html [Accessed 12 Nov. 2019].

Dostal1, V., Lu1, J., Fueger1, P. T., & DeVerse, J. S. (n.d.). JoVE. Retrieved from
https://www.jove.com/science-education/5041/introduction-to-light-microscopy.

APPENDIX

1. Dilution factor for set B

D = 11/1

=1

2. Concentration set A

= (11x24268.8)/(5x1E-10)

3. Concentartion set B

= (11x6124.6)/(5x1E-10)

Where K (volume),

= 1 mm x 1 mm x 0.01 mm

= 100 nl

= 0.0001 ml x 10^-6

= 1E-10 m3

11