CHAPTER 1:

PRACTICAL 1.1

USING A MICRSOCOPE

KEY WORDS: magnification

Introduction
Light microscopes have been in use since the seventeenth century. Antonie van
Leeuwenhoek observed many single-celled creatures and Robert Hooke also made many
observations. Amongst other things, Hooke studied cork from an oak tree. He noticesd
that the structures resembled the rooms in which monks lived. These rooms were called
cells and so he introduced the term into Biology. During the eighteenth century the main
use of microscopes was by upper class ladies for recreation – to look at butterfly wings
and flower structure. The nineteenth century saw the rise of science and by the mid
1800s Schleiden and Schwann had put forward their ‘cell theory’; that all living things
consisted of cells.

A huge amount of information can be obtained from the light microscope if it is used
properly.

Fig. 1.1.1 shows a typical light microscope.

However, different types of microscope differ in their features. Some have sub-stage
illumination and some do not have a condenser.

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 1. Always carry the microscope carefully. Hold the spine with one hand and keep
the other hand under the base of the microscope.
2. Remove all clutter from your bench.
3. Set up the microscope so that it is not too near the edge of the bench.
4. On some you may need to rotate the head so that as you use the microscope the
lead is railing away from you.
5. Before you view any specimens, clean the objective and eyepiece lenses with lens
tissue.
6. Rotate the coarse focus knob until the objective lenses are about 2 cm above
the stage.
7. Rotate the nosepiece so that the low power objective lens is in line with the body
of the tube and above the aperture (hole) in the stage. It should click into place.
8. Place the microscope slide that you wish to view on the stage. Make sure that
the specimen is above the aperture in the stage. Clip the slide in position, using
both clips.
9. Looking at the microscope from the side, rack down the coarse focus until the
low power objective is about 5 mm above the slide.
10. Now look down the microscope and rack the coarse focus upward until the object
is in focus.
11. You may need to use the fine focus to bring the image into sharp focus.
12. Adjust the sub-stage condenser and iris diaphragm to obtain optimum
illumination. If you are viewing a transparent object you will need to reduce the
amount of light passing through the object by adjusting the iris diaphragm.
13. Examine the whole specimen, moving the slide if necessary.
14. Find a structure that you wish to examine more closely and position then slide so
that this structure is in the centre of your field of view.
15. Rotate the nosepiece so that the next power objective is above the slide and
make sure that it clicks into place.
16. If the object is no longer in focus look at the microscope from the side and rack
the fine adjustment until the objective lens is about 1 mm from the slide.
17. Look down the microscope and rack up the fine adjustment knob until the object
is focussed.
18. Adjust the illumination for maximum clarity.
19. The total magnification is the magnification of the eyepiece lens x magnification
of the objective lens.
20. Don’t use a higher power than is necessary. You will see more if the image is
clear at a lower power than if viewing a blurred image at a higher power. At
lower powers you will also see more of the specimen.

Adjusting the illumination

1. If your microscope does not have sub-stage illumination you will need to use a
lamp. You may need to place a thin sheet of white paper between the bulb and
the microscope to give a more diffuse light.
2. Use the flat side of the mirror and adjust the angle of the mirror so that the
light from the lamp is transmitted through the microscope.
3. The purpose of the condenser is to bring rays of light from a wide angle onto the
object. To focus the condenser, adjust its height until an object such as a pencil

2
 placed in front of the lamp, reflectd by the mirror, is seen in focus at the same
time as the object on the microscope slide.
4. If you are not using a condenser then use the concave side of the mirror to
focus the light.
5. Adjust the aperture of the diaphragm to obtain the optimum light.

Use of oil immersion

If your microscope has a x100 objective lens you will need to use oil immersion.
The slide does not have a coverslip placed on it.
A drop of immersion oil is placed on the specimen on the slide.
Looking at the microscope from the slide rack down the x100 objective lens so that it is
in the oil and just above the microscope slide.
Now look down the microscope and using the fine adjustment knob, very slowly rack up
the objective lens until the specimen comes into view.

Trouble shooting

If you do not have a clear image, think about the following:

Is the slide clean?

Is the objective lens centred?
Is the source of illumination satisfactory?
Are the objective and eyepiece lenses clean?
Is the diaphragm adjusted correctly?
Is the condenser focussed?
Are you using the correct side of the mirror?

When you have finished make sure that you do not leave a slide on the stage of the
microscope. Clean the objective lenses and the stage of the microscope.
Replace the hood over the microscope or put the microscope away in its box.

3
Requirements
Per group

Microscope
Lamp
Thin white paper
Pencil
Lens tissue
Prepared slides: e.g. amoeba, TS leaf, lung tissue, blood smear

4
 PRACTICAL 1.2.

MEASUREMENT OF SPECIMENS SEEN WITH A MICROSCOPE

KEY WORDS: eyepiece graticule stage micrometer calibrate

All microscopic measurements are in micrometres (µ m). These used to be called

microns, so if you see that term anywhere, it is the same size as a micrometre.

There are 1000 µ m in 1mm.

1 µ m = 0.001mm or 10-3mm.

If your microscope has an eyepiece graticule (sometimes rather confusingly known as an

eyepiece micrometer – note the different spelling, this micrometer is a measuring device
not a unit of metric measurement) you will be able to measure any specimens that you
view. The eyepiece graticule has a 1mm distance marked out in divisions; 10 large
divisions, each of which is sub divided into ten smaller divisions.

However, the scale on the eyepiece graticule is arbitrary and needs to first be
calibrated. For this you will need a stage micrometer (see page 2).

When you look at a specimen you should see the eyepiece graticule/micrometer
superimposed over the magnified image of the specimen. Some people find it hard to
see the two things at once; if they focus on one, they cannot see the other. This comes
with practice.

Fig.1.2.1 Eyepiece graticule

Eyepiece graticules on acetate can be purchased quite cheaply from Philip Harris and
inserted into the eyepiece of the microscope.

5
 Fig. 1.2.2. Inserting the eyepiece graticule

When you look at a specimen at low power magnification, with a x10 eyepiece lens and x4
objective lens, the image is magnified 40 times (x10 x4). It may cover 20 small divisions
on the eyepiece graticule.

When you look at the same specimen at higher magnification, say with the x10 eyepiece
lens and a x10 objective lens, the image is now magnified 100 times. It will now cover 50
small divisions on the eyepiece graticule.

We want to find the actual size of the specimen. As it has been observed under a
higher magnification, it has not actually become larger.

Therefore the size of the divisions of the eyepiece graticule is arbitrary. The
divisions will represent a different length at each magnification and we first have to
calibrate the eyepiece graticule.

We do this using a stage micrometer. This is a special slide with a 1mm distance
marked into 10 large divisions; each subdivided into 10 smaller divisions. In other
words, the 1mm is divided into 100 smaller divisions. A micrometer slide can be made by
sell taping an acetate eyepiece graticule onto a microscope slide.

Fig.1.2.3 Micrometer slide

If 1mm is 1000 µ m, then each of the small divisions on the stage micrometer is 1/100
mm
or 10 µ m.

Procedure for calibrating

6
• Place the stage micrometer slide on the stage of the microscope and focus on the
1mm line using your lowest power objective (probably x4)
• Line up the eyepiece graticule with the stage mm line – you can swivel the eyepiece
and move the slide to bring them parallel to each other.

Fig. 1.2.4 Appearance of stage micrometer and eyepiece graticule at x40 magnification

• Count how many eyepiece divisions (epd) are equal to the 1mm (1000 µ m) of the
stage micrometer.

On many modern microscopes 40 epd = 1000µ m at this magnification

Therefore 1epd = 1000/40 µ m

So 1epd = 25 µ m. (At a magnification of x40)

• Keeping the stage micrometer in the centre of the microscope stage, focus on it
using the x10 objective and line up the eyepiece scale on the graticule with the scale
on the stage micrometer.

Fig. 1.2.5 Appearance of stage micrometer and eyepiece graticule at x100 magnification

7
• Count how many eyepiece divisions are equal to the whole scale of the stage
micrometer.

On most modern microscopes 100 epd = 1mm or 1000µ m

Therefore 1 epd = 1000/100 = 10 µ m (At a magnification of x100)

• Now repeat this process using the x40 objective but remember that you will not be
able to see the entire stage micrometer.
• If you have a x100 objective lens (total magnification x100) you can try to calibrate
that as well. This may be difficult and you may need to use a drop of immersion oil
on the slide. If you fill in the table below you may see a pattern emerging and can
calculate the value of 1 epd at x1000 magnification.
• If your microscope has different objective lenses, you can still carry out this
exercise, but your values for the epd will differ from those given above.
• If you do not have a stage micrometer, you can focus on a piece of 1mm graph paper
and calculate the value of 1epd with x4 and x10 objective lenses. You can then
calculate the values with x40 and x100 objective lenses.

Table. 1.2.1 Values of eyepiece divisions at different magnifications.

Magnification by Magnification by Total Value of 1 eyepiece
eyepiece lens objective lens magnification division/µ m

X10 X4 X40 25

X10 X10 X100 10

X10 X40

X10 X100

• Now that you have calibrated your microscope, you do not want to have to do that
each time you use it to measure something. Stick the information on a small label
and stick it onto the base of the microscope.
• When you want to measure something viewed with this microscope, just measure in
epd and convert it to µ m using that calibration chart.

For example if a chloroplast seen under x100total magnification is 1epd long,

then its actual measurement is 10µ m using the scale in this example.

8
• When observing cells or tissues, measure as many structures as possible, for
example:

The length of a cheek or liver cell

The diameter of the nucleus in a cheek or liver cell
The length of a plant cell – onion epidermis cell
palisade mesophyll cell
spongy mesophyll cell
the diameter of the nucleus in these cells
the diameter of a nucleolus
the dimensions of a chloroplast
the thickness of the cellulose cell wall
the length of a prokaryote cell
the thickness of a leaf
the diameter of a vascular bundle in a leaf
the thickness of the palisade layer
the thickness of the spongy layer

Risk assessment
List all the possible hazards and risks of this experiment and indicate the precautions
that you should take.

Requirements
Microscopes
Microscope lamps if microscopes do not have sub-stage illumination
Eyepiece graticules
Stage micrometers (if these are not available, make them using acetate eyepiece
graticules sellotaped to slides)
Freshly made slides of cheek or liver cells (see Practical 1.3), onion epidermis (see
Practical 1.4), prokaryote cells (see Practical 1.7)
Prepared microscope slides: TS leaf

9
 PRACTICAL 1.3

EXAMINING ANIMAL CELLS

Key words: cheek cells liver cells

Introduction
The procedure outlined below is a safe procedure recommended by the Institute of
Biology. It should, like all other practicals, only be carried out under the supervison of a
teacher or lecturer.

Risk assessment
List all the possible hazards and risks associated with this practical and identify the
precautions that you will need to take.

Procedure
1. Take a cotton bud from a newly opened pack. Move it over the inside of your
cheek on one side of your mouth.
2. Smear the cotton bud over a small area in the centre of a clean microscope slide.
3. Immediately place the cotton bud into a container of 1% sodium hypochlorite
solution.
4. Allow the smear to air dry.
5. Heat-fix the smear by passing the slide quickly through the blue flame of a
Bunsen burner.
6. Place two drops of 1% methylene blue stain onto the smear and leave for one
minute.
7. Rinse off the methylene blue.
8. Dry the slide and add a drop of distilled water to the stained smear.
9. Cover with a coverslip and observe the cheek cells under the microscope. Use
low power first.
10. If you wish to observe the cells using x1000 magnification, instead of adding a
drop of water and a coverslip, add a drop of immersion oil and observe using the
x100 oil immersion objective. Make sure that you wipe the oil off this objective
lens when you have finished.
11. Draw three cheek cells as seen under high power. Label cytoplasm, cell
membrane, nucleus. Measure the diameter of a cheek cell and the diamter of a
nucleus.
12. When the cells have been observed, place your slides with their coverslips into a
pot of Virkon.

10
Alternative cells

If you do not want to examine your own cheek cells you can use some liver cells.

1. Using a pipette, place a drop of water on a clean microscope slide.

2. Using fine forceps put a speck of liver tissue into that drop of water.
3. Using a mounted needle, gently macerate the tissue to make a smear over the
slide.
4. Allow it to dry.
5. Add a drop of methylene blue dye and leave for 60 seconds.
6. Rinse off the dye.
7. Place a drop of water over the smear and then place a coverslip over this.
8. Gently blot off any excess liquid, using fine tissue.
9. Examine the cells, using low power first.
10. Make sure that you measure some of the structures that you can see, using the
protocol that you learnt from worksheet 1.2.

11
Requirements
Per group
Cotton buds in sealed pack
Microscope slides
Coverslips
Bunsen burner and mat or a hotplate
Dropper pipettes
Distilled water
Fine forceps
Mounted needle
1% methylene blue stain
Small beaker on which to rest slide whilst adding stain
Tissue for blotting slides
Microscopes
Microscope lamps (if no sub-stage illumination with the microscopes)
Fresh liver
Immersion oil
Pot of Virkon
Small container of 1% sodium hypochlorite

Safety

If the above safe procedure is followed, there is no risk of transmission of pathogens.

After the practical the used cotton buds should be placed in a polythene bag which can
then be sealed and autoclaved.
Slides and coverslips should be washed thoroughly and dried for reuse according to
normal practice.

12
 PRACTICAL 1.4

EXAMINATION OF PLANT CELLS

Key words: cell wall vacuole middle lamella

Introduction
If you cut an onion bulb in half and then separate the fleshy leaf bases, you will be able
to peel off the thin, one-cell thick epidermis layer that is between them. As the
epidermis is one-cell thick, provided that it is laid flat on a microscope slide, the
transparent cells will be seen. Iodine/KI solution will stain the cells and enable you to
more clearly see the nucleus, nucleoli, cytoplasm and cell wall. You may be able to
distinguish the large vacuole inside the cell. You may also be able to see the middle
lamella between adjacent cells.

The shape of each cell in the epidermis layer is partly determined by the pressure of
other cells around it. If a single cell were isolated it would be rounded or oval. In some
cells the nucleus will appear near to one edge of the cell and is some the nucleus will
appear in the centre of the cell, but it is still surrounded by cytoplasm connected by
strands to that lining the wall.

Risk assessment
List all the possible hazards and risks associated with this practical and identify the
precautions that you will need to take.

Procedure

1 Place 2 drops of iodine/KI solution onto a clean microscope slide.

2 Peel off a small piece of onion epidermis from between two fleshy leaf bases of
an onion bulb and place it, flat on the slide, in the iodine solution.

3 Carefully add a coverslip and blot off any surplus liquid.

4 Observe the slide. Use the lowest power objective first and then use the x10
and x40 objectives.

5 Draw two or three cells. Label as many structures as you can see.

6 Measure: the length of a cell, the width of a cell, the diameter of a nucleus and
of a nucleolus.

13
Requirements

Per group:
Microscope
Lamp
Mounted needle
Iodine/KI solution with dropper pipette
White tile
Knife
Onion Fine forceps
Tissue for blotting surplus iodine/KI solution from slide
Microscope slides
Coverslips
Lens tissue

14
 PRACTICAL 1.5

EXAMINATION OF ANIMAL TISSUES

Key words: epithelial tissue connective tissue cartilage Smooth muscle

Introduction
Although single-celled organisms carry out all metabolic reactions within one cell, in
multi-celled organisms there is a division of labour. Cells become specialised to perform
particular functions – this is cell differentiation. Such specialised cells are grouped
together. A collection of similar cells (they may be of one type or more than one type)
that perform a specific function is known as a tissue. Examples of tissues in animals
include epithelial tissues (such as ciliated epithelium and squamous epithelium).

You can examine ready-prepared slides showing squamous and ciliated epithelial tissues.
On permanent slides the specimens have been fixed, dehydrated by immersion in alcohol,
cleared to remove the alcohol so that the material is transparent, embedded in a
substance so that thin sections can be cut, stained to show the different structures and
mounted so that the material is protected and can be viewed over a long period of time.

Procedure

1. Read page 22 of your textbook so that you understand the features of epithelial
tissues.
2. Examine prepared slides showing arteries and veins in transverse section. The
endothelium (innermost layer of the wall) is a layer of one-cell thick squamous
epithelial tissue.
3. Examine a prepared slide showing trachea or bronchi in longitudinal section.
Note that the epithelia here consist of columnar (long narrow) cells attached to
a basement membrane. At the end opposite to the basement membrane the cells
possess cilia.
4. Using the information and diagrams on p 22 of your textbook, make annotated
drawings of squamous epithelial cells and of ciliated, columnar epithelial cells.

Risk assessment
There is really no risk attached to this practical, although damage could arise from
broken slides.

15
Requirements
Per group
Microscope
Lamp
Prepared slides e.g. LS trachea, bronchi
TS artery and vein

For use with the whole class

35mm slides of LS trachea, TS artery and vein.
Slide projector
Screen

16
 PRACTICAL 1.6

EXAMINATION OF PLANT TISSUES

Key words: epidermis parenchyma collenchyma chlorenchyma

Sclerenchyma

Introduction
Although single-celled organisms carry out all metabolic reactions within one cell, in
multi-celled organisms there is a division of labour. Cells become specialised to perform
particular functions – this is cell differentiation. Such specialised cells are grouped
together. A collection of similar cells (they may be of one type or more than one type)
that perform a specific function is known as a tissue. Examples of tissues in plants
include vascular tissue such as xylem and phloem.

You can examine ready-prepared slides showing vascular tissues.

On permanent slides the specimens have been fixed, dehydrated by immersion in alcohol,
cleared to remove the alcohol so that the material is transparent, embedded in a
substance so that thin sections can be cut, stained to show the different structures and
mounted so that the material is protected and can be viewed over a long period of time

Procedure

1. Read pp 22 – 23 of your textbook. You may also need to refer to sections 10.4.1
and 10.2.1 for more details of xylem and phloem cell types.
2. Examine prepared microscope slides of TS stem and root (dicotyledonous plants).
3. In root sections you will see the vascular tissue in the centre and in stems
sections the vascular bundles, containing xylem and phloem, are arranged near
the outer part (see fig. 10.1 p 137 of your textbook).
4. Draw a low power plan of TS stem and TS root, showing the position of vascular
tissue – xylem and phloem. Do not draw any individual cells.
5. Now make a high power drawing of one vascular bundle seen on the TS stem
slide. Annotate your drawing and indicate the magnification used.

Risk assessment
There is really no risk attached to this practical, although damage could arise from
broken slides.

17
Requirements
Per group

Microscope
Lamp
Prepared slides of: TS dicot root
TS dicot stem

For use with the whole class

35mm slides of TS dicot stem and TS dicot root

18
 PRACTICAL 1.7

EXAMINING PROKARYOTE CELLS

KEY WORDS: simple stain oil immersion bacteria

Bacteria and cyanobacteria are prokaryote cells. They lack a membrane-bound nucleus.
They lack all membrane-bound organelles, and their ribosomes are the smaller 70s
ribosomes.

They are small, some are rod shaped (bacillus), some are round (coccus) and some have
other shapes such as spiral/corkscrew.

As they are small they will need to be viewed using a x100 objective lens (total
magnification of x1000), used with oil immersion.

You will use a simple stain that binds to the bacteria to make them visible.

Risk assessment
List all the hazards and risks associated with this experiment and indicate the
precautions that you should take.
Always follow the aseptic procedure for working with micro organisms.

Procedure

1. You should use aseptic technique as this is good practice when carrying out any
microbiology practical. Soak the bench (if you work at a wooden bench it should
ideally be covered with benchcoat) with a disinfectant spray such as 1% Virkon and
leave for 10 minutes. If you need to wipe off surplus, do so with paper towels and
place them in the autoclave bag that should be taped to the end of your bench.
2. Wear a plastic apron that can be disposed of in the autoclave bag. This bag will be
sealed and autoclaved at the end of the practical.
3. You can wear safety spectacles if you wish.
4. Work in the vicinity of a lit Bunsen burner. This creates an updraft to prevent
bacteria from the air contaminating your work, and you will need to sterilise your
wire loop. When not being used to sterilise the loop the flame should be on yellow.
This is cooler and more visible.
5. Hold your loop in the Bunsen burner flame as shown, so that much of the wire is
flamed, not just the loop, and just above the inner blue cone of the flame. Heat the
base of the wire first, near the handle and then slowly draw the wire through the
flame so that the loop is last to be flamed.

19
Fig. 1.7.1. Flaming a loop

6. Take the loop out of the flame and allow it to cool.

7. Hold onto the loop in one hand. Hold the culture bottle in the other hand and using
your loop hand, use your fourth and fifth fingers to remove the top from the culture
bottle of bacteria. Keep hold of the lid using those fourth and fifth fingers. See
practical 15.1 for diagrams.
8. You will have the culture bottle in your other hand and just pass the mouth of the
bottle through the Bunsen burner flame.
9. Dip your loop into the culture and then remove it. There will be a thin film of culture
adhering to the loop.
10. Pass the neck of the culture bottle through the Bunsen burner flame again.
11. While carefully holding the loop, replace the lid on the culture bottle and put the
bottle down, away from the flame.
12. Rub the loopful of culture onto a clean microscope slide. See fig. 1.7.2 (a)
13. Move the loop in a circular motion until the smear is about the size of a 20p-piece.
See fig. 1.7.2. (b). Flame the loop and place it on your heatproof mat.

Fig. 1.7.2 (a) and (b) Fig. 1.7.2. (c)

14 Allow the smear to air dry. See fig 1.7.2 (c) and then quickly pass the slide over
the blue flame of a Bunsen two or three times. See fig. 1.7.2 (d). Do NOT hold
the slide still in the flame.

20
Fig. 1.7.2. (d) Fig.1.7.2. (e)

15 Place the slide over a small beaker or over staining rods that are over a sink or
small container. Place a drop of methylene blue onto the dried smear and leave it for
60 seconds.
16 Using a wash bottle of distilled water, gently rinse off the methylene blue. See fig.
1.7.2 (e). Allow the slide to air dry or dry it in the Bunsen burner flame as before.
17 Place a drop of the immersion oil on the slide over the dried and stained smear.
18 Examine the slide under low power and locate the stained area.
19 Now put the oil immersion, x100 objective lens in place and carefully lower it down
as far as it will go, so that it is in the oil and nearly touching the slide.
20 Now look down the microscope and slowly manipulate the objective lens upwards,
until the bacteria come into view. They will be about 1epd (1µ m) in diameter if
round or about 2-3epd long if rod shaped. If the lens is above the oil you have
moved it too far and need to repeat this step again.
21 When you have finished, place the slides in a beaker of disinfectant, and wipe the oil
immersion objective with lens tissue. Swab your bench again and then put all
instruments used in disinfectant. Place any lens tissues plus your apron and bench
swabs in the autoclave bag and wash your hands.

21
Requirements

48 hour culture of E. coli

48 hour culture of S. albus
Bacteriological loop
Bunsen burner
Mat
1% Virkon disinfectant solution for soaking and swabbing benches
Pots of 1% Virkon disinfectant for used slides
Autoclave bag - 1 per bench
Plastic disposable aprons or lab coats
Safety spectacles
Slides
Microscope with x100 oil immersion objective
Microscope lamp if microscope does not have sub-stage illumination
Immersion oil
Lens tissues
Methylene blue dye 1%
Staining rods or small beaker
Distilled water and pipette

The above will be per pair/group/student depending on resources available.

Safety note:
Students should be given two copies of the Science/Biology department code of conduct
for working with microorganisms and be asked to sign them. They should return one
copy to the senior Curriculum Manager and keep the other copy in their file.

Gloves should NOT be used for this work as they hamper dexterity and a melted latex
glove will cause severe burning as it melts onto the skin.

Recipe for methylene blue

Methylene blue 0.3 g

Distilled water 100 cm3

22
 PRACTICAL 1.8

EXAMINING LEAVES

KEY WORDS: dicot transverse section spongy mesophyll

palisade mesophyll vascular bundle

Introduction
Tissues are grouped together into organs. An organ is a collection of tissues that are
coordinated to perform a variety of functions, although they often have one major
physiological function. In plants, the leaf is an organ made up of the following tissues:
• Palisade mesophyll – carries out photosynthesis
• Spongy mesophyll – adapted for exchange of gases
• Epidermis – protects the leaf and allows gases to diffuse in/out
• Phloem – transports the products of photosynthesis away from the leaf
• Xylem – transports water and ions to the leaf

Procedure

1. Read p 23 of your textbook

2. Examine a prepared slide showing a dicotyledonous leaf in transverse section.
3. Use fig. 1.21 on page 23 of your textbook to help you identify the different
tissues in the leaf.
4. Make a low power plan diagram showing the distribution of the various tissues in
the leaf. Label your plan diagram. Do not draw any individual cells.
5. Observe a vascular bundle using high power magnification and draw one or two
xylem vessels and one or two phloem sieve tube elements. Draw two palisade
mesophyll cells, two spongy mesophyll cells, one upper epidermal cell and one
lower epidermal cell..
6. Measure the thickness of the leaf (not at the midrib).
7. Measure the length of a palisade mesophyll cell.
8. Measure the diameter of a spongy mesophyll cell.

Risk assessment
There is really no risk attached to this practical, although damage could arise from
broken slides.

23
Requirements
Per group

Microscope
Lamp
Eyepiece graticule
Prepared microscope slide: TS dicot leaf

24
 PRACTICAL 1.9

EXAMINING ELECTRON MICROGRAPHS

KEY WORDS: cells organelles

Introduction
The light microscope is relatively cheap and easy to use. Whole and living specimens can
be used and, using dark ground illumination, transparent objects can be viewed without
staining. However, above magnification of x1500 the image may become blurred. This is
due to limits on the resolution of the light microscope. Visible light has a range of
wavelengths of 400 – 700nm. Some of the organelles inside cells are smaller than this
so the light cannot get in between these objects. Hence, two small objects, closer
together than 700nm will be seen as one blurred object.

Electron microscopes are very expensive and require a high degree of skill and training
to operate. Preparation of materials is lengthy and often involves the use of highly toxic
stains. Specimens have to be viewed in a vacuum so living material cannot be observed.
Images are always in black, white or grey, although false colour can be added using
computers.

Instead of a beam of light, a beam of electrons is used. In light microscopes the beam
of light is focussed onto the specimen by a condenser, which is a thick glass lens
mounted under the stage. In electron microscopes the condenser is a vertical magnetic
field produced by the cylindrical electromagnet, which straightens and intensifies the
electron beam. The radiation passes through the specimen and is focussed by an
objective lens. The final image is focussed, by a projector lens, onto a viewing screen
coated with a fluorescent compound, such as zinc sulphide. When irradiated with
electrons the fluorescent substance emits light visible to the human eye. The final
image is photographed to produce an electron micrograph. (Photographs taken of
specimens seen under the light microscope are called photomicrographs.)

The wavelength of electrons is 0.05nm. This should produce a corresponding increase in

resolution but there are technical difficulties that prevent this increase being realised.
The resolving power is actually about 0.5nm, which is 400 times greater than that of the
light microscope. Clear images of 200 000 – 300 000 magnification can be obtained
using the electron microscope.

The transmitting electron microscope uses very thin sections of specimens. The
scanning electron microscope bounces electrons off a specimen.

Sets of electronmicrographs are obtainable from Phillip Harris Ltd. And you can observe
the fine structure of organelles such as mitochondria, chloroplasts, rough endoplasmic
reticulum, nucleus, ribosomes, nucleolus and plasma membrane. The magnification will be
shown so you can measure these structures.

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