VCE Biology

AN IMPORTANT TECHNIQUE - USE OF THE MICROSCOPE

Name:_________
Contents
Page/s Content
2-9 and 11-14 Introductory Practical Skills
9-11 Notes on Measuring field diameters, estimating size
and calculating scale
14 Appendix 1: Setting up microscope and Obtaining a
focus
16 Appendix 2: Cleaning slides and coverslips
16-17 Appendix 3: Biological Drawings
17-18 Appendix 4: Estimating Size and Scale

-1-
VCE Biology

AN IMPORTANT TECHNIQUE - USE OF THE MICROSCOPE

PURPOSE:

a) To learn the parts of the light microscope and to understand their

functions.
b) To learn how to use the light microscope.
c) To learn how to make slides for use with the microscope.
d) To learn how to estimate the size of microscopic objects.
e) To learn the conventions for recording microscope observations in the
form of clear diagrams.

INTRODUCTION:

Because so much that is studied in biological work is too small to be seen with the naked eye; the
various types of microscope are essential tools of the biologists.

A microscope has two functions:

1. To resolve fine details

2. To magnify the image so that it is large enough for the detail to be seen easily.

Magnification without resolving power is of little value. Many cheap microscopes provide high
magnification but the image is indistinct- it lacks clarity, and fine details are not visible. A good quality
light microscope, used well, can provide a clear, sharp image at magnifications up to 1000 times the
diameter of the object being examined.

Resolution

Lenses don't produce perfect images of objects. If an object contains much fine detail, the lens may not
reproduce all this detail in the image. The ability of a lens to reproduce fine detail is called RESOLVING
POWER or RESOLUTION. For example, take 2 lines i.e. . With less resolution they are seen as
ONE line and with more resolution they are TWO DISTINCT LINES. If the resolving power of the
microscope only allows us to see them as one line, we cannot see two lines by increasing the
magnification. If we increase the magnification we well see a larger single line that is blurred, not two
distinct lines. The objective lens is responsible for the resolution of the light microscope.

MATERIALS and EQIPMENT:

 CX21 Olympus Binocular Microscope

 eye dropper
 Mini grid
 lens tissue
 microscope slides
 fine forceps
 coverslips
 a mini grid
 piece of newspaper with letter ("e")
 a prepared slide of cheek cells
 scissors
 a length of paper toweling about 40 cm in length
 dissecting needle

-2-
VCE Biology

PROCEDURE:

PART A FAMILIARISATION

Familiarise yourself with the following components and controls: eye piece (ocular), objective lenses
(your microscope has three), specimen holder, specimen holder Y-axis adjustment knob, specimen
holder X-axis adjustment knob, coarse adjustment knob, fine adjustment knob, condenser, condenser
adjustment knob, aperture iris diaphragm ring, main switch, light intensity adjustment, globe
Complete the names of the parts of the microscope on the diagram below.

2. Binocular observation tube

1.
DO NOT adjust either of
these knobs.

4.

3.

7. Microscope
5. frame

6. 8.

10. 9.
11.

13.
17.

14.
16.

15.

12.
11. 10.

-3-
VCE Biology

Components of the Binocular Light Microscope and their Functions

Number Name Function

1 Eye piece (ocular) To magnify the image

2 Binocular observation To hold the ocular and objective lenses in the correct position
tube in relation to each other
3 Objective lens
(three different To magnify and resolve
magnifications)
4 Revolving nose piece To support the objective lenses and allow easy change of
(Turret) magnification
5 Specimen holder To hold slide in place on the stage

6 Stage Supports the slide and allows light to pass onto the slide

7 Microscope frame To support the other components of the microscope

8 Coarse adjustment To move the body tube rapidly to allow focussing using the
knob scanning and low power magnification.
9 Fine adjustment knob To move the body tube slowly to allow focussing under High
Power magnification.
10 Aperture iris To adjust the size of the diaphragm and thus to control the
diaphragm ring amount of light passing through the slide. Allows for the
control of contrast.
11 Condenser Focuses light from source onto slide to improve resolution

12 Condenser adjustment Rotate to lower or raise the condenser. The condenser is

knob usually used in the highest position. DO NOT alter the
condenser position unless it has been lowered. For the work
we are doing it should be at the highest position.
13 Specimen holder Y axis Controls the vertical movement of the specimen holder. In
adjustment knob conjunction with the X-axis adjustment knob, enables you to
easily reposition the specimen on the stage
14 Specimen holder X axis Controls the horizontal movement of the specimen holder. In
adjustment knob conjunction with the Y-axis adjustment knob, enables you to
easily reposition the specimen on the stage
15 Light intensity Adjust the brightness of the light. Rotating clockwise
adjustment knob increases the brightness and anticlockwise decreases the
brightness.
16 Main switch Switches the light on. “|”=(ON)
17 lamp Provides light to illuminate specimen

-4-
VCE Biology

PART B: PREPARING THE MICROSCOPE FOR USE

1. Holding the microscope by the frame and supporting the base, take the microscope out of the
cupboard and carefully remove its cover.
2. Collect a power cord from the storage tub. Position of dot to the side of this plate enables
3. Place the microscope on the bench so that the microscope interpupillary distance value to be read
frame faces away from you and plug in the power cord.
4. Click the scanning (x4) objective into position.
5. Ensure that the condenser is at the top of is movement. It
should already be in this position and should be not adjusted by
you.
6. If necessary adjust the aperture iris diaphragm ring (10) so that
the X4x10 setting is facing forward. (This setting will need to be
readjusted if you click the x40 objective into place)
7. Flick the main switch on. Fig 1.
8. Adjust the interpupillary distance.
 The interpupillary distance adjustment consists of
regulating the two eyepieces according to your eyes so
that you can observe a single microscope image through two eyepieces. This greatly helps to
reduce fatigue during observation.
 While looking through the eyepieces, move both eyepieces until the left and right fields of
view coincide completely. At this point the two separate circular fields overlap and appear as
one.
 The position of the index dot indicates the interpupillary distance value.
(Note the interpupillary distance so that it can be quickly duplicated in future)

My Interpupillary distance: L:_________ R:_________

9. The microscope is now ready to use.

C. MAKING A WET MOUNT OF A “SPECIMEN” FOR EXAMINATION

 Place the rest of the materials and equipment in an orderly arrangement on the length of paper
towelling.
 Obtain one letter ‘e’ from newpaper.
It is important to use a lower case ‘e’ since it has clear orientation. You can clearly tell top from bottom
and left from right.
 Using an eyedropper put 2 or 3 drops of water on a clean slide. (fig. Fig. 2
2) Using fine forceps, place the piece of newspaper in the centre of
the water drop, printed side up. Push it down into the water with
dissecting needle until it is saturated with water and fully immersed.
 Hold the coverslip at an angle of about 45 to the slide; then bring
the coverslip down to the slide until the lower edge touches the drop
of water. (fig.3)

Using the dissecting needle, slowly lower the outer edge of the coverslip
until it is parallel to the surface of the slide. (fig.4) Even when this is
done carefully, air bubbles will occasionally be trapped in the mount. You have now prepared a wet
mount of the letter e.

-5-
VCE Biology

Fig. 4
Fig. 3

D. USING THE MICROSCOPE TO EXAMINE A SPECIMEN

Placing the Slide

 Turn the power on.

 Rotate the coarse adjustment knob (8) in an 5b

anticlockwise direction to fully lower the stage. 8

 Open the bow shaped lever of the specimen holder

(5a) outwards and place your prepared slide by 5a
gliding the glass slide on the stage from the front .
to the rear. Gently return the bow shaped lever
(5b).

 Rotate the upper knob (13), the Y-axis adjustment

13
knob, and the lower knob (14), the X-axis 14
adjustment knob, to move the specimen holder so
that the letter “e” sits centrally over the opening in
Fig. 5
the microscope stage.

Focusing Procedure

 Make sure that the scanning (x4) objective 3.

(3) is in place.
 Look from the side and rotate the coarse
adjustment knob (8) in a clockwise direction
so that the objective lens is as close to the
specimen as possible. (Our microscopes have
an automatic stop which should prevent you 8.
from accidentally hitting the coverslip with the
objective lens however you still need to take
care in case this device fails)
 While observing the specimen through the 9.
eyepieces, slowly rotate the course
adjustment knob (8) in an anticlockwise
direction (towards you) to lower the stage.
15. Fig. 6
 When coarse focusing of the specimen is
obtained rotate the fine adjustment knob (9)
to adjust to a fine focus if required.
 Use the light intensity adjustment knob(15) to adjust brightness if required.
 You should now have a clear sharp image.

-6-
VCE Biology

Working distance:

The Working distance (WD) refers to the distance between each objective lens and the specimen when
precise focus of the specimen is obtained.
The table below indicates the working distance for each objective lens. Note that the higher the
magnification of the objective lens, the small the working distance. This means that when using the high
power objective lens, the tip of the lens will be very close to the specimen.

Objective Magnification 4x 10x 40x

WD (mm) 22.0 10.5 .56

 The brightness of the light should be adjusted so that the image you see is just brighter than the
level of lighting in the room. Use the light intensity adjustment knob (15) to alter the light intensity.

 Try the effect of adjusting the iris diaphragm to alternative magnification settings (x10, x40) as you
look at the letter ‘e’. This alters the size of the aperture (opening) through which light passes.

This procedure has two very useful effects. One is to increase the depth of field. "Depth of field"
refers to the thickness of the plane of focus. With a broad depth of field, parts of a specimen in
different planes can be in focused at the same time. With a narrower depth of field, only parts of the
specimen in one narrow plane can be focused at one time, everything else will be out of focus .
Increasing depth of field means that you can view several “planes” of a thick specimen fairly
effectively simultaneously. This is very useful when viewing unicellular organisms, which are quite
large, or samples that are quite thick.

(1) What is the other useful effect of closing the iris diaphragm?

The image below illustrates the effect of partially closing the iris diaphragm

 Find an air bubble in your own or another student's preparation and examine it under the
microscope, focusing up and down as you do so.

(2) What happens to the appearance of an air bubble when you focus up and down?

-7-
VCE Biology

(3) Draw the orientation of the ‘e’ as it appears on the slide and when viewed through the
microscope.
(don’t worry about size difference or the details of the image. Just record the orientation)

e as it appears on the actual e as it is seen when viewing it

Slide through the microscope

 Compare the position of the image of the letter "e" in the ocular with the position of the actual
printed "e" on the slide.

(4) Describe the change in its orientation.

Moving the slide:

(5) While looking into the ocular, use the x-axis adjustment knob to slowly move the slide
from right to left. Which way does the image move?

(6) While looking into the ocular, use the y-axis adjustment knob to slowly move the slide
away from you. Which way does the image move?

(7) Predict the direction in which you would have to move the slide to make the image move
to the bottom left of the field of view.

 Test your prediction be moving the slide as you suggested .

The image seen under the microscope:

 Turn the rotating nosepiece to bring the low power (x10) objective lens into place.
(Ensure that you feel it click into place. If it doesn’t you will not be able to see anything when you look
down the microscope.)

 View the specimen and sharpen using the fine focus if required
Look at the image of the ‘e’ closely. Besides its orientation, think about how it looks different to the
‘e’ view with the unaided eye.

(8) What PROPERTY of the microscope (apart from the magnification) is illustrated by the
image? Explain briefly.

-8-
VCE Biology

Fine focusing:

Obtaining a focus under High Power

4
 Centre the ‘e’ in the low power field of view, refocus under low .
power if required, and then rotate the revolving nosepiece (4)
so that the high-power (longer) objective is in line with the
ocular. You should feel it 'click' into position. In doing this,
make sure that the lower end of the objective does not touch
the coverslip. Fig. 7

(NOTE: When the high-power objective is in correct focus its lower end will be very close to the slide.
There is only a very small working distance and this is why you need
to be careful)

 Use fine focus to sharpen the image if necessary

 Adjust the aperture iris diaphragm ring(10) so that the X40 12.
setting is facing forward. This opens the diaphragm slightly so 11. 10.
that the brightness of the high-power image is about the same
as when low power is used.
Fig. 8

 Microscopes which remain in focus, or very nearly so, when the nosepiece is rotated, are said to be
'parfocal'

(9) Is your microscope parfocal? Explain

___________________________________________________________________________

(10) Why must you be careful when rotating the nosepiece to bring the high power
objective into place?

THEORY: USING THE MINIGRID AND MEASURING SIZE AND SCALE

-9-
VCE Biology

Field Of View

-10-
VCE Biology

E. MEASURMENT WITH THE MICROSCOPE

Because objects observed with the microscope are usually quite small, biologist find it convenient to use
units of length smaller than centimetres or millimetres for microscopic measurement. One such unit
commonly used is the micrometre (micron) for which the symbol is 'm'. One micrometer is equivalent
to one thousandth of a millimetre. (1m=0.001mm AND 1000 m = 1 mm)

We can estimate the size of a microscopic object by comparing it with the size of the circular field of
view. Details are show in the preceding slides and are summarized in APPENDIX 4.

-11-
VCE Biology

Field diameters

The standard field diameter or various magnifications of the school binocular microscopes are given in
the table below:

MICROSCOPE NUMBER:________

X10 Field Diameters Verify field

Eyepiece Magnifications diameter(tick)
with Or record the
……….. Micrometres/ field diameter
mm microns if it varied
Power used Objective (m) from expected
lens value
scanning X4 4.5mm
Low power X10 1.8mm 1800 m
High power X40 .450mm

Calculate the field diameter in micrometres for the scanning objective and high power objective and
record these in the table above.

Check the field diameter for your microscope at low power using a mini-grid and either verify its
measurement or record its measurement if it varies to what was expected.

To measure the diameter of the low power (x100) field of view:

 Place the 10x objective in position. The eyepiece lenses have a magnification of 10x. The eyepiece
lens and the objective lens together produce a magnification of X100. (ie. 10x10) (This is called
"low power" magnification”). RECORD this in the table above.
 Now place a mini-grid on the stage and obtain focus using previously outlined technique. It will be
necessary to move the mini grid on the stage until a measurement is possible. Your
teacher will assist you if necessary.

Determine the total magnification and check the field diameters for the scanning and high
power objectives.

(11)
a) Compare the diameter of the field of view under high and low power magnifications.

b) Explain why this is the case by referring to the magnification achieved by each objective
lens.

Remember always to obtain focus under low power before attempting to focus under high power.
Always observe the procedures set out above. Now you have these skills, proceed to use the microscope
as a tool for making observations of organisms.

For additional information about microscopes and to play with the virtual scanning microscope go to the
following website: http://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.html

-12-
VCE Biology

F. USING THE MICROSCOPE TO VIEW A PREPARED SLIDE

 Ensure that the microscope is ready for use. Check APPENDIX 1 for a summary of the necessary
steps.
 Collect one of the prepared cheek slides and place it cover slip side up on the stage.
 Obtain focus using first the scanning objective and then using the low power and then the high
power objective. APPENDIX 1 lists the necessary steps.
 Read APPENDIX 3 in order to become familiar with the conventions for drawing biological
diagrams.
 In the space below draw a representative sample of the cells that you see using the high power
objective. This may mean 2 or 3 cheek epithelium cells or one single celled organism.

 Complete your diagram by including a title, magnification, scale and labels as outlined in
APPENDIX 3 and 4. Refer to “Final Drawing” slides on pages 11 and 12 to see how to
set it out.

-13-
VCE Biology

 Skills practise 2: Use Biological drawing conventions to make a high power drawing
of a human white blood cell.(leukocyte)

-14-
VCE Biology

APPENDIX 1

HOW TO SET UP AND USE A MICROSCOPE

A SUMMARY
SETTING UP

1. Holding the microscope by the frame and supporting the base, take the microscope out of the
cupboard and carefully remove its cover. Leave its cover on the teacher’s bench at the front of the
classroom. The laboratory staff will replace these later.
2. Collect a power cord from the storage tub.
3. Place the microscope on the bench so that the microscope frame faces away from you and plug in
the power cord.
4. Click the low power (x4) objective into position.
5. Adjust aperture iris diaphragm ring so that the X4x10 setting is facing forward.
6. Check that the condenser is at the top of its movement.
7. Switch the main switch on.
8. Set the interpupillary distance to suit your eyes.
9. The microscope is now ready to use.

HOW TO OBTAIN AN IMAGE IN FOCUS USING THE MICROSCOPE

1. Make sure the microscope is set up correctly as described above.

2. Rotate the coarse adjustment knob in an anticlockwise direction to fully lower the stage.
3. Open the bow shaped lever of the specimen holder outwards and place your prepared slide on the
stage. (The cover slip must face up!) Make sure that the specimen on the slide is over the opening
of the stage.
4. Use the x-axis and y-axis adjustment knobs to move the slide on the stage until you find the portion
of the specimen that you wish to view. Ensure that this portion of the slide is in the centre of your
field of view.
5. Click the scanning objective (the scanning objective is the shortest of the three objectives) into
position and ensure that the x4x10 magnification is facing forward on the aperture iris diaphragm
ring.
6. Look from the side and rotate the coarse adjustment knob in clockwise direction so that the objective
lens is as close to the specimen as possible.
7. While observing the specimen through the eyepieces, slowly rotate the course adjustment knob (8)
in an anticlockwise direction (away from you) to lower the stage.
8. When coarse focusing of the specimen is obtained rotate the fine adjustment knob (9) to adjust to a
fine focus. You should now have a clear sharp image.
9. To increase the magnification, click the low power objective into position. The microscope is parfocal
and you should be able to see an image.
10. Adjust the focus using either the coarse or fine adjustment knob. At this stage, you can either focus
up or down as the low power objective has an automatic stop; you should only have to alter the
working distance a small amount. Again, centre the image if you want to view the slide using high
power magnification.
11. Carefully rotate the high power objective into position. Adjust the aperture iris diaphragm ring so
that the x40 magnification is facing forward.
12. The image should appear. The focus should be adjusted using the fine focus only. (You will
need to ensure that the objective does not hit the slide) Again, it may be necessary to centre the
image. Adjusting the light intensity and the fine focus may enhance the quality of the image. Slight
alteration of the aperture iris diaphragm ring may assist with obtaining maximum contrast in your
specimen.

-15-
VCE Biology

APPENDIX 2 : CLEANING SLIDES AND COVER SLIPS

Materials to be studied are placed on a piece of glass of standard size called a microscope slide. In most
cases the material is covered with a small, thin piece of glass, the coverslip. Both slide and coverslip
should be as clean as possible before use.

 To clean glass slides, hold them by the edges, between finger and thumb, and dip in water. Wipe
them clean and dry use paper towel.
 Cover slips are much more fragile than slides. Hold
them carefully, by the edges, using a finger and
thumb of one hand, and dip into water. Lens tissue
should then be folded and held between the finger
and thumb of the other hand. Next, insert the wet
coverslip in the fold and gently wipe both surfaces at
the same time by bringing thumb and finger together
(fig 2). A gently circular wiping motion is most Fig. 9
effective. Avoid touching the flat surfaces of slides
and coverslips with your fingers. Always handle them
by the edge.

APPENDIX 3

BIOLOGICAL DRAWINGS

1. Drawing materials:
 A sharp HB pencil. Do not use pen or coloured pencils.
 Good quality unlined paper.

2. Positioning:
 Centre the diagram on the page. This leaves space for titles, labels and annotations to be
added.
 Do not put the diagram in a corner of a page.

3. Size:
 The diagram should be large enough to represent all the details in the specimen without
crowding them.
 The minimum size is about 1/3 of a page. Rarely is a diagram too large.

4. Accuracy: Your diagram should:

 Be accurate and demonstrate your understanding of what you have observed
 Be representative of what you see. You do not have to draw the entire field of view but
rather you should draw enough to show the detail of the specimen and the relationships of
the various parts of the specimen to each other.
 If asked to draw ONE cell, make it a representative cell of those viewed, don’t pick out an
unusual cell.
 Sometimes it is necessary to draw the entire field of view but usually your teacher would
indicate this.
 You will often be asked to draw a sufficient number to show their arrangement. You may
have to use your judgement here, but usually up to four cells is sufficient.
 Show accurate proportions of the specimen

5. Technique:
 Lines should be simple, narrow, sharp and firm.
 When lines indicate an outline then the ends of the line should meet.

-16-
VCE Biology

 Represent depth only when necessary by stippling(dots). Do not use shading or colour.

6. Labels:
 Leave plenty of margin for labels. (NB. You were asked to centre the diagram on the page)
 Label lines must be ruled and they should not have arrow heads on the end.
 Label lines should sit ON the structure they are indicating, not beside it.
 The labels should appear at the end of the label line, they should not sit on the line.
 As far as possible labels should be parallel and horizontal and vertical.
 Names of structures must he horizontal.

7. Title, Size and Scale conventions
Microscope drawings should include the following (unless told otherwise):
 A title, which should identify the material (organism, tissues or cell/s) and if appropriate the
stain that was used in preparation. Singular/plural forms of words matter here since the title
should indicate precisely what the viewer is seeing. For example if one cell is drawn and the title
says cells, then this will confuse the viewer who will be trying to make out where the cells are.
 The magnification under which it was observed.
 A scale to indicate the size of the object. (see APPENDIX 4 for instructions how to calculate
scale)
 In the case of living materials, a brief description of any movement that was observed
 Other annotations neatly made beside the drawing that communicate observations made.

APPENDIX 4
ESTIMATING SIZE AND SCALE

1. Estimate the actual size of the cell you are viewing using known field diameters .
The field diameter when using the x10 ocular and the x10 objective is approximately 1800µm. A
hypothetical x100 field of view is shown below. (Note that you are not required to draw the entire field
of view in your diagram.)Estimate the number of cells that can fit across the field of view.

Field of
view

cells

In the above case approximately three cells can fit lengthways across the field diameter
The length of the cell can thus be estimated as 1800µm÷3=600µm
The width of the cell can be estimated in a similar manner.
This method can also be applied to the high power and scanning magnification using the appropriate
field diameters.

2. Draw your diagram using the appropriate conventions as described previously.

-17-
VCE Biology

3. Measure the actual size of your diagram.

In this case, the diagram is 4.75 cm long.

4. Calculate the scale: To calculate the scale, divide the estimated length of the cell by the length of
the diagram. In this case, 600÷4.75= 126 µm (Round off 126.3. You can approximate as using
decimals would imply an accuracy that we cannot claim)
You are in fact performing an exercise in direct proportion and saying, “ If 4.75 cm represents 600µm
then 1cm represents 126µm”

5. Indicate the scale on your diagram: Draw a 1cm line near your diagram and below it write the
length that the line represents
Magnification x100

126µm

6. Finish your diagram: Remember to include a title and labels as described in APPENDIX 3.

8. Magnification must not be confused with scale; both need to be included in your diagrams.
9. The size of the specimen does not change with magnification. In a diagram made using low power,
1cm will represents a greater distance than in a diagram made using high power magnification.

ADDITIONAL FUNCTIONS

Specimen Holder Scales

These scales allow the position (coordinates) being observed on the specimen to be identified. Even
after the specimen is moved it can be returned easily to the
original position. (assuming that the specimen has not move on A
the slide)

1. The horizontal coordinate can be read at position (A) on

the specimen holder
2. The vertical coordinate can be read at the position of the
index line (B) B

-18-