Introductory Practical

USING THE MICROSCOPE

Welcome to the Biology Laboratory

Ø This class gives you the chance to practice using a microscope.

Ø Take your time, work carefully and ensure that you know how all the parts work.
Ø Practice finding, focusing, changing magnification and re-focussing on objects on a slide.

Before the prac:

Go to the LMS (Learning Management System) for BIOL10004
? Complete ILT: Microscope Training and Biological Drawing
Watch TechTips: ‘Welcome to the laboratory’
Watch TechTips: ‘Making a wet mount’
Read through all these Introductory Practical Using the Microscope Student Manual
& instructions

Bring to prac:
P Hard copy of Introductory Practical Using the Microscope Student Manual notes
P Lab coat, safety glasses and appropriate shoes
P Sharp HB pencil, ruler, eraser, pen, calculator
P Student Card (to scan in)

After the prac:

Complete the ILT Microscope Training and Biological Drawing if you have not already
?
done so.

All references for pre-reading refer to:

th
Knox, B., Ladiges, P., Evans, B., & Saint, R. (2015). Biology: an Australian focus (5 ed.). North Ryde,
Australia: McGraw-Hill Education (Australia) Pty Ltd.

Your safety in the laboratory is very important:

• At all times wear a lab coat and suitable shoes with enclosed heel and toe and safety glasses.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Microscope slides and coverslips - use caution. The coverslip glass is very fine and easily
broken. Dispose of used glass slides in the ‘Waste Glass’ bins on your bench.
• Microscopy stains are used in this practical. Wear gloves and do not contact stain

A risk assessment has been carried out for the practical classes and identified risks minimised. Please
observe the safety signs displayed and ask your demonstrator if you would like to know more, MSDS
(Material Safety Data Sheets) are available in the laboratory. Further information can be found at:
http://safety.unimelb.edu.au
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Using the Microscope

The light microscope is an important tool in the study of biology. The scale or size of the object that
you wish to view determines which type is the most appropriate to use. The light microscopes you
will use today can provide up to 400x magnification. Thus, they are the types of microscopes used
to observe bacteria as well as animal and plant cells. Another kind of light microscope, called a
dissecting microscope, produce lower magnifications and are used especially to give a 3-
dimensional view of opaque objects.

Figure IP.1. ‘The Scale of Life’. Reproduced with permission from Sadava et al. 9th Edition 2009.

Biological Concepts covered in this prac:

• All living things are made from cells and the products of cells
• Cells vary in size, with most cells ranging between 1 and 100 micrometres (µm) in diameter.

At the end of this prac you will be able to:

• Find and focus on cells under a microscope
• Observe cell structures using a microscope and make accurate scientific diagrams of what you
see.

Important to note:

• Bring these Introductory Practical pages to all subsequent practicals.

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Part A: Features of a Light Microscope

The light microscopes we use are also called compound microscopes as they have two kinds of
magnifying lenses: objective and ocular (= the eyepiece). Light is transmitted through a very thin
object and is then focussed via the lenses into your eyes. The image you see is inverted.
The microscopes are parfocal, meaning that little or no focus adjustment should be needed after
changing from one objective lens to another. Light microscopes today are generally binocular (with
twin eyepieces), but in the past were often monocular.
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Activity 1: Initial Setting up of the Microscope

1. Place the microscope on the bench with the limb toward you.
Use the coarse focus knob to ensure the stage is fully lowered.
2. If necessary, move the scanning power objective lens (red band) into position above the
condenser. You will feel a ‘click’ when it comes into its proper position.
3. Turn the light switch on. Rotate the light intensity knob to maximum intensity.
4. The amount of light passing through the condenser can be varied by opening and closing the
iris diaphragm. These adjustments are important for achieving good contrast. Locate the iris
diaphragm lever and use it to change the iris diaphragm aperture.
5. The condenser should be almost fully raised. Use the appropriate dial to adjust its position.

Activity 2: Slide Preparation and Use of the Microscope

Equipment • Slides and coverslips
• Plant stems in pond water • Forceps & probes
(collect one Egeria leaf per person) • Blotting paper

Procedure:
First prepare a microscope slide of Egeria.
1. Place a single leaf of the aquatic plant Egeria on a glass slide and add a drop of water.
2. Lower a coverslip over the material.
(Follow steps 1-4 of the diagram.)
The coverslip is lowered gently so that air
bubbles are not trapped.
Use tissues/paper towels to dry the underside of
the slide if necessary, and to mop up any
excess fluid outside the coverslip. This method of preparation is called a ‘wet mount’.
Now observe your freshly prepared slide using the compound microscope.
3. Start with the scanning power objective lens in position.
4. Place your slide correctly on the stage. Pull back the slide clip to fit the slide into position.
5. Use the stage adjustment knobs to position the leaf over the condenser lens.
6. Use the coarse focus to raise the stage as close as you can to the objective lens.
7. Look through the eyepieces. You should be able to see a blurry image. Use the coarse focus
knob to slightly lower the stage until the material comes into focus. Note: if examining material
that is not visible on the slide, the edge of the coverslip can be used to find the focal plane.
8. Adjust the iris diaphragm to see the effect this has on contrast.
9. After examining the leaf at scanning power, swing the revolving nosepiece so the low power
objective lens (yellow band) clicks into place. Use the fine focus knob to improve the focus.
Examine leaf as before. Use stage adjustment knobs to see different areas of the leaf.
10. Slowly rotate the revolving nosepiece until the high power objective lens (blue band) clicks into
place. If you were in focus at low power, the lens WILL click into place without contacting the
slide. Use the fine focus knob to improve focus. If the contrast is too high (i.e., the material
looks too dark) open the iris diaphragm.
Note: Before removing slides, rotate the nosepiece to put the scanning power objective lens in
position.
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Question 1: What detail can be seen in the Egeria leaves at

a) Scanning power (x40)

b) Low power (x100)
c) High power (x400)

Calculating Magnification
The magnification you see is the product of the magnification of the objective lens
(either 4x, 10x or 40x) and that of the ocular (eyepiece) lens which in our microscopes
is 10x.
eg. scanning power provides a magnification of 40x (4 times 10).

Estimating Size of Structures and Scale Bars

The approximate size of microscopic structures such as cells can be determined by
estimating how many of a chosen object (e.g. a cell) would fit across the field of view
Fields of view have known diameters (see below). This technique for estimating size
will be used this semester so that you can draw a scale bar beside a drawing of this
object.
Name and magnification Diameter of field of view
Scanning power (40×) 4.5 mm (4500 µm)
Low power (100×) 1.8 mm (1800 µm)
High power (400×) 0.45 mm (450 µm)

Size of object* (S) = the diameter of field of view (D) divided by an estimation of how
many (N) of the selected object would fit in a series across this diameter. S = D ÷ N
*this could be its length or width or, if it is circular, its diameter.

Question 2: Calculate the width of a typical Egeria cell under low power.
Then repeat this exercise for the same cell at high power.
Compare the results. Why might they have yielded different results?
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Draw an outline of an Egeria cell
Produce a heading (see appendix 5) and scale bar (see box on previous page) for your
drawing.
No labelling is required for this drawing since it is only an outline.

Activity 3: Human Epithelial Cells

Equipment
• Sterile toothpick
• Slides and coverslips
• Methylene Blue stain
• Blotting paper/tissues

Ensure your hands are clean before beginning this exercise.

Procedure
1. Scrape the inside of your cheek using a sterile toothpick.
2. Smear what you have collected onto a slide, spreading it over an area about the size of a
coverslip. Dispose of the toothpick in the general rubbish bin.
3. Place a drop of the stain Methylene Blue on the material and leave for 2 minutes.
4. Place a coverslip on the slide. Remove excess stain using the method described in Activity 1.
[Alternatively you can place a small drop of stain on the slide first and then add the cheek
material by stirring the end of the toothpick in the drop.]
5. Search for cells under the microscope at scanning power. Have the iris diaphragm closed at
this point to improve contrast. Once cells are located, examine them at higher magnifications.
6. In the space provided on the following page, draw a single epithelial cell. Label the
cytoplasm and the nucleus.
7. Use the checklist on the following page to determine whether your presentation is good.
Note: You cannot see cell and nuclear membranes with a light microscope, so it is not
appropriate to apply labels for these structures. For your heading, the scientific name of
humans is Homo sapiens and the preparation type is a smear.
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Drawing

Question 3: Can you see any bacterial cells on the epithelial cells? They will usually be very
small and rod-shaped.
If you can, estimate their size.

Checklist for drawings & diagrams

detailed realistic scale, magnification all required ruled label lines to label lines structures not large
heading scale bar ruled labels label centre of not crossed, shaded etc. drawing
lines structure without
arrowheads

Dispose of the slides you prepared in the Waste Glass bin.

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Appendix 1: Identifying Parts of the Compound Microscope

With the aid of the photograph on page IP - 3 locate the following parts:

Base - the bottom of the microscope, used for support.

Condenser - a lens that focuses light onto the slide, located beneath the stage opening.

Focus adjustment knobs - used to focus the microscope by raising and lowering the stage.
Coarse focus - the large control moves the stage strongly (a high gear). Use this at scanning
power and very carefully at low power, NEVER at high power.
Fine focus - the smaller control moves the stage subtly. It is best used when at low power but
must be the ONLY focus control used at high power.

Iris diaphragm - this is similar to the iris diaphragm found in cameras. Adjusting the iris diaphragm,
with the projecting lever, varies how much light passes from the light source to the slide. Use
this to adjust the contrast of the image. For example, you will often need to shut down the iris
diaphragm (i.e. make the opening smaller) when viewing unstained material.

Light source - light is directed from here, through the lenses and the object on the slide, to your
eyes. You can adjust the intensity of this light by rotating the light intensity knob.

Objective lens - the lens that focuses light (and so an image) from the slide up toward the
eyepiece (and ultimately your eyes). Different objectives provide different magnifications. Your
microscope is equipped with three objectives. Note that the higher the magnification, the
smaller the field of view (the circle of light that you see).
4x lens - the ‘scanning power’ lens. This should be used first to search the slide to find the
region of interest.
10x lens - the ‘low power’ or LP lens.
40x lens - the ‘high power’ or HP lens.

Ocular lens (eyepiece) - the lenses closest to your eyes. These provide a magnification of 10x in
our microscopes

Revolving nosepiece - the rotating portion to which the objective lenses are attached.

Slide clip - a clip mounted on the stage, which holds the microscope slide in place.

Stage - the platform upon which material to be studied is placed. An opening in the centre of the
stage permits light to pass from below through the material being examined.

Stage adjustment knobs – these knobs move the slide clip left-right and forwards-backwards.
This enables you to move the slide around and search for the region of interest.
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Appendix 2: Rules for Using the Microscope

• Always treat your microscope with care. These are expensive pieces of equipment.

• Carry your microscope upright using both hands: one on the limb and the other firmly on
the base.

• Keep all parts clean, particularly the lenses (ocular, objective and condenser). To clean,
use special lens tissue paper provided by staff in the prep room. DO NOT use paper towels or
cloth as these contain coarse fibres that can scratch lens surfaces. If an objective seems
particularly dirty report this to your demonstrator.

• Keep both eyes open while observing materials through the microscope. The distance
between eyepieces can be adjusted to suit your eyes.

• Always use the scanning power objective lens to locate an object. Then, if necessary, switch
to a higher power.

• Only use fine focus at low and high power.

• Rotate the nosepiece slowly when changing magnification.

• Always lower the stage (use the coarse focus adjustment) away from the objective lenses
before placing a slide on the stage and AGAIN before removing the slide.

• Putting the microscope away at the end of a laboratory session:

- turn the revolving nosepiece to put the scanning power lens in position
- remove slide
- if needed, clean all lenses with moist lens tissue paper
- put the cover on the microscope
- push the microscope into the centre of the bench
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Appendix 3: Microscope Troubleshooting Guide

Problem Possible Solution
No image Check:
- Power cord plugged in
- Light switch on
- Light intensity knob turned up
- Iris diaphragm open
- Objective lens clicked into position
- Specimen centred under objective lens
Double image or partial image Check:
- Objective lens clicked into position
- Specimen centred under objective lens
- Slide not upside down
Poor focus or cannot focus Check:
- Both eyepieces are in focus
- Lens is clean (objective/ocular lens)
- Coverslip is clean
- Light intensity knob adjusted correctly
- Iris Diaphragm adjusted correctly
- Slide not upside down
Image too bright Check:
- Light intensity knob adjusted correctly
- Iris Diaphragm adjusted correctly

Appendix 4: Cleaning your Workspace

You are expected to leave your workspace in a neat and tidy condition for the following
classes to use.

• Microscopes - return to the centre of the bench.

• Return all material / equipment to the location from where you collected it.
• Waste Glass: dispose of in the ‘Waste Glass’ bins on your bench.
- Waste Glass includes slides that you have prepared and any broken glass.
- This DOES NOT include watchglasses, test tubes, or pre-prepared slides provided to you
by Biology. These must be returned for reuse.
• Sharps (razor blades): dispose of in the yellow ‘Sharps’ bins on your bench.
• General Rubbish: dispose of in the bins located under the sinks.
- for paper towel, tissues, plastic, plant material and other general waste

DO NOT PUT GENERAL RUBBISH INTO THE WASTE GLASS OR SHARPS BINS!!
• Wipe down your bench with damp paper towel to remove spills, stains and eraser rubbings.
• Push your stool fully under your bench.
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Appendix 5: Biological Drawings

This is a checklist of what every drawing you do in first year Biology should contain.
The terms drawing and diagram have different meanings, but in both cases, it is important to be
accurate in terms of the proportions of structures.
A DRAWING is a portrayal of a cell or cells showing internal detail of the cells.
A DIAGRAM is a plan of structural variation within tissues or organs. It shows recognisable zones
as outlines. Individual cell outlines are not usually drawn.

Use a sharp HB or B grade pencil.

Draw regions/structures that you can distinguish as areas.
Label all structures bolded in the text unless otherwise advised.
Label lines must be ruled, without arrowheads, not crossing over, and extending well into the
structure (i.e., not stopping at the boundary).
Don’t use any shading or stippling.
Headings should contain the name of the organism, name of organ and/or tissue and, if a
drawing, name of cell type. It should also state the type of preparation (eg. wet mount,
section, smear etc.) and type of stain, if any. Species names and genus names must be
underlined (or written in italics if typed) to distinguish them from the rest of the heading.
Indicate the magnification you required to enable you to make the diagram/drawing.
Provide a scale (positioned vertically or horizontally) with the measurement rounded to a
realistic number. The size indication you obtain is a rough estimate, so decimal places are
inappropriate usually.
Make the drawing/diagram as large as possible.

Example of a biological drawing

Broad bean (Vicia faba) root-tip cell with outlines

of adjacent cells. Thin section. Stained with
toluidine blue (400x)