LABORATORY 1: Cell Structure & Organisation - I
(September 09 - 13, 2024)
*Required Pre-Lab Viewing:
1. “Preserving the History of the Microcosmos with Prepared Slides” by Journey to the
Microcosmos.
Link: https://www.youtube.com/watch?v=lw8TvhnlMH0
2. “Gram Staining” by Bio-Rad Laboratories.
Link: https://www.youtube.com/watch?v=sxa46xKfIOY
In this lab, we will build on our understanding of light microscopy by preparing samples for
observation.
Before specimens can be examined under the microscope, they usually have to be prepared in
some way. The simplest method is to put the living organism in some suitable medium on a
microscope slide, and to then place a coverslip on top. This is only applicable to certain thin
objects, and such a preparation is only of a temporary nature. More often, the object has to
be stained, using certain dyes, in order to make the object more visible.
It is often desirable, however, to make a permanent preparation – one that will last for many
years. This means that the animal must be killed, fixed, stained and mounted. With small
animals, the whole animals can be mounted – this is known as a whole mount. With larger
animals, and in order to examine the internal structure, it is necessary to cut a thin slice of the
animal. Such a slice is called a section. In yet other cases, it is possible to make a thin smear
of the tissue or culture that one wishes to observe. This smear is then fixed and stained.
In this laboratory class, you will make unstained and stained temporary mounts of both plant
and animal cells and stained permanent smears of bacterial cells. In addition, you will examine
permanent preparations of blue green algae.
EUKARYOTIC CELLS:
Eukaryotic cells are those cells which are organized into complex structures by internal
membranes and a cytoskeleton. The most characteristic membrane bound structure is the
nucleus.
1. Plant cells
Temporary Stained Preparation of Plant Epidermal Cells
You have been provided with epidermal tissue from an onion. Use the following procedure to
stain the tissue:
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1. Place a drop of iodine in potassium iodide on a slide.
2. Use a pair of forceps to strip the transparent "skin" from the inner (concave) surface
of the onion. This inner lining should be the thickness of one cell.
3. Carefully spread the epidermal strip in the stain and apply a coverslip.
4. Examine the preparation at x4 and x40.
Assignment:
a. Calculate the size of a single onion cell at x4.
b. Identify and label the following components on your drawing of the onion (epidermal)
cell at x40:
i. Nucleus: located centrally or along the cells' margin;
ii. Nucleoplasm: the interior of the nucleus;
iii. Nucleolus: tiny, faint body within the nucleoplasm;
iv. Nuclear membrane: the boundary around the nucleus;
v. Vacuole(s): a relatively transparent space that occupies much of the central
portion of cell (vacuoles actually contain a watery solution, or cell sap, in which
sugars, etc., are dissolved);
vi. Cytoplasm: a narrow, lightly-stained band situated along the cells' periphery
(strands of cytoplasm may criss-cross the vacuole);
vii. Cell membrane: the outer boundary of the cytoplasm. This is NOT visible in the
onion cell;
viii. Cell wall: the rigid wall surrounding the cells.
a) b)
Figure 2.1. Onion cells shown under a) x4, and b) x40 objectives.
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2. Animal cells – Human cheek cells
You will use our own cheek cells for this exercise. These cells are very thin and almost
transparent. The cells will be made more visible for observation under the microscope by
staining with methylene blue. The following procedure will be followed to stain the cells, as
will be demonstrated by the instructor.
Staining with methylene blue:
» Scrape the inside of your cheek with a clean scalpel handle or a finger nail.
» Put the resulting tissue on a slide with a drop of tap water.
» Gently place a cover slip on top of the preparation, and then examine under the
microscope.
You should see epithelial cells from the mucus membrane lining the mouth. These cells are
squamous (i.e., they are either square or diamond-shaped in outline). Notice that each cell
has a definite shape, with a cell membrane, a nucleus and cytoplasm.
• Now put a drop of methylene blue stain on the left side of the slide near the coverslip
(do not get stain on the coverslip).
• Pull the stain under the coverslip by using a piece of filter paper to suck out the water
on the right side of the coverslip. The methylene blue will be drawn via capillarity
across and under the coverslip.
• Observe the cells while doing this, and notice that the nucleus of the cell becomes
more easily seen. This is because methylene blue is a vital stain (i.e., a stain that will
colour living cells).
The resulting stained cheek cells are shown in Figure 2.2.
Assignment:
1. Calculate the size of a cheek cell at x4 (= scale bar of your drawing!).
2. Identify and label the following components on your drawing of the cheek cell:
a. Nucleus: a spherical, stained body located in the central portion of the cell;
b. Nucleoplasm: the interior of the nucleus;
c. Nucleolus: a tiny, faint body within the nucleoplasm;
d. Nuclear Membrane: the boundary around the nucleus;
e. Cytoplasm: the lightly-stained portion of the cell outside the nucleus;
f. Cytoplasmic Organelles and Inclusions: tiny, sometimes darkly-stained bodies in the
cytoplasm.
g. Cell (Plasma) Membrane: the outer, boundary of the cytoplasm.
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a)
b)
Figure 2.2. Cheek cells a) x4, and b) x40.
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Prokaryotic Cells:
Prokaryotic cells lack a distinct membrane-bounded nucleus and most of the organelles found
in eukaryotes. However, they do possess a cell membrane and an outer, non-living cell wall.
The cells of bacteria and blue-green algae are prokaryotic.
Bacteria
Most bacteria are one of three shapes (although there are a few other possibilities):
1. coccus (sing.), cocci (pl.): are spherical (coccus = a berry)
2. bacillus (sing.), bacilli (pl.): are rod-shaped (bacill(um) = a little stick)
3. spirillum (sing.), spirilla (pl.): are spiral (spiro = spiral, coil)
While many bacteria live singly, others are found in aggregates or clusters. These aggregates
are named based on the arrangement of the bacterial cells of which they are composed. Using
cocci as an example:
a. diplococcus: are in sets of two
b. streptococcus: are in chains
c. staphylococcus: are in clusters
Figure 2.3
Making stained bacterial smears using Gram stain
With Gram staining, bacteria appear purple or pink. Cells that retain the violet stain are Gram
positive. Gram negative cells are pink (since the cells are destained with acetone-alcohol and,
therefore, show the safranin counterstain).
The following procedure will be followed to stain bacterial cells, as will be demonstrated by
the instructor.
Bacteria collected from our own teeth will be used for this exercise.
1. Using the wide end of a clean toothpick that has been dipped in alcohol, scrape your
teeth near the gums.
2. Spread the scrapings in a small drop of water in a thin film on a clean glass slide.
3. Allow the smear to dry then fix the dry film by passing the slide, film side up, through
the upper part of the flame of a burner.
Stain the smear using the Gram staining technique, which is as follows:
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1. Stain the smear for 2 min with 2 drops of gentian violet and 1 drop of sodium
bicarbonate.
2. Rinse off the stain with iodine, then allow the smear to stand 2 min covered with iodine
solution.
3. Rinse with water.
4. Destain with acetone-alcohol until washings are colourless (usually 30 s).
5. Rinse with water.
6. Counterstain with safranin for 30 seconds and wash with water.
7. Blot dry with filter.
8. Examine under oil immersion.
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A B
C D
Figure 2.4. Bacterial cell types.
Assignment:
a. Identify the major bacterial cell shapes seen in Figure 2.4 A - D.
b. Which of the four examples shown are Gram stained? How did you know this? Specify
which examples are Gram positive or negative?
c. Observe another cyanobacyterium, Anabaena sp. on the demonstration microscopes
located around the laboratory.
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