Experiment 2: BIO 111

LIGHT MICROSCOPIC OBSERVATION OF ONION PLANT CELLS AND ANIMAL

CHECK CELLS, THE UNSTAINED AND STAINED
Aim
• To do microscopic observation of onion plant cells and human check cells (epithelial cells)
and then calculate total magnifications and lengths of each of the individual cells observed.
Introduction
The cell theory states that, all living things are composed of cells which are basic units of life, and that
all cells do arise from existing cells. The cell in this case, is the fundamental unit of life and every
living organism is composed of them. Modern cell theory states that, all living matter is composed of
cells and that, all new cells do arise from other cells. In addition to this theory, it is known that, all
metabolic reactions of any organism do take place inside the cells and that; cells contain hereditary
information of those organisms of which they are a part of. All cells are similar in one-way or another
in that, they have a cell membrane and a nucleus at some stage of their existence. Cells show
remarkable diversity in structure and function. Modification in cell structures is very important as it
leads to cells diversity and differences in cell functions.
There are two types of cells, prokaryotic and eukaryotic cells. Prokaryotic cells do not have a nucleus
and membrane true organelles, and are typically significantly smaller than eukaryotic cells.
Prokaryotic organisms are found in domains of bacteria and archaea. While eukaryotic cells have a
nucleus and other membrane organelles. Although there are differences between the cell types, few
features are common to all cells and these are plasma membrane, cytoplasm, ribosomes, and
cytoskeleton. In eukaryotic cells, DNA is found in a nucleus, while in prokaryotes, it is found in the
nucleoid region of a cytoplasm. Generally, prokaryotes have a cell wall made of peptidoglycan and
some have flagella or fimbriae, which are used for movement or attachment. On the other hand,
eukaryotic cells have several more organelles, and among eukaryotic cells, are plant and animal cells.
Organelles common to eukaryotic cells include mitochondria, peroxisomes, vesicles, lysosomes,
smooth and rough endoplasmic reticulum, and Golgi bodies. In animal cells, there are no cell walls
and chloroplasts. While plant cells have chloroplasts and cellulose cell walls.
During microscopic examination of plants and animal cells, transmitted light is used to observe
sample specimen of cells. In this case, specimen to be observed under a microscope need to be
prepared in such a way that, it is thin enough to allow light to pass through it. While to improve on the
clarity of the specimen details and to prevent the specimen from drying out, as well as the protoplasm
of the cell from becoming deformed, small drops of water are usually dropped on the specimen once it
is put on a slide to prepare a wet mount. After observation of wet mounts, then the prepared cells on
the slide are further stained with stains to make some of their structures easier to be examined. In this
case, staining is a technique used in microscopy to enhance contrast in the microscopic image.
Biologists often use stains that bind to various macromolecules and structures in cell to make
structures more visible. Living cells are stained to show cilia, flagella, nucleus, or other cell
organelles. Some stains destroy cells immediately. While others, called vital stains, kill cells more
slowly. In this case, cells absorb stains and continue to carry on their life functions for some time.
In laboratory, there are two main categories of staining techniques, which are direct staining and
negative staining. Under direct staining, cells are stained and the background is left unstained. While
in negative staining, the background is stained and the cells are left unstained. Examples of stains
commonly used in the lab, are the methylene blue, dilute carbol fuchsine and polychrome methylene
blue. In this laboratory activity, we are going to observe the structures of the onion plant cells and the
animal check or epithelial cells using a compound light microscope. In this case, wet mounts will be
observed, and then after that, stained slides of methylene blue or iodine solution will also be observed
and then study the importance of staining when it comes to microscopic observation.
Apparatus/Materials
a. Sharp knife, Petri dish, tissue paper/absorbent paper, onion bulb, dropper, chopping board,
disposable gloves, beaker (250 ml) for used slides
b. Compound light microscope, Iodine solution, methylene blue 1%, 2 microscope slide, cover
slips, toothpick, disinfectant, wash bottle and disposal container for used gloves, tissues,
swabs, toothpicks and disposable mouth swabs.
Procedure Part A-Wet Mount (Unstained)
1. Set up the microscope and place a drop of water on the clean slide.
2. Cut the onion bulb in half and then separate two fleshy leaves of the bulb. Locate the
epidermis between them and after finding the epidermis, peel off the epidermis and cut it into
a small piece.
3. Transfer a piece of this epidermal onto the slide and add a drop of water using a dropper..
4. To avoid creating water bubbles, cover the cover slip on a slide having an onion sample
specimen with a water droplet by placing it at an angle of 45 O to 60 O to cover the specimen
slowly on the slide.
5. Using blotting paper, dry a slide if necessary or if there is too much water and label the slide.
6. Observe the sample slide under the microscope, starting from a scanning objective x 4, then
x10 and finally use the high power objective x4O.
7. Draw well labelled diagrams of your observations. Note: For today, drawings should be done
at only objective x40.
8. Calculate the length of an individual cell sketched/drawn from microscopic using the
following formula Below;
9. Length of Cell = Diameter of Field of View / Number of Cells Lying along the Diameter
of Field of View.
Procedure Part B-Methylene Blue Stain
1. Prepare another slide of onion epidermal cell as you did above for the wet mount.
10. Stain the prepared slide by placing a drop of methylene blue on the well prepared piece of
onion epidermal layer on the glass slide and then cover it with a cover slip at an angle of 45 O
to 60 O slowly to avoid creating air bubbles on the slide of the sample.
2. Using blotting/tissue/absorbent paper, dry the slide carefully if excess stain on the slide.
3. Observe the prepared sample slide under the microscope starting from a scanning x4
objective, then x10 and finally, use high power objective of x40.
4. Draw well labelled diagrams of what you have observed at only objective x40 and make sure
that, you draw only three (3) representative cells for this lab and then calculate the length of
an individual cell sketched/drawn from microscopic using the following formula Below;
5. Length of Cell = Diameter of Field of View / Number of Cells Lying along the Diameter
of Field of View.
Procedure Part C- Iodine Stain
1. Prepare another slide of onion epidermal cell as you did above for Methylene Blue Stain, but
this time use iodine stain by placing a drop of it on the prepared piece of onion epidermal on
the slide then cover it with a cover slip at an angle of 45 O to 60 O slowly in order to avoid
creating air bubbles on the slide of the sample prepared.
1. Using blotting/tissue/absorbent paper, dry the slide carefully if excess stain on the slide.
2. Observe the stained sample slide under the microscope starting from a scanning objective of x
4, then a low power x10, and finally the high power objective of x40.
3. Draw well labelled diagrams of the observations at objective x40 for this lab and then.
calculate the length of an individual cell sketched/drawn from microscopic using the
following formula Below;
4. Length of Cell = Diameter of Field of View / Number of Cells Lying along the Diameter
of Field of View.
Procedure Part D
1. Put a drop of methylene blue on the slide.
2. Gently scrape inside of your cheek with a flat side of the toothpick/mouth swab/ your finger
lightly.
3. Smear the end of a toothpick/finger/ mouth swab on the center of the glass slide and then add
a drop of methylene blue stain on a center of a slide where there is a sample and then safely
throw a toothpick/mouth swab in a designated disposable container and wash your hands.
4. Place a coverslip on the slide having a swab mixed with methylene blue and put the slide on
the microscope stage for observation.
5. Observe the stained sample slide under the microscope starting from a scanning objective of x
4, then a low power x10, and finally the high power objective of x40.
6. After observations, sketch or draw 2 to 3 cells as representatives at x40 objective, and label
their parts observed and against each drawing indicate total magnification by calculation.
7. Without removing the refocused image of cells at x40 objective, observe the cells in a circle
field of view of a microscope eyepiece by targeting those cells laying along the diameter of
the field of view of a circle and count the number of cell stretching along the diameter only.
8. Sketch the number of cells observed that are stretching along the diameter of field of view
and then label them as number of cells lying along the diameter of field of view.
9. Calculate the length of an individual cell sketched/drawn from microscopic using the
following formula; Length of Cell = Diameter of Field of View / Number of Cells Lying
along the Diameter of Field of View.
Instructions
Besides each drawing neatly, record the following information.

 Calculation of total magnification and length of an individual cell

 Estimated number of cells that will stretch across the diameter of the field of view.

 Title of results for microscopic observation which is clear and representing the results.
Task

 Write a full laboratory report and discuss it in details according to the collected results.
Due Date

 Tomorrow at the same time when you came to attend the laboratory

Additional Technical Notes for the Lab

Please make sure you study the Technical Notes below as well before coming to the lab;

Magnification is the product of eye piece and objective lenses as shown below in the table

Field of View and Estimating the Size of Specimens

When you view an object under the microscope you observe that, it lies inside a circular field of
view. Each different magnification of the objective has a different field of view. If you determine the
diameter of the field of view, you can estimate the size of an object seen in that field. As you
increase the magnification, the field of view (and diameter) gets proportionately smaller. As a
consequence, a critter that appears small under scanning power (X4 objective) may appear to be
large under high power objective. The actual size of the critter did not change only the space in
which you placed it for viewing. Refer to the procedure for a discussion on how to determine the
diameter of field of view and how to estimate the size of a specimen viewed with a microscope.

To calculate, the length or size of the cell been observed, first you need to know the objective lens
that you are using. Then after that, use the diameter of field of view of that particular lens u are
using to calculate the size or length of the cell. The table below, shows different diameter of field of
view for different objective lenses, X4, X10, X40 and X100.

Actual Calculations of Size or Length of the Cell is illustrated below;

a. First put your eyes on the eyepiece, focus and observe cells that are laying along the diameter
of the objective lens that you are using

b. Count the number of cells laying along the diameter. Note: In the figure below, the number of
cells laying along the diameters is 8.
c. Since the objective lens that is in use in the figure is X10 with diameter of field of view 2.0
mm. Therefore, to calculate the length of the cell use the following calculations; Length =
Diameter of Field of View/ Number of Cells Laying Along the Diameter (L of Cell = D.F.V/No of
Cells, D) =2/8 = 0.25 mm

1.0 mm = 1000 um
0.25mm = (1000) (0.25) um
= 250 um

d. Note: In the illustration above, 2.0 mm is the field of view since we are using objective X10
(with a total magnification X100). This means that, if 8 of any of the cells have to lay along or
across the diameter of field of view 2mm, then each of those cells should be 2/8 (0,25mm) long.
Since diameter of field of view depends on the power of objective used it is important to know
the diameter of field of view for each of the lens used as indicated above in the table for
diameter of field of view values.