MBG 113- LAB MANUAL 4

MICROSCOPIC INVESTIGATION OF VARIOUS PLANT CELLS AND SUBCELLULAR

STRUCTURES

1. INTRODUCTION

1.1. PLANT CELLS

Plant cells are usually rectangular shaped eukaryotic cells. They are adjacent to each others with the middle
lamella (Figure 1).

Figure 1. The structure of a plant cell.

Plant cells differ in some aspects from other eukaryotic cells of other organisms. These distinguishing
features of plant cell are;
• They have a large central vacuoles: Vacuoles are surrounded by a membrane called tonoplast, and
the contents are filled with water. Thus, it is responsible for ensuring turgor pressure in the cell, transporting
substances between the cytosol and the plant extract, storing useful materials, and disrupting waste proteins
and organelles. While the vacuole is small in young cells, it grows wide enough to cover the entire cytoplasm
as the cell ages.
• They have a cell wall outside the cell membrane: it consists of cell wall, cellulose and hemicellulose,
pectin and lignin. The cell is called the protoplast to the plant cell, surrounded by cell membranes.

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 • The presence of pores in the cell wall called plasmodezmata: Thus, the cytoplasm of neighboring
cells is continuous and communication between cells is ensured.
• They have plastids: Plastids are protoplasmic structures in the cytoplasm that have different
structures, pigment contents and functions. They are divided into two according to whether they carry color
matter or not. Chromoplast is plastids carrying color matter and leukoplast for colorless ones. Chlorophyll,
which is a green pigment from chromotophores, is called chromoplast in those containing chloroplast,
xanthophyll (yellow), carotene (orange) and lycopene (red) pigments. Leucoplasts have varieties such as
starch-storing amyloplasts, oil-storing elaioplasts.
• Fragmoplast formation during cell division: Fragmoplast provides the formation of a cell plaque in
late cytokinesis.
• The male reproductive cell (sperm) does not carry the flagellum.

1.2. SECTIONING FOR SPECIMEN PREPARATION

The certain tasks in the cell metabolism are performed by cell compartments or organelles. For
example, cell nuclei, mitochondria, plastids, dytosomes, endoplasmic reticulum, vacuoles, lysosomes,
peroxisomes and centrioles are the major organelles. We can examine these organelles in detail only by
electron microscopy. But it is possible to see some of these organelles with light microscopy by taking thin
sections from plant and animal organs. If the plant or animal material to be examined is too large and too thick
to pass the light, thin sections should be taken from these materials. Cutting thin pieces so that they can easily
pass through the light from thick materials is called sectioning. There are mainly three sectioning types (Figure
2):
Cross-section: The tissue is cut from the plane perpendicular to the long axis of the plant organs
(Figure 2A).
Longitudinal section: The tissue is cut from the plane parallel to the long axis of the plant organ.
Oblque section: The tissue is cut at an angle between a cross and longitudinal section.

Figure 2. The sectioning types.

The sections from the materials can be taken in two ways, either manually or with microtome.
1- Manual sectioning: Manual sections are taken straight from fresh or fixed materials. Immediately
put the section in the water to prevent from shrink. The surface of the material is smoothed before taking the
section. Then, the material is held firmly with the left hand and is cut inward from the outside with a sharp
razor blade. At this time, be careful not to crush the material, otherwise the tissues will deteriorate. If the
material to be investigated is too small and too thin to be hand held, the elderberry fruit should be used. In this
case, the elderberry fruit is divided into two vertically, and the material is interlaced and the elderberry and the
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material are cross-sectioned together. Thus, it becomes easier to take the section by increasing the surface.
Then, the elderberry is discarded and the material is examined under a microscope.
2- Sectioning with microtome: Uniform, thin, homogeneous sections can only be taken by
microtome. There are many types of microtomes. The main ones; hand microtome, rotary sleeve microtome,
skid microtome, frozen microtome, ultra microtome. Each of these has its own characteristics, useful or
useless to the work to be done. In order to be able to section through soft or very small materials with the
microtome, the material to be cut must be placed in a solid wrapping, such as paraffin or celloid or the material
must be frozen or hardened.

1.3. MOUNTING A MICROSCOPE SLIDE

There are four common ways to mount a microscope slide as described below:
Dry Mount: In a dry mount, the specimen is placed directly on the slide. A cover slip may be
used to keep the specimen in place and to help protect the objective lens. Dry mounts are suitable for
specimens such as samples of pollen, hair, feathers or plant materials.
Wet Mount: In a wet mount, a drop of water is used to suspend the specimen between the
slide and cover slip. Place a sample on the slide. Using a pipette, place a drop of water on the
specimen. Then place on edge of the cover slip over the sample and carefully lower the cover slip
into place using a toothpick or equivalent. This method will help prevent air bubbles from being
trapped under the cover slip.
Your objective is to have sufficient water to fill the space between cover slip and slide. If there
is too much water, the cover slip will slide around. Take a piece of paper towel and hold it close to
one edge of the cover slip. This will draw out some water. If too dry, add a drop of water beside the
cover slip. Practice this until you get used to it.
Wet mounts are suitable for studying water-bound organisms such as paramecium or bodily
fluids such as saliva, blood and urine.
Section Mount: In a section mount, an extremely thin cross-section of a specimen is used.
Using a microtome, cut a thin slice of your selected specimen such as an onion, and carefully set it
on your slide. Then follow the instructions for a dry or wet mount. A stain can often be applied directly
to the specimen before covering with a cover slip.
Section mounts are suitable for useful for a wide variety of samples such as fruit, vegetables
and other solids that can be cut into small slices.
Smear: A smear is made by carefully smearing a thin layer of the specimen across a slide and
then applying a cover slip. Typically, a smear should be allowed to air dry before applying a stain.

1.4. STAINING OF THE MOUNTED A MICROSCOPE SAMPLE

Stains are used to help identify different types of cells using light microscopes. They give the
image more contrast and allow cells to be classified according to their shape (morphology). By using
a variety of different stains, you can selectively stain different areas such as a cell wall, nucleus, or
the entire cell. Stains can also help differentiate between living or dead cells.
Stains tend to be grouped as neutral, acidic or basic, depending upon their chemical makeup and will
attract or repel different organisms accordingly. For example, scientists and health professionals use Methylene
Blue, a slightly alkaline stain, to reveal the presence of deoxyribonucleic acid, more commonly known as
DNA.
Iodine is one of the more commonly available stains and is used to identify starch in a variety of
samples. It will stain carbohydrates in plants and animal specimens brown or blue-black. Glycogen will show
as red.
Methylene Blue is an alkaline stain useful in identifying acidic cell nuclei and DNA in animal, bacteria
or blood samples. It’s also useful in aquariums to prevent the spread of fungal infections in fish.
Eosin Y is an acidic stain which stains pink for alkaline cells (cytoplasm, for example). It colors red
for blood cells, cytoplasm and cell membranes. Eosin's most important medical uses are in blood and bone-
marrow testing, including the PAP smear.
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 Gram's Stain is one of the most frequently used processes in identifying bacteria – used daily in
hospitals. It is a primary test that quickly and cost effectively divides bacteria into one of two types: Gram
positive or Gram negative.

STAINING STEPS:
1. Prepare a wet mount slide.
2. Collect a drop of stain with an eye dropper or pipette.
3. Put a drop of stain on an outer edge of your cover slide.
4. Place a piece of napkin or paper towel against the opposite side of your cover slip, right up against the edge.
This will help draw the stain under the cover and across the specimen.
5. You may need to add another drop to ensure complete coverage.
6. The slide is now ready for viewing.

2. AIM: To get familiar with the eukaryotic plant cells and their subcellular structures, to prepare of wet mount
samples from sevaral plant part by sectioning tecnique, to identify macromolecule in cells by using a reagent
and to gain mastering in observing of cells under the microscope.

3. PROTOCOLS
3.1. EXAMINING OF Allium cepa (onion) CELLS

MATERIALS:
 Allium cepa (onion)
 Microscope slide/cover slip
 Razor blade
 Droplet
METHOD:
The preparation of wet mount sample for microscopy: Before you start building your slides, make sure
you have everything you will need, including slides, cover slips, droppers or pipets and any chemicals or stains
you plan to use. Wet slides will use a cover slip or cover glass, a very thin square piece of glass (or plastic)
that is placed over the sample drop. Without the cover in place, surface tension would cause the droplet to
bunch up in a dome. The cover breaks this tension, flattening the sample and allowing very close inspection
with minimal focusing. The cover also serves to protect the objective lens from interfering with the sample
drop.
 Take a microscope slide and put 1 drop of water in the center.
 Take a thin superficial piece from the pinkish outer epidermis of the fleshy leaves of the onion
and place on the drop, subsequently place the cover with 45º angle by preventing air bubbles
 Examine the sample at 10 X 40 magnification.
 Draw the observed sample on your notebook by indicating size of the sample.

RESULT:

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 a) b)

Figure 2. General view of cells in Allium cepa (a) and an enlarged view of a cell (b).

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3.2. EXAMINING OF Pyracantha sp.( fire thorn) CELLS

Cells found in free form in fire thorn fruit will be examined. These cells were liberated by the melting
of the middle lamella.
MATERIALS:
 Red fruit of Pyracantha sp.
 Microscope slide/cover
 Razor blade
METHOD:
 Take a fruit and cot into half
 Take a microscope slide and put 1 drop of water in the center.
 Scrape the cut surface with a razor blade and place the sample on the drop
 Examine the sample a at 10 X 40 microscopic magnification.
 Draw the observed sample on your notebook by indicating size of the cells.
RESULT:

Figure 1. Pyracantha sp. mesocarp cells in its fruit.

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 4X 10X 40X

3.3. EXAMINING OF CROMOPLAST in Daucus carota (carrot) CELLS

As it is known, carotene, which gives the carrot root its orange color, transforms into vitamin A in the
body and gives resistance to diseases, prevents eye and skin diseases, and gives vitality to the skin. Carrot
fruits contain resin, essential and fixed oils. The fruits, which are pounded into flour, are used as stomachic,
diuretic and worm reducer.

MATERIALS:
 Daucus carota (carrot) root,
 Microscope slide/cover
 Razor blade

METHOD:
 Cut a handleable piece from carrot and trim the surface with a razor blade
 Take a microscope slide and put 1 drop of water in the center.
 Take a cross section from the sample and place it on the drop.
 Examine the sample a at 10 X 40 microscopic magnification.
 Draw the observed sample on your notebook by indicating size of the sample.

RESULT:

Figure 3. Chromoplasts and carotin crystals in Daucus carota root cells.

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 4X 10X 40X

3.4. EXAMINING OF CROMOPLAST in Lycopersicon esculentum (tomato) CELLS

MATERIALS:
 Lycopersicon esculentum (tomato) fruits
 Microscope slide/cover
 Razor blade

METHOD:
 By taking a cross section from the fleshy part of the red colored tomato (Lycopersicon esculentum)
fruit, it is examined under a microscope at 10 X 40 magnification in a drop of water, between the microscope
slide and lamella.
 There are long, narrow crystalloids structures inside parenchyma cells. These structure contain red
colored lycopene pigments. Examine them and draw their shapes.

RESULT:

Figure 4. Chromoplasts and lycopene crystals in cells of the fleshy part of Lycopersicon esculentum
fruit.

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3.5. EXAMINING OF LEAF ANATOMY
The ground tissue of a leaf, a region called the mesophyll (from the Greek mesos, middle, and phyll,
leaf), is sandwiched between the upper and lower epidermal layers. Mesophyll consists mainly of
parenchyma cells specialized for photosynthesis. The mesophylls of many eudicots have two distinct layers:
palisade mesophyll and spongy mesophyll. Palisade mesophyll consists of one or more layers of elongated
parenchyma cells on the upper part of the leaf. Spongy mesophyll is below the palisade mesophyll. These
parenchyma cells are more loosely arranged, with a labyrinth of air spaces through which CO 2 and oxygen
circulate around the cells and up to the palisade region. The air spaces are particularly large in the vicinity of
stomata, where CO2 is taken up from the outside air and O2 is discharged.

MATERIALS:
 Hoya carnosa (wax flower) leaf
 Microscope slide/cover
 Razor blade

METHOD:
 Take a cross section from Hoya carnosa (wax flower) leaf and prepare microscope sample
 Observe the sample at 10X and 40X magnifications and draw on notebook

RESULT:

Figure 5. The cross section of the leaf of Hoya carnosa.

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3.6. EXAMINING OF LEUKOPLAST in Solanum tuberosum (potato) TUBER CELLS
MATERIALS:
 Solanum tuberosum (potato) tubers
 Microscope slide/cover
 Razor blade

METHOD:
• Scratch some sample off the surface of a potato with a sharp razor blade
• Prepare a microscope sample and observe at 10X and 40X microscopic magnification
• Add 1 drop of diluted iodine solution through one side of the coverslip then absorb excess dye
via placing tissue paper on other side of the slide
• Observe the changes before and after iodine application
• Draw the observed samples on your notebook.

RESULT:

Figure 6. Potato starch grains. The pink oval structures are starch grains in potato tuber cells.
(http://www.microbehunter.com/starch-grains-of-a-potato/).

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