Introductory Practical

USING THE MICROSCOPE

Welcome to the Biology Laboratory

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

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

Before the prac:

Go to the LMS (Learning Management System) for BIOL10002
 Complete ILT: Microscope Training and Biological Drawing
Watch TechTips: ‘Welcome to the laboratory’ and ‘Making a wet mount’
Read through all these Introductory Practical Using the Microscope Student Manual

instructions

Bring to prac:
 Hard copy of Introductory Practical Using the Microscope Student Manual notes
 Lab coat, safety glasses and appropriate shoes
 Sharp HB pencil, ruler, eraser, pen, calculator
 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 are to your textbook:

Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, The Science of Biology (11th Ed.). Sunderland,
USA: Sinauer Associates Inc

Your safety in the laboratory is very important:

 At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
 Wear safety glasses at all times.
 Always work to ensure your safety and the safety of those around you.
 Immediately report any injuries or spills to a demonstrator.
 Wash your hands after class.
 Microscope slides and coverslips - use caution. The coverslip glass is very fine and easily
broken. Dispose of all used glass slides in the ‘Waste Glass’ bins on your bench.
 Microscope stains are used in this practical. Avoid contact with skin, use gloves if appropriate.
A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions used in
class are dilute and present a low risk hazard when laboratory safety rules are followed. 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

Safety information specific to each practical class will be presented at the beginning of that class.
Because of this, students who arrive late to the class will not be permitted to complete the class at
that time.
IP - 2 Introductory Practical: Using the Microscope BIOL10002: 2020

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 (m) 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.

During this practical there will be a Virtual Reality program called “The Cell” for you to
access

Important to note:
 These Introductory Practical pages will be helpful in all subsequent practicals.
BIOL10002 2020 Introductory Practical: Using the Microscope IP - 3

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.

x 10 magnification

x 4, x 10 and x 40
magnification
IP - 4 Introductory Practical: Using the Microscope BIOL10002: 2020

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. Adjust the width of the eyepieces so that you can comfortably see only one circle of light.
5. 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.

Activity 2: Slide preparation and Use of the Microscope

Equipment
 Plant stems in pond water (collect one Egeria leaf per person)
 Slides and coverslips
 Forceps and probe
 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 below).
3. The coverslip is lowered gently so that air
bubbles are not trapped.

4. 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.
5. Start with the scanning power objective lens in position.
6. Place your slide correctly on the stage. Pull back the slide clip to fit the slide into position.
7. Use the stage adjustment knobs to position the leaf over the condenser lens.
8. Use the coarse focus to raise the stage as close as you can to the objective lens.
9. 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, focus on the edge of the coverslip first, then scan around for your
sample.
10. Adjust the iris diaphragm to see the effect this has on contrast.
11. 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.
BIOL10002 2020 Introductory Practical: Using the Microscope IP - 5

12. 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 and the iris diaphragm to improve contrast.
Note: Before removing slides, rotate the nosepiece to back to scanning power.
Question 1: What detail can be seen in the Egeria leaves at;
a) Scanning power (x40)?
b) 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 to create a Scale Bar

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)

Question 2: Calculate the width of a typical Egeria cell under low power (value in whole
numbers). Then repeat this exercise for the same cell at high power. Explain which
magnification would give you the most accurate estimate?
IP - 6 Introductory Practical: Using the Microscope BIOL10002: 2020

Draw an outline of one cell

Present the scientific name of the organism correctly in the heading and include everything
in the check list.

Checklist for drawings & diagrams

detailed realistic scale, magnification all required ruled label lines to label lines clear drawing large
heading scale bar ruled labels label centre of not crossed, (no shading, drawing
lines structure no no fine lines)
arrowheads
BIOL10002 2020 Introductory Practical: Using the Microscope IP - 7

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. Note: You cannot see cell and nuclear membranes with a light microscope,
so it is not appropriate to apply labels for these structures
7. See Appendix (P1 - 12) for an example of a satisfactory heading. The scientific name for
humans is Homo sapiens. The preparation you have done is called a smear.
8. Use the method described previously to estimate the size of your cells.

Question 3: What is your estimate of the size of a human epithelial cell?

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

Dispose of any slide that you make into the waste glass bin.
IP - 8 Introductory Practical: Using the Microscope BIOL10002: 2020

Checklist for drawings & diagrams

detailed realistic scale, magnification all required ruled label lines to label lines clear drawing large
heading scale bar ruled labels label centre of not crossed, (no shading, drawing
lines structure no no fine lines)
arrowheads
BIOL10002 2020 Introductory Practical: Using the Microscope IP - 9

Appendix 1: Identifying Parts of the Compound Microscope

With the aid of the photograph of the compound microscope, 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 or cautiously at low power.
 Fine focus - the smaller control moves the stage subtly. It 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 - ‘scanning power’ lens. This should be used first to search the slide to find the
region of interest.
 10x lens - ‘low power’ or LP lens.
 40x lens - ‘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.
IP - 10 Introductory Practical: Using the Microscope BIOL10002: 2020

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

BIOL10002 2020 Introductory Practical: Using the Microscope IP - 11

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
IP - 12 Introductory Practical: Using the Microscope BIOL10002: 2020

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.

BIOL10002 2020 Introductory Practical: Using the Microscope IP - 13

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).
Do not 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 put 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.
Make the drawing/diagram as large as possible.

Example of a biological drawing

Broad bean (Vicia faba) root-tip cell with outlines of
Observed cells under a adjacent cells. Thin section. Stained with toluidine blue
microscope (400x)

50𝜇m
 Practical 1
CELL STRUCTURE
Ø The most successful students in Biology 10002 Practicals come very well prepared.
Ø It may be some time since you have done practical work so it is important you complete
these set tasks before you come to the laboratory.
Ø There will be no time during the Practical class to ‘catch up’ if you are not prepared.
Before the prac:
P Attend the Introductory Practical (microscope familiarisation)
& Read Sadava et al. 11th Ed., Eukaryotic Cell, pp. 88-108, Biological Membranes, pp.111–
113, Allosteric Enzymes, pp. 166–170.
& Read through all Practical 1 Student Manual instructions
Watch BioBytes: ‘Molecules of Life’ and ‘The cell’
Watch TechTips: ‘Making a wet mount’ and ‘Staining plant cells’
? Complete the Introductory ILT: Microscope Training and Biological Drawing (and the
associated test)
? Complete Pre-practical Quiz (A score of at least 80% is worth 1 mark out of the 10 marks
available for the practical)

Bring to prac:
P Hard copies of
• Introductory Practical Student Manual notes
• Practical 1 Cell Structure 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)

All references for pre-reading refer to:

Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, the science of Biology (11th Ed.). Sunderland,
USA: Sinauer Associates Inc.
Your safety in the laboratory is very important:
• At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
• Wear safety glasses at all times.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Wash your hands after class.
• Microscope slides and coverslips - use caution. The coverslip glass is very fine and easily broken.
Dispose of all used glass slides in the ‘Waste Glass’ bins on your bench.
• Microscope stains are used in this practical. Avoid contact with skin, use gloves if appropriate.

A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions used in
class are dilute and present a low risk hazard when laboratory safety rules are followed. 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

Safety information specific to each practical class will be presented at the beginning of that class. Because
of this, students who arrive late to the class will not be permitted to complete the class at that time.
P1 - 2 Practical 1 : Cell Structure BIOL10002 : 2020

In this practical class you will use the compound light microscope to study different types of cells
and their internal structure.
_____________________________________________________________________________
Biological Concepts covered in this practical:
• Living things are made from cells and their products.
• Cells are organised and have complex internal structures.
• Cells separate their contents from the environment with selectively permeable membranes.
• Cells are active and control movement and functions of their organelles.
_______________________________________________________________________________________
At the end of this practical you will be able to:
• Prepare living material for observation under a microscope.
• Understand the purpose of staining cells in microscopy.
• Observe cell structures using a microscope and make accurate scientific drawings of what
you see.
• Carry out a simple experiment to investigate osmosis, including developing a hypothesis
and drawing a conclusion from your observations.
• During this practical there will be a Virtual Reality program called “The Cell” for you to
access.
________________________________________________________________________________
Assessment
• Pre-practical Test
• In-practical assessment
• Post-practical Test 1

Practical Timeline
Estimated Time
Use this as a guide to how much time you should spend on each activity
Introduction 10 mins
Activity 1: Staining Plant Cells 25 mins
Activity 2: Osmosis in Plant Cells 25 mins
Activity 3: A Living Plant Cell 45 mins
Clean up 10 mins
BIOL10002 : 2020 Practical 1 : Cell Structure P1 – 3

Activity 1: Staining Plant Cells

Staining is an important histological* technique used to enhance contrast and to show the chemical
composition of cells. In this activity you will make and examine a wet mount of potato tissue. The
tissue will then be stained with different stains.
*histology is the study of cells and tissues

Equipment
• Thin shavings of potato tissue
• Slides and coverslips
• Paper towel/tissues
• Distilled water
• Stains: Toluidine Blue, Iodine
Procedure
1. Take a piece of potato tissue (thinner pieces are best) and place it on a microscope slide.

2. Add a drop of distilled water.

3. Lower a coverslip over the material

following the method shown in the
diagram and in the video. Use tissues to
ensure the underside of the slide is dry,
and mop up any excess fluid outside the
coverslip.

4. Examine your slide under the microscope and record your findings in the table on the next
page. Remember to vary the diameter of the iris diaphragm to obtain the best contrast.

5. Remove the slide from the microscope. Lift the coverslip with a dissecting needle and hold
the coverslip in one hand while you add Toluidine Blue with the other. Replace coverslip and
leave for 4 minutes.

6. Rinse the excess stain. To do this, add a drop of water to one side of the coverslip while at
the same time drawing off the fluid under the coverslip by using paper towel at the opposite
side.

7. Re-examine your slide. Take note of any structures that have taken up the stain and their
colour as a result of staining.

8. Repeat steps 5-7 using Iodine.

P1 - 4 Practical 1 : Cell Structure BIOL10002 : 2020

Activity 1: Results
Water List cell structures that are identifiable.

Toluidine Blue Effect of staining.

Iodine Effect of staining.

Question 1: Why would you use different stains on the same tissues?

Dispose of slides that you have made into the Waste Glass containers
BIOL10002 : 2020 Practical 1 : Cell Structure P1 – 5

Activity 2: Observing the Effects of Osmosis in Plant Cells

Osmosis is the net movement of water through a selectively permeable membrane from a region of
higher water potential to a region of lower water potential. Adding solutes, e.g., salts, to one side of a
membrane will lower the water potential in that region and create a water potential gradient across
the membrane. The net movement of water will be from the region with higher water potential (and
lower solute concentration) to the one with the lower water potential (and higher solute concentration).

Aim: The aim of the experiment is to observe the effects of osmosis on rhubarb cells placed in a
saturated salt solution
Equipment
• Rhubarb stems (one each)
• Forceps
• Slides and coverslips
• Paper towel
• Saturated Salt Solution
• Distilled water

Hypothesis:
A hypothesis is a possible explanation of observations. Hypotheses can be used to make predictions,
which can then be tested using experiments.
Before proceeding with the experimental part of this activity, a hypothesis first needs to be developed.
Consider the following questions when writing your hypothesis:

• What is the experimental treatment or biological process/ phenomenon I am investigating?

• What is the variable I am manipulating (i.e., what is being altered)?
• What is the pre-treatment status or control condition for the variable being manipulated?
• What is the change I predict?
• How will I measure/observe this change?

Sample hypothesis: If carrot root tissue was stained with Toluidine Blue, then I predict that the cell walls will
appear blue/purple in colour and be easier to see under light microscopy as compared to unstained tissue.

Incorporating your answers to the questions above, write a hypothesis that includes your prediction of
the observable outcome that saturated salt solution will have on the rhubarb cells:

Guide to writing your hypothesis

The hypothesis includes clear reference to the experimental treatment or biological process/
¨
phenomenon being investigated.
¨ The hypothesis clearly states which variable is being manipulated in the experiment.
¨ The hypothesis includes a comparison to the pre-treatment status or control condition.
¨ The hypothesis includes a prediction of the outcome of the experiment (which is observable).

¨ The hypothesis clearly states how any change will be observed/measured in the experiment.
P1 - 6 Practical 1 : Cell Structure BIOL10002 : 2020

In the table below, draw cells styled like in fig. 1.2 to show how you think the cells will appear when
placed in distilled water and saturated salt solution. Use the faint illustrations showing cell walls as
a guide for your drawing. Redraw the cell walls if you think they will change shape

Cytoplasm
Cell wall In reality this is a
very thin layer that
is not generally
identifiable
Nucleus
Vacuole
Pink due to
pigment. In reality
it appears to fill
the entire cell

Figure 1.2. Stylised cell under electron microscope

Prediction and Observation Table

Experiment Predicted appearance of cells Changes observed (describe in words)

Rhubarb cell
mounted in
distilled water

Rhubarb cell
mounted in
saturated salt
solution
BIOL10002 : 2020 Practical 1 : Cell Structure P1 – 7

Equipment
• Rhubarb stems (one each)
• Forceps
• Slides and coverslips
• Paper towel
• Saturated Salt Solution
• Distilled water

Procedure

1. Use forceps to peel a thin strip of rhubarb epidermis.

Divide the strip into thin pieces that will fit under a cover slip.

2. Mount pieces of rhubarb on a slide and add a coverslip

(NO water at this stage).

3. Observe under the microscope. At low power, search

the strip until you find an area where you can clearly
distinguish cells containing pink pigment.

Figure 1.3. Diagram of making a slide

sample for Activity 2.

Addition of distilled water.

4. Remove the slide from the microscope by only lowering the stage without moving it
from side to side (this will allow you to find the same cells after addition of distilled water).
5. On one side of the cover slip, add 1 or 2 drops of distilled water, while on the opposite side,
use paper towel to draw off the water.
6. Carefully return your slide to the microscope stage and observe the same cells as before.
Record your observations in the table.

7. Repeat steps 4-5 using the saturated salt solution.

Dispose of slides that you have made in the Waste Glass containers

Clean your microscope of any spilled water or salt solution

P1 - 8 Practical 1 : Cell Structure BIOL10002 : 2020

Discussion questions
Question 2: Why does the pigment appear darker in the cells in a saturated salt solution?

Question 3: Explain what happens to the cells in the two treatment conditions.
Try to include some of these words in your explanation: water potential, solute, cell membrane,
vacuole, cytoplasm cell wall, osmosis, plasmolysis, turgid

a. Distilled water:

b. Saturated salt solution:

Question 4: Why don’t the rhubarb cells burst when water enters them?

Question 5: What do you think could happen to an animal cell if it was placed in water? Explain.

Conclusions
When writing conclusions always refer to your predictions (hypotheses). Have the results supported
your predictions? Explain.
 BIOL10002 : 2020 Practical 1 : Cell Structure P1 – 9

Activity 3: A Living Plant Cell

Living cells are active and control the movement and functions of their organelles. Stained cells die
and so this normal cell activity cannot be observed in them. It is possible to observe unstained,
living plant cells under the light microscope if they are kept wet and you adjust the light to increase
the contrast.
______________________________________________________________________________

The surfaces of many leaves are covered with trichomes (hairs). In

pumpkins, each trichome consists of only a few cells. The trichomes you
will look at today are from the leaf stalk (petiole). The cells nearer to
where the trichome attaches to the leaf stalk are called basal cells (see Distal
Figure 1.1). They are cylindrical (rectangular in side view). trichome
cells

Most of the volume of a trichome cell is taken by a central vacuole. The

nucleus can be located anywhere in the cell, and fine strands of
cytoplasm can generally be seen radiating from it. Chloroplasts are Basal trichome
evident either in these strands or at the margins of the cell. They often cells
cluster around the nucleus. The cytoplasm at the cell
margins is generally indistinct, i.e., no boundaries are Stalk cells

evident. Movement of the cytoplasm in plant cells is Figure 1.4. Side view of
pumpkin trichome showing
called cytoplasmic streaming. In cells that are suitable to draw, you cell outlines.
will be able to see this streaming.

The cells nearer the tip of the trichome typically die soon after they develop.
They may persist or they make break off. Do not draw these.

Equipment
• Pumpkin leaves (one each) • Blotting paper/tissues
• Slides and coverslips • Water in beakers, pipettes
Procedure: Preparing a wet mount of pumpkin (Cucurbita pepo) trichomes
1. Add a few drops of water on slide.
2. Take a pumpkin leaf and carefully peel off several
thin, transparent epidermal strips from the leaf stalk.
3. Place strips on the slide and, when properly arranged,
cover with a coverslip
4. Add more water under the coverslip if necessary to
ensure the strip sample is completely covered in
water. Do not allow the material to dry out.
5. Under the microscope, use scanning power until you
find a large trichome in a side-on orientation similar
to Figure 1.4. Adjust your slide so that your desired
cell is within the center of your field of view. Then Figure 1.5. Peeling off a thin epidermal strip
move to low power to closely examine its basal cells. from a pumpkin leaf stalk.
Finally, move to high power to visualise additional
structures within the cell.
6. At high power, spend some time using the fine focus to move the plane of focus through different
levels of the cell. The large central vacuole accounts for why most of the cell is clear. Adjust the
iris diaphragm to get the right contrast, the nucleus will look like a slightly darker area within the
cytoplasm. The cytoplasm should be visible as fine strands. The boundary between the vacuole
and cytoplasm at the cell margins will not be evident.
P1 - 10 Practical 1 : Cell Structure BIOL10002 : 2020

Biological drawing of a living plant cell

Draw a detailed basal trichome cell and the outline of two adjoining trichome cells
(above and below, 3 cells in total, refer to Figure 1.4).

Label the following 5 structures in the cell drawn in detail:

cytoplasmic strand (cytoplasm), nucleus, cell wall, chloroplast, position of vacuole.

Refer to the appendix on the next page for guidance about appropriate presentation of a biological
drawing and mark the checklist below.

Dispose of slides that you have made into the Waste Glass containers

Checklist for drawings & diagrams

detailed realistic scale, magnification all required ruled label lines to label lines structures not large
title scale bar ruled labels label centre of not crossed, shaded etc. drawing in
lines structure without pencil
arrowheads
BIOL10002 : 2020 Practical 1 : Cell Structure P1 – 11

Appendix: 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).
Do not 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 put 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.
Make the drawing/diagram as large as possible.

Example of a biological drawing

Broad bean (Vicia faba) root-tip cell with outlines
Observed cells under a microscope of adjacent cells. Thin section. Stained with
toluidine blue (400x)

50𝜇m
 Practical 2

Cells and Tissues

Ø In this second practical you will need to be familiar with the use of a compound microscope.
Ø You will be expected to plan and carry out a controlled experiment and investigate the
movement of substances in and out of cells
Ø You will use careful observation to describe differences seen in sections of tissue.
Ø This practical is very busy so it is very important you are organised and prepared before you
come.

Before the prac:

Read Sadava et al. 11th Ed. Cells: Chapter Summary, pp. 108, Movement across membranes:
& Section 6.3, pp. 118-123, Glycolysis: Section 9.4, pp. 184-186
& Read through all Practical 2 Cells and Tissues Student Manual instructions
Watch BioBytes: ‘The Cell’, ‘The Scientific Method: Show me the evidence’ and ‘Applying
the Scientific Method’
Complete Pre-practical Quiz (A score of at least 80% is worth 1 mark out of the 10
?
marks available for the practical)

Bring to prac:
P Hard copy of Practical 2 ‘Cells and Tissues’ Student Manual notes
P Lab coat and appropriate shoes
P Sharp HB pencil, ruler, eraser, pen
P Student Card (to scan in)
All references for pre-reading refer to:
Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, the science of Biology (11th Ed.). Sunderland,
USA: Sinauer Associates Inc.

Your safety in the laboratory is very important:

• At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
• Wear safety glasses at all times.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Wash your hands after class.
• Microscope slides and coverslips - use caution. The coverslip glass is very fine and easily
broken. Dispose of all used glass slides in the ‘Waste Glass’ bins on your bench.
• Bromothymol blue indicator solution may be disposed of in the general sink.

A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions
used in class are dilute and present a low risk hazard when laboratory safety rules are followed. 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

Safety information specific to each practical class will be presented at the beginning of that class. Because
of this, students who arrive late to the class will not be permitted to complete the class at that time.
P2 - 2 Practical 2: Cells and Tissues BIOL10002 : 2020

Cells and Tissues

Cells and their products constitute all living things. Cells are constantly exchanging molecules with
their environment across their cell membranes. This exchange is essential to ensure efficient
functioning of the cell through supply of substrates for metabolic reactions and the removal of toxic
or unwanted molecules. Within eukaryotic cells, movement of molecules across the internal
membranes of their cellular organelles is also essential to their function.
In multicellular organisms cells are organised into complex arrangements called tissues to carry out
their necessary functions. Disruption of cell function and organisation can have serious
consequences to the organism as a whole.

In this practical you will consider how the movement of molecules across living cell membranes
affects cell function.

Biological Concepts covered in this prac:

• Cells are surrounded by a selectively permeable membrane and movement of substances across
this membranes can occur in different ways.
• Cells are active and in most cases control the movement of substances across their membranes.
• Movement of water molecules across a selectively permeable membrane is a special case of
diffusion called osmosis.
• Hypotonic, isotonic and hypertonic solutions will affect the functioning of animal cells.
• Cells breakdown complex organic molecules to gain energy for their own cell processes.
• Glycolysis is the initial conversion of glucose to pyruvate.
• Fermentation occurs in the absence of oxygen and in the process converts pyruvate to either
lactic acid or ethanol and carbon dioxide.
• Cells in multicellular organism are organised into tissues and structures which serve a particular
function.

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

• Design a simple experiment.
• Infer the movement of substances across a cell membrane.
• Recognise differences between normal and abnormal tissue
_________________________________________________________________________________
Assessment
• Pre-practical Test
• In-practical assessment
• Post-practical Test 1

Practical Timeline
Estimated Time
Use this as a guide to how much time you should spend on each activity
Introduction 15 mins
Activity 1: Testing Artificial Plasma 40 mins
Activity 2: Molecules for Energy Up to 60 mins run time
Clean up 10 mins
BIOL10002: 2020 Practical 2: Cells and Tissues P2 - 3

Activity 1: Testing Artificial Plasma

The development of artificial plasma for use in emergency medical situations has many advantages:
• There is no need to determine the blood type of the patient prior to administering blood.
• The product is not ‘living’ and so can be stored and transported without refrigeration.
• The product need not have a short expiry date.
• There is no risk of disease transmission from donor to recipient.

One important criterion for an artificial plasma product is that it

must not cause damage to the patient’s red blood cells
(erythrocytes).
The membrane of red blood cells is relatively permeable to
water but fairly impermeable to salts. Net movement of water into
cells causes them to swell and then rupture; this is called
haemolysis. Net movement of water out of cells causes them to
shrink; this is called crenation. If either of these osmotic effects
occurred, it could lead to the death of a patient.

Figure 1. Appearance of normal

erythrocytes.
Red blood cells suspended in an isotonic solution in a test tube, do not transmit light very well and
so the solution appears cloudy. It is difficult to read print through the tube. If the cells swell and burst
(haemolysis) the suspension appears clear and it is possible to read print through the tube. This
process may occur immediately or may take some time. Crenation will not produce this effect but
may be observed using a light microscope.

Your Task
You are working as a medical scientist and you have been asked by a biomedical company to
determine which of their artificial plasma products deserve further development.
1. You are supplied with three plasma samples to test. These are labelled X, Y and Z.
2. You have access to three ‘known’ solutions:
• ‘normal’ saline (which is isotonic to blood plasma)
• distilled water (which is hypotonic to blood plasma)
• saturated salt solution (which is hypertonic to blood plasma)
3. You also have 10mL of mammalian blood, which has been treated to prevent it from clotting.
You may dilute this with normal saline if you wish.

Work in pairs to design and conduct an experiment to determine which artificial plasma product (X,
Y or Z) you would recommend for further development based on its compatibility with mammalian
blood. Make notes and record findings on the following two pages and then write a neat final report
on the hand-in sheet.
Remember to use controls in your experiment and to work with the volumes of blood and solutions
supplied.
P2 - 4 Practical 2: Cells and Tissues BIOL10002 : 2020

Record of Experiment

Question 1: State the purpose of your experiment.

Question 2: List all the factors that you will need to keep consistent to ensure your experiment will
show only the effect of the different artificial plasmas on mammalian blood.

To start, set up controls:

• A positive control should confirm the observable effect of a known solution(s) on your blood
sample.
• A negative control should confirm the appearance of blood in a known solution(s) that has no
effect on your blood sample.

Question 3: Detail your proposed materials and method (your demonstrator must check this
before you proceed with the experiment):
(Hint: this information must ensure that someone else could repeat your experiment. You may
like to use a flowchart to show the sequence of steps you will follow).
BIOL10002: 2020 Practical 2: Cells and Tissues P2 - 5

Record your results:

Test Tube ID Contents of Test Tube Observations

Complete the hand in sheet with the results and conclusions of your investigation.
P2 - 6 Practical 2: Cells and Tissues BIOL10002 : 2020

Activity 2: Molecules for Energy

Glycolysis is the initial metabolic process that all eukaryotic cells use to release energy from
carbohydrates. The final product of glycolysis is pyruvate. If not enough oxygen is available,
fermentation occurs. In fermentation animal cells convert pyruvate to lactate. Plant cells and yeast
cells convert pyruvate to ethanol and CO2.
In an anaerobic environment it is possible to determine the rate of fermentation in yeast cells by
measuring the amount of CO2 released by the fermenting yeast.
In this experiment you will determine which carbohydrate molecule(s) yeast cells are able to readily
utilize. You will be able to measure the CO2 production by monitoring the colour change of
Bromophenol Blue indicator in the solution. CO2 forms an acid in solution and the change in pH will
turn Bromophenol Blue from blue to a pale yellow.
Work in groups of four to set up this experiment. You will need to allow time for the yeast to start
fermenting and to record observations at regular time intervals up to 60 minutes.

Equipment
• 5 McCartney bottles
• 5 solutions: starch, sucrose, fructose, glucose, and distilled water
• Bromophenol Blue indicator solution
• Fresh yeast (1 block per McCartney bottle. Each block = approximately 1 gram yeast)

Procedure
1. Each group will need 5 McCartney bottles, labelled 1 to 5.
2. Using a measuring cylinder, add 20 mL of the appropriate carbohydrate solution into the
corresponding bottle (see table below).
*Rinse measuring cylinder at least three times between each use to avoid cross
contamination of the solutions.
3. Add 10 drops of Bromophenol Blue indicator to each bottle. Gently invert the bottles to mix
the solutions.
4. Add one small portion of the fresh yeast to each bottle and record the time of dispensing
for each bottle.

Bottle Contents
1 starch suspension + indicator
2 sucrose solution + indicator
3 fructose solution + indicator
4 glucose solution + indicator
5 distilled water + indicator

5. Screw on the lid.

6. Place the bottles together in a water bath at 37˚C.
7. Send one member of your group to make observations on the bottles at regular intervals
(how often you observe your vials is up to you, but if there are no changes early on you can
reasonably lengthen the time intervals). Record the time at which the colour changes in
each bottle.
BIOL10002: 2020 Practical 2: Cells and Tissues P2 - 7

Purpose: What is the purpose of this experiment?

Results

Table 1: Record of colour changes in each bottle.

Bottle Contents Results

(note the time to initial colour change)

1 starch suspension

2 sucrose solution

3 fructose solution

4 glucose solution

5 distilled water

Question 4: Which carbohydrates have the yeast been able to take up from the solution and use in
metabolism? Which one was the most readily used in metabolism?

Question 5: What evidence do you have to support this?

Discussion
Question 6: What is the purpose of bottle number 5?

Question 7: What criticisms can you make of the design of this experiment?

Question 8: Using your knowledge of movement across membranes and cell metabolism, suggest
reasons why some carbohydrates are utilised faster than others.

Conclusion
Make a statement about the outcome of your experiment in relation to its original purpose.
P2 - 8 Practical 2: Cells and Tissues BIOL10002 : 2020
 Practical 3
HEART and LUNGS:
GETTING the ESSENTIALS
Ø It takes some practice to understand biological structures from the dissection of real
organs. The diagrams in textbooks may explain structures clearly but in reality the
organs themselves very rarely look like a text book diagram.
Ø What we currently understand about the structure and function of organs has been
built up almost entirely by this very process - careful observations of a dissection.
Ø It is important you complete these set tasks below before you come to the laboratory
for much of this prac class you and a partner will be working independently.

Before the prac:

& Read Sadava et al. 11th Ed., Gas Exchange, pp. 1023-1026, Circulation pp. 1044-1056.
& Read through all Practical 3 Student Manual instructions.
Watch BioBytes: ‘Exchange surfaces’.
Watch TechTips: ‘External features of the heart’, ‘Opening the heart’ & ‘Opening the left
side of the heart’.
Explore: ‘Virtual Heart’ http://thevirtualheart.org ILT 1: Cell Biology (and associated test).
Complete Pre-practical Quiz (A score of at least 80% is worth 1 mark out of the 10
? marks available for the practical).

Bring to prac:
P Hard copy of Practical 3 ‘Heart and Lungs Getting the Essentials’ Student Manual notes.
P Lab coat and appropriate shoes.
P Sharp HB pencil, ruler, eraser, pen.
P Student Card (to scan in).
All references for pre-reading refer to: Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, the science
of Biology (11th Ed.). Sunderland, USA: Sinauer Associates Inc.

Your safety in the laboratory is very important:

• At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
• Wear safety glasses at all times.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Wash your hands after class.
• Dissecting instruments are used in this practical. Use blunt probes and avoid cuts.
• If you have an existing cut or abrasion on your hands please speak to your demonstrator
A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions used in
class are dilute and present a low risk hazard when laboratory safety rules are followed. 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

Safety information specific to each practical class will be presented at the beginning of that class. Because
of this, students who arrive late to the class will not be permitted to complete the class at that time.
P3 - 2 Practical 3 : Heart and Lungs: Getting the Essentials BIOL10002 : 2020

All living things require an energy source and all the organisms we will look at require oxygen in
order to access this energy at a cellular level. The gas exchange surfaces and the circulation
systems of vertebrate animals are responsible for supplying this oxygen and removing the waste
gas carbon dioxide.
In this practical you will investigate the heart and lungs of a typical mammal.

Biological Concepts covered in this prac:

• Complex multicellular organisms require specialised exchange surfaces and transport systems
in order to meet the needs of all their cells.
• The rate of diffusion across exchange surfaces increases as permeability, surface area, and
pressure differences increase but is inversely proportional to diffusion distance.
• The structure of biological exchange surfaces increases diffusion rates.
• Closed circulation systems allow blood to be moved under higher pressure compared to open
circulation systems.
• The double circulation of the mammalian heart delivers blood to both gaseous exchange
surfaces (lung) and the tissues of the body under pressure.

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

• Recognise the distinguishing features of vascular tissue in animals.
• Recognise the features of an efficient gas exchange surface.
• Identify structures of the mammalian heart and understand their function.

Assessment
• Pre-practical Test
• In-practical assessment
• Post-practical Test 2

Practical Timeline
Estimated Time
Use this as a guide to how much time you should spend on each activity
Introduction 10 mins
Activity 1: Exchange Surfaces (sheep pluck, lung tissue, Fick’s Law) 45 mins
Activity 2: Heart Dissection and assessment 50 mins
Clean up 15 mins
BIOL10002 : 2020 Practical 3 : Heart and Lungs: Getting the Essentials P3 – 3

Activity 1: Exchange Surfaces

Part A: Lung Inflation

Your demonstrator will show you the sheep’s pluck. The lungs occupy
a large proportion of the thoracic (chest) cavity of a mammal. If
possible, and with your demonstrators help, inflate the lungs using the
pump provided.
Observe how the heart of the mammal is closely associated with the
lungs.

Question 1: Describe the appearance and texture of the inflated

sections of the lungs.

Figure 1: Diagram showing blood

vessel connections between a heart
and lungs of a mammal.

Part B: Calculating Relative Density of Lung Tissue

Work in a group to complete this activity.

Equipment:
• 1 piece of lung tissue (weighing at least 10 grams)
• 1 piece of liver tissue (ensure the pieces of lung and liver are a similar size)
• Weighing scales (1 per tutorial group)
• Graduate measuring container
Step 1: Weigh the pieces of lung and liver tissue. Record the mass of each.

Step 2: Calculate the volume of each piece of tissue. To do this, half-fill your measuring container
with water and record the water level. Immerse each piece of tissue in water and record
the new water level. Determine the volume of water displaced for each piece of tissue.

Step 3: Use your measurements to determine the relative density of liver tissue and lung tissue.
(Density = mass/volume)

Question 2: Based on your results, what would you expect the microscopic structure of lung
tissue to be like?
P3 - 4 Practical 3 : Heart and Lungs: Getting the Essentials BIOL10002 : 2020

Part C: Cellular Structure of Lung Tissue

Equipment:
• Compound microscope
• Slide M25 - Lung tissue
Examine the cellular structure of lung tissue under high power. Use the diagrams provided to help
you identify features such as alveoli (singular is alveolus), alveolar ducts, capillaries and (if
possible) bronchioles. Remember that you are looking at a 2D slice from a 3D structure as shown
by the diagram of part of a lung below:

Bronchiole

If the histological section is taken here,

only those structures that the dotted line
passes through will be visible on the slide
Alveolar sacs
Alveolar duct

Figure 2: Structure of lung tissue. (Modified from Knox et al. 4th Edition 2010)

Draw a diagram of a section of the lung tissue in the space below, at magnification x100. Clearly
label the structures that are visible.

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
BIOL10002 : 2020 Practical 3 : Heart and Lungs: Getting the Essentials P3 – 5
Question 3: In the lung tissue, how do you distinguish between a capillary and an alveolar sac?

Question 4: How do you distinguish between a vein and an artery or arteriole? What is the
significance of these differences?

Question 5: Bronchi and bronchioles are lined with cilia. Suggest a reason for this.
P3 - 6 Practical 3 : Heart and Lungs: Getting the Essentials BIOL10002 : 2020

Question 6:

If the diameter of a red blood cell is Air space

in alveolus
6-8μm, estimate the distance gas
travels from the alveolus to the red
blood cell.
Red blood
cell

Air space
in alveolus
10 µm

Figure 3. Electron micrograph of human lung (Magnified x1800)

The rate of movement of molecules across a membrane or exchange surface such as the lung
depends on several factors. Fick’s Law describes the relationship between diffusion rate and these
factors and states:
Rate of diffusion ∝ (permeability) x (surface area) x (pressure difference)
(diffusion distance)

Question 7: Having observed the lung tissue in the pluck and the histological section, describe
how the structure of the lung maximises the rate of diffusion according to Fick’s
Law above. Refer specifically to the variables of surface area and diffusion
distance.
BIOL10002 : 2020 Practical 3 : Heart and Lungs: Getting the Essentials P3 – 7

Activity 2: Abnormal Tissue

Cancer is an example of abnormal tissue development. Rapidly proliferating cancer cells have a
different metabolism to normal cells. Cancer cells use increased glycolysis for energy and can build
up high levels of lactic acid because of this.
Hexokinase is a enzyme in the glycolysis pathway that is overexpressed in liver cancer cells. In the
future, targeting this metabolic pathway and the inhibition of this enzyme could be one way to check
the growth of some cancers.
Most patients with suspected cancers will have a biopsy taken. This means a small amount of tissue
is removed from the patient and observed under the microscope.

In this exercise you will be working individually to compare samples of normal and abnormal tissue.
You should make detailed observations of abnormal lung tissue that could lead to a diagnosis.
If you want a further challenge compare normal and abnormal liver tissue

Procedure: Examining abnormal tissue

1. Observe SLIDE M25/B (human abnormal lung) under low power.
2. Observe the arrangement of tissue and note any differences when compared to the normal
tissue.
3. If you have time examine and compare SLIDE M27 (human liver) and SLIDE M27/B
(human abnormal liver).

As you observe your samples, list the differences between the two with respect to both tissue
organisation and, if time permits, cell structure (you will need to use high power for this).
Use this space to list the observed differences:

Question 8: How would the changes in structure observed in slide M25/B affect gas exchange in the
lung?
P3 - 8 Practical 3 : Heart and Lungs: Getting the Essentials BIOL10002 : 2020

Activity 3: Heart Dissection

The heart of a vertebrate is myogenic, meaning that it is able to beat in the absence of nerve
stimulation. Cardiac muscle cells have the intrinsic property of spontaneously contracting, and can
rapidly conduct electrical signals. The heart has a pacemaker region that triggers an electrical wave
to move across the atria and then the ventricles in an orderly sequence. This ensures that the atria
contract simultaneously, filling the ventricles, and then the ventricles contract, expelling blood from
the heart to either the lungs or the rest of the body.

Work in pairs to complete the dissection of the sheep’s heart.

Equipment:

• 1 heart per pair

• wooden dissecting board
• dissecting scissors (blunt ended)
• dissecting probe (blunt ended)

External Features

Lay the heart ventral side up as shown in the photograph

(Figure 5).
Note: Some of the major blood vessels normally protruding
from the base of the heart (which is at the top of Figure 5)
may be missing.
Use your finger or a probe to work out which vessel is
connected to each chamber of the heart. This information
can be used to identify the vessels.

Figure 4: Sheep heart, ventral view

Identify the following:

• Interventricular and atrioventricular grooves - mainly filled with fat.

• Pulmonary artery – emerges from right ventricle.
• Aorta – emerges from left ventricle.
• Venae cavae - posterior and anterior, open into right atrium.
• Pulmonary veins – open into left atrium.
• Coronary vessels - visible on the surface of the heart. These supply blood to the heart wall.
BIOL10002 : 2020 Practical 3 : Heart and Lungs: Getting the Essentials P3 – 9

Procedure: Opening the right side of the heart

!
Step 1: !
!
Cut off the apex of the heart at the line in the diagram to !
expose the two ventricular cavities. ! "#$%
&'#&!)
Note that the right ventricle does not reach to the apex. 4%.&*
Make the first cut at an angle so that it opens both the left
ventricle and right ventricle.
Identify the thin-walled chamber (this is the right ventricle). 1

Apex of right
ventricle
Apex of left
ventricle

Step 2:
Cut through the ventral wall of the right ventricle parallel to
and close to the inter-ventricular groove.

Step 3:
Continue the cut to the left, along the atrioventricular groove
as far as the pulmonary artery, then cut through the wall of
the right atrium and anterior vena cava.
Turn back the V-shaped flap to expose the lumen of the
right ventricle and the right atrium.

Step 4:
Cut open the pulmonary artery.
Note the three parts of the pulmonary semilunar valve,
which prevents back-flow of blood when the ventricle
relaxes.

Identify the following features: Figure 5. Sheep heart, dissected ventral view
• Venae cavae (if present).
• Interatrial septum – divides the right and left atrium and contains a thin area (fossa ovalis).
Before birth blood flows from right to left atria through a hole called the foramen ovale. The
fossa ovalis is created when this hole becomes sealed.
• Coronary sinus posterior to the fossa ovalis. This is the main vessel returning blood from the
heart wall.
• Septomarginal trabecula, between the interventricular septum and ventricle wall. It has a
function in electrical conduction.
• Tricuspid valve – three thin, transparent flaps of tissue which, when the ventricle contracts, are
forced up to close the atrioventricular opening.
• Chordae tendineae, which attach the valve flaps to papillary muscles on the ventricle walls.
These are the ‘strings’ of the ‘parachute valves’.
• Pulmonary artery leaving the ventricle.
• Pulmonary semilunar valve, which prevents back-flow of blood when the ventricle relaxes.
P3 - 10 Practical 3 : Heart and Lungs: Getting the Essentials BIOL10002 : 2020

Procedure: Opening the left side of the heart

Step 5:
Open the left ventricle: make an incision in the ventral
wall of the ventricle parallel to and 1cm from the
interventricular groove as shown in the diagram.

Step 6:
Open the left atrium: make a vertical cut on the ventral
surface to expose the left atrium. Note the opening of the
two pulmonary veins (these have no valves).

Step 7:
Open the aorta. Push a probe from the left ventricle
upwards until it emerges from the aorta. Use this as a
guide to cut through the muscle and open the aorta as
shown.

Figure 6. Sheep heart, dissected

Identify the following features:

• Bicuspid (mitral) valve - similar in appearance and function to the tricuspid valve, but with 2
flaps rather than 3.
• Aorta leaving the ventricle.
• Chordae tendineae, which attach the valve flaps to papillary muscles on the ventricle walls.
• The 3 ‘pockets’ of the aortic semilunar valve at its base.
• Opening of two coronary arteries just beyond the semilunar valve.

Question 9: How can the difference between the thickness of the right ventricle and the left
ventricle wall be explained in terms of heart function?

At the conclusion of your dissection your demonstrator will assess your knowledge of heart
anatomy by asking you questions. Keep your dissected heart in front of you for this
assessment. The terms in bold are those you will be expected to identify and know. You will
also be assessed on your knowledge of lung anatomy during the practical.

******Do not clean up before you have been assessed******

BIOL10002 : 2020 Practical 3 : Heart and Lungs: Getting the Essentials P3 – 11

Heart Dissection / Anatomy

Identifying the structures of the heart:

• Aorta
• Atrioventricular groove
• Aortic semilunar valve
• Bicuspid (mitral) valve
• Chordae tendineae
• Coronary arteries (openings to)
• Coronary sinus
• Coronary vessels
• Interatrial septum (AND fossa ovalis)
• Interventricular grooves
• Interventricular septum
• Left atrium
• Left ventricle
• Papillary muscles
• Pulmonary artery
• Pulmonary semilunar valve
• Pulmonary veins
• Right atrium
• Right ventricle
• Septomarginal trabecula
• Tricuspid valve
• Venae cavae

IMPORTANT: You will also be expected to be familiar with the function of each structure
for your assessment
 Practical 4
STRUCTURE and FUNCTION of the
MAMMALIAN DIGESTIVE SYSTEM
Ø In this practical you will set up and run a controlled experiment and collate the
results. Your understanding of this enzyme activity must be linked to the
anatomy of the mouse digestive system.
Ø It is important you complete these set tasks below before you come to the
laboratory.
Ø For part of the class you will be working in a team, then you will be working
independently to dissect the mouse digestive system.

Before the practical class:

& Read Sadava et al. 11th Ed, pp. 1076-1085.
& Read through all Practical 5 Structure and Function of the Mammalian Digestive System
Student Manual instructions (especially Dissecting Skills and Anatomical Terms).
Watch TechTips: ‘Opening the mouse’ and ‘Digestive system of the mouse’.
? Complete Pre-practical Quiz (A score of at least 80% is worth 1 mark of the total available
for the practical).

Bring to the practical class:

ü Hard copy of Practical 5 Structure and Function of the Mammalian Digestive System
student manual notes.
ü Lab coat, safety glasses and appropriate shoes.
ü Sharp HB pencil, ruler, eraser, pen, calculator.
ü Student Card (to scan attendance).
All references for pre reading are to your textbook:
Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, the science of Biology (11th Ed.). Sunderland,
USA: Sinauer Associates Inc.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 2
All living things require nutrition. Heterotrophs cannot photosynthesise and so must breakdown
other organisms or organic matter and absorb the nutrients in a simple form.
Digestion of the different classes of macromolecules uses both a variety of enzymes specific to
that macromolecule and a system of complex organs in which to carry this out.
In this practical you will investigate some of the digestive enzymes produced in these organs, and
then investigate the structure of the mouse (mammalian) digestive system.
______________________________________________________________________________
Biological Concepts covered in this practical:
• Complex multicellular heterotrophs require specialised digestive systems and absorption
surfaces in order to meet the nutritional needs of their cells.
• Common features of biological exchange surfaces increase diffusion rates.
• Breakdown of complex macromolecules in preparation for absorption requires many
specific enzymes functioning under different physiological conditions.
______________________________________________________________________________
After completing this practical you should be able to:
• Dissect a mouse to locate and identify the important structures in the digestive system.
• Relate the structures of a mammalian digestive system to their function.
• Compare the activity of digestive enzymes from different parts of the digestive system
______________________________________________________________________________
Assessment
• Pre-practical Test
• In-practical assessment
• Post-practical Test 2

Practical Timeline Estimated Time

Introduction 10 mins
15 mins set-up
Activity 1a: Digestive Enzymes setup (working in groups of 4)
60 mins run time
Activity 2: Mouse Dissection 60 mins
Activity 1b: Digestive Enzymes data collection (group results) and collation 20 mins
(working individually)
Clean up 5 mins
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 3

Your safety in the laboratory is very important:

• At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
• Wear safety glasses at all times.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Wash your hands after class
• If you have an existing cut or abrasion on your hands please speak to your demonstrator.
• Solutions of Iodine and Phenol Red are used in this class. Avoid contact with skin, use gloves
if appropriate.
• The animals used in this class are disease-free, control animals previously disposed of by
researchers with Animal Ethics Committee approval for their work. The animals are not bred
specifically for Biology classes. Please respect the animal and dispose of all material into the
specifically marked waste bins. Students acting inappropriately will be asked to leave the
laboratory.

Ethics
The mice that are used in these dissections are not specifically bred for first year Biology
practicals. The mice are reused, disease-free control animals donated by researchers who have
Animal Ethics Committee Approval for their studies. You must respect these animal at all times and
dispose of all animal material in the bin at the front of the class. Any student acting inappropriately
will be asked to leave the laboratory.

A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions used in
class are dilute and present a low risk hazard when laboratory safety rules are followed. 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

Safety information specific to each practical class will be presented at the beginning of that class.
Because of this, students who arrive late to the class will not be permitted to complete the class at
that time.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 4

Activity 1: Digestive Enzymes

Meals of a typical mammal contain a mixture of foodstuffs that must be broken down into simpler
molecules and then absorbed.
There are two things to consider in the breakdown process: the type of macromolecule that needs
to be digested and the part of the digestive system where each breakdown occurs. The breakdown
of food in the mammalian digestive system begins very early so that absorption from the small
intestine can occur very soon after the chyme (partly digested food) arrives from the stomach.
AIM: In this activity you will investigate which kinds of enzymes are produced in three different parts
of the digestive system: salivary glands, pancreas and stomach.
You will be supplied with:
• Mixture 1: enzymes derived from the salivary glands.
• Mixture 2: enzymes derived from the pancreas.
• Mixture 3: enzymes derived from the stomach wall.
• Mixture 4: a mixture of all of the above enzyme extracts denatured by heat.

Presence of enzyme activity is detected by mixing substrates with the enzyme extracts and
detecting either the loss of the substrate or the formation of a product. You are given three different
macromolecules to use as substrates in separate experiments:
• Protein (gelatine blocks)
• Lipids (pure cream)
• Starch (0.5% solution)

You will also investigate the activity of these enzymes at room temperature and 37oC

Work in teams of 4 to set up this activity.

Notes:

After all experiments have been completed, produce a summary of results (Question 1) and
answer Questions 2 to 4 on page P5 - 7.

Note: To transfer enzyme mixtures into test tubes you must use the appropriately labelled
pipette. Do not cross-contaminate the enzyme mixtures by using the wrong pipette.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 5

Amylase Test

Amylases initially break the straight chain portion of the starch molecule leaving a highly branched
molecule called dextrin. Amylases then continue to break the dextrin down to the disaccharide
maltose.
The starch test consists of adding Iodine (I2) to the solution to be tested.
• Starch gives a dark blue/black reaction with iodine.
• Dextrins give a red/muddy brown reaction with iodine.
• In the absence of starch or dextrin the iodine remains yellow.

Equipment
• Glass test tubes • Starch solution
• Marker pen • Enzyme mixtures 1-4
• Test tube rack • Iodine dropper bottle

Amylase Test Method

1. Label enough tubes to test each enzyme mixture at two temperatures.
2. Add 0.5 ml starch solution to each tube.
3. Add 1 ml of each enzyme mixture into the corresponding tube
4. Mix each tube gently by flicking each tube with your fingers.
5. Incubate the tubes accordingly at room temperature and 37°C for 60 minutes. Water baths
are located in the fume hoods at the end of the laboratory.
6. At the end of the incubation time, add 2 drops ONLY of iodine to each tube and record the
colour of each solution.
7. Record your results in Table 1 below.

Table 1. Amylase Test Results

Colour of
Temperature Mixture Interpretation
Iodine Test

Room 1 (salivary gland)

Temperature
2 (pancreas)

3 (stomach wall)

4 (denatured)

37°C 1 (salivary gland)

2 (pancreas)

3 (stomach wall)

4 (denatured)
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 6

Lipase Test

Lipase breaks down lipids by splitting the fatty acid from the glycerol. The appearance of the fatty
acid in solution is detected by Phenol Red, an indicator that turns yellow in very acidic conditions.

Equipment
• Glass test tubes • Diluted cream
• Marker pen • Phenol Red dropper bottle
• Test tube rack • Enzyme mixtures 1-4
Lipase Test Method
1. Label enough tubes to test each enzyme mixture at two temperatures.
2. Add 0.5 ml of diluted cream to each tube.
3. Add 2 drops ONLY of Phenol Red solution to each tube.
4. Add 1 ml of each enzyme mixture into each tube as follows
5. Gently mix the solutions in each tube by flicking each tube with your fingers.
6. Incubate the tubes accordingly at room temperature and 37°C for 60 minutes. Water baths
are located in the fume hoods at the end of the laboratory.
7. Record your results in Table 2 below.

Table 2: Lipase Test Results

Initial Final
Temperature Mixture Interpretation
Colour Colour

Room 1 (salivary gland)

Temperature
2 (pancreas)

3 (stomach wall)

4 (denatured)

37°C 1 (salivary gland)

2 (pancreas)

3 (stomach wall)

4 (denatured)
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 7

Protease Test
Proteases are enzymes that break down proteins. We can detect the presence of this enzyme by
observing reductions in the size of blocks of gelatine (a protein).

Equipment
• Glass test tubes • Gelatine blocks (dyed blue) of equal
• Marker pen size (NOTE: record the initial size of the
• Test tube rack blocks)
• Enzyme mixtures 1-4
• Paper towel
Protease Test Method
1. Label enough tubes to test each enzyme mixture at two temperatures.
2. Take a block of gelatine and record the approximate dimensions before adding it to each
tube
3. Add 1 ml of each enzyme mixture into the corresponding tube.
4. Ensure there is equal amount of mixture in each tube to cover the gelatine block.
8. Incubate the tubes accordingly at room temperature and 37°C for 30 minutes. Water baths
are located in the fume hoods at the end of the laboratory.
5. At the end of this time, tip each tube onto a paper towel and measure the dimensions of each
gelatine block.
6. Record your results in Table 3 below.

Table 3. Protease Test Results

Size of
gelatine
Temperature Mixture block Interpretation
compared to
original size

Room P1 (salivary gland)

Temperature
P2 (pancreas)

P3 (stomach wall)

P4 (denatured)

37°C P1 (salivary gland)

P2 (pancreas)

P3 (stomach wall)

P4 (denatured)
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 8

Activity 1: Digestive Enzyme Investigation

Question 1: Summarise the results of the three experiments in a table.

Ensure that the summary can be easily understood by using appropriate headings.

Consider your results and answer these questions:

Question 2: What was the negative control in these experiments? What is its purpose and why
is it called a negative control?

Question 3: What do you think the results of a positive control would have been?

Question 4: Consider the aims of this experiment, what conclusion/s can you draw from your
results?
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 9

BOX 1 DISSECTING SKILLS

Before you begin your dissection, it is important to realise that the aim of the dissection is not simply
to find the organs, but rather to observe the location of organs within the body and determine the
anatomical relationship between the component organs of each system. Finally, throughout your
dissection, “think function!”
Points to note when dissecting:
1. The aim of a dissection is to identify, display and show the relationships of the various
structures of the animal.
2. By gently separating the various organs from one another and cleaning away the connective
tissue that binds and conceals them, the finished dissection should have all the parts of the
system clearly visible.
3. Certain structures will have to be moved or even pinned away from their natural position in
order that everything can be displayed, but this should be kept to a minimum. The key is to
avoid gross displacement of organs from their natural position.
4. Do not remove organs or tissues from the animal unless it is absolutely necessary, as this may
destroy the relationship of one system to the others.
5. Separate structures, rather than cut, wherever possible. Forceps, probes and the non-cutting
outer sides of scissors are most suitable for this.

BOX 2 ANATOMICAL TERMS

These terms are useful for describing the locations of structures in a body. They are valid for
a mouse and many other animals. The terminology is somewhat different, however, for
humans as we are bipedal (walk upright).
distal: further from the beginning/attachment point
proximal: nearer to the beginning/attachment point
dorsal: on or towards the back
ventral: on or towards the front/underside
lateral: towards the sides of the animal
medial: towards the mid-line
anterior: towards the head end
posterior: towards the tail end
ascending towards the head
transverse from one side to the other, across the body
descending towards the tail
transverse section: a section cut at right angles to the long axis of the structure.
longitudinal section: a section cut parallel to the long axis of the structure.

Note: combinations of these terms are also used

eg. antero-dorsal = towards the head and back
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 10

Activity 2: Structure of the Mouse Digestive System

The digestive system of mammals is essentially a tube, with a mouth at the anterior (head) end and
an anus at the posterior (tail) end. Along the way there are specialised compartments for digestion,
both mechanical and chemical, and absorption. In herbivores in particular, there are large
compartments for storage and specialized digestion by bacteria and protists.
Equipment
• 1 mouse • dissecting probes (blunt ended)
• dissecting board • dissecting forceps
• dissecting scissors (blunt ended)

Procedure: Mouse Dissection

1. Lay the mouse on the board, ventral side up.

2. Use forceps to lift the abdominal skin from the

body, snip the raised skin to make an incision.

3. Insert the blunt side of your scissors under the

skin, pulling upwards, cut the skin as far
upwards as the mouth and down to the urinary
opening.

4. Pull the skin away from the body wall and lay it
back, cut the skin towards the legs until your
mouse looks like this.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 11

5. Repeat steps 2, 3 and 4 to open the body wall, but Your mouse should look
only for the abdomen. See the diagram below. Be similar to this when you have
careful not to damage the organs underneath. finished.

6. Identify as many parts of the digestive system as you can before you disturb the organs of
the abdomen.

The digestive system begins at the

mouth (buccal cavity) with the
associated salivary glands.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 12

The liver is a very conspicuous, dark reddish-brown organ that lies directly posterior to the
diaphragm and covers the stomach.

7. Lift the liver to find the entry of the oesophagus into the stomach, just posterior to the
diaphragm. You will need to remove a small liver lobe to reveal it.
8. Take note of the shape of the stomach. Pay careful attention to where the oesophagus
enters the stomach. The duodenum is the first part of the small intestine. At the junction
of stomach and duodenum you will see a slight constriction. This is the pyloric sphincter,
which controls the movement of food out of the stomach.

The diffuse dark pink tissue in the ‘loop’ formed by the duodenum just past the stomach is the
pancreas.
Note: Be careful when handling the duodenum as you may damage the pancreas.
9. Lift the duodenum and find the pancreas between the duodenum and the transverse
colon. Digestive enzymes formed in the pancreas enter the lumen of the small intestine
through a fine duct (hepato-pancreatic duct) that also brings bile from the liver.
10. Gently lift the small intestines out of the abdominal cavity and lay to the side.
To do this you may need to cut the thin transparent membrane called the mesentery.
Beginning at the oesophagus, follow the pathway that food would take as it moves through
the stomach and small intestine.

11. Locate the caecum, a large sac-like structure that is the first part of the large intestine.
Identify where the small intestine and the colon connect to the caecum.

12. The next section of the large intestine is the colon. It has three sections named for their
orientation in the body: ascending (towards the head), transverse (across the body) and
descending (towards the anus).

13. The last section of the large intestine is the rectum (not visible in the photograph), which
terminates at the anus (a sphincter that controls the exit of faeces from the digestive
system).
 BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 13

Liver
(has been lifted to expose the
stomach and small intestine)

Duodenum
(the first part
of the small
intestine) Oesophagus

Pancreas

Stomach

Small intestine
Colon

Caecum
(has been
removed from
body cavity)
Anus

If time permits you should observe the reproductive system of your mouse. The testes of the male
will be in the scrotal sac outside the body cavity. You can push the testis back into the body cavity
and find the associated epididymis, vas deferens, seminal vesicles and penis. The uterus of the
female mouse is bi-horned and can be found lying dorsally to the intestines. A very small ovary and
oviduct can be found, usually embedded in fatty tissue, at the tip of each uterine horn.
BIOL10002 2020 Practical 4: Structure & Function of the Mammalian Digestive System P4 - 14

Drawing of Dissected Mouse Digestive System

Draw the digestive system as you have displayed it and label the structures listed in bold. Make
your drawing larger than real life. The outline of the mouse is not required. When you have
completed your drawing of the digestive system you may cut through the ribs to observe the heart
and lungs in the thoracic cavity.
Ø On your diagram, indicate where the enzymes identified in Activity 1 perform their functions
in the mouse’s body.
Ø You must draw from your dissection. The photographs provided in the workbook do not show
all the organs and connections that you need to draw.

• mouth
• salivary glands
• oesophagus
• oesophageal
sphincter
• stomach
• pyloric sphincter
• pancreas
• hepato-
pancreatic duct
• small intestine
- duodenum
- ileum
• large intestine
- caecum
- ascending colon
- transverse colon
- descending colon
- rectum
• anus

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
 Practical 5

COMPARATIVE REPRODUCTION
Ø This is the last practical in the semester and you are expected to be competent with the
microscope and dissection.
Ø You will use careful observation to compare reproductive systems of two different vertebrate
animals.
Ø This practical is very busy so it is expected that you will show initiative and work through the
activities relatively independently.
Ø In order to complete many of the practical activities you will need to observe the specimens
of other students; however your answers to questions must be your own.
Ø Work as a team and make sure every member accesses the information needed to
complete the tasks.

Before the prac:

& Read Sadava et al. 11th Ed. Getting eggs and sperm together: pp. 894-898.
& Read through all Practical 5 Comparative Reproduction Student Manual instructions.
Watch: ‘The Human Body: An Everyday Miracle’ at this website:
http://www.cornel1801.com/bbc/HUMAN-BODY/An-Everyday-Miracle-movie-film.html
Watch TechTips: ‘Opening the mouse’ and ‘Cane Toad Dissection.
? ILT 5: Toad Development.
Complete Pre-practical Quiz (A score of at least 80% is worth 1 mark out of the 10
?
marks available for the practical).
Bring to prac:
P Hard copy of Practical 5 ‘Comparative Reproduction’ Student Manual notes
P Lab coat and appropriate shoes
P Sharp HB pencil, ruler, eraser, pen
P Student Card (to scan in)
All references for pre-reading refer to
Sadava, D., Hillis, D., Heller, C., & Hacker, S. (2017). Life, The Science of Biology (11th Ed.). Sunderland,
USA: Sinauer Associates Inc.
k

Your safety in the laboratory is very important:

• At all times wear a lab coat and suitable shoes with enclosed heel and toe that cover the foot.
• Wear safety glasses at all times.
• Gloves are required when handling cane toads.
• Always work to ensure your safety and the safety of those around you.
• Immediately report any injuries or spills to a demonstrator.
• Wash hands carefully after the class. If you have an existing cut or abrasion on your hands
please speak to your demonstrator.
• Dissecting instruments are used in this practical. Use blunt probes and avoid cuts.
A risk assessment has been carried out for the practical classes and identified risks minimised. The solutions used in
class are dilute and present a low risk hazard when laboratory safety rules are followed. 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
P5 - 2 Practical 5: Comparative Reproduction BIOL10002 : 2020

The mice used in this class are disease-free, control animals previously disposed of by researchers
with Animal Ethics Committee approval for their work. The cane toads used in this class are also
disease-free and are sourced from the wild as part of efforts to control their population in
Queensland. The animals are not bred specifically for Biology classes. Please respect the animal
and dispose of all material into the specifically marked waste bins. Students acting inappropriately
will be asked to leave the laboratory.

Safety information specific to each practical class will be presented at the beginning of that class. Because of
this, students who arrive late to the class will not be permitted to complete the class at that time.

Sexual reproduction is characteristic of almost all vertebrate animals. It allows the redistribution of
parental genes into offspring and generates variation within populations.
In this practical you will make observations of the reproductive systems of vertebrates where
fertilisation and development occurs within the female body, and compare these to the reproductive
systems of vertebrates where fertilisation and development occurs external to the female body.

Biological Concepts covered in this prac:

• Sexual reproduction requires the production of haploid gametes (sperm and ova) by specialised
tissues in gonads.
• The union of sperm and egg is fertilisation which gives rise to a diploid cell known as a
fertilized egg or zygote.
• Different reproductive strategies require different reproductive organs and behaviours in
vertebrate animals.
• Some vertebrates use external fertilisation and external development of the young, some use
internal fertilisation and external development of the young, and others use both internal
fertilisation and internal development of the young

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

• Identify and name the structures of the reproductive tract of the mouse and the toad and how they relate
to function.
• Appreciate the similarities and differences in structure and function of the reproductive tracts of animals
adapted to either external or internal fertilisation.
• Understand the adaptive significance of internal and external fertilisation strategies for reproduction.
________________________________________________________________________________
Assessment
• Pre-practical Test
• In-practical assessment
• Post-practical Test 2

Practical Timeline
Estimated Time
Use this as a guide to how much time you should spend on each activity
Introduction 10 mins

Activity 1: Dissection of Mouse Reproductive System 20 mins

Activity 2: Observation of the Mouse Ovary 10 mins

Activity 3: Dissection of the Toad Reproductive System 25 mins

Activity 4: Histology of Ovaries 15 mins

Activity 5: Histology of Testes 10 mins

Summary Table 15 mins

BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 3

Activity 1: Mouse Reproductive System

In this activity we will use the mouse as an example of a placental mammal. The anatomy of its
reproductive system is typical of this group. You will be able to observe some differences between
species during the practical class.

Equipment • dissecting board

• 1 mouse per student (ensure that your • dissecting scissors (blunt ended)
group or table has at least one male and
one female mouse) • dissecting probes (blunt ended)
• dissecting microscope • dissecting forceps

Procedure: Mouse Dissection

Use the diagrams below to observe the external features of your mouse and to determine its sex.
Open the abdominal cavity of the mouse as you did in Practical 3: Structure and Function of the
Mammalian Digestive System.
You should compare the external differences between male and female mice prior to dissection to
fill in your summary table found at the last page of Practical 5.

Opening the body cavity

1. Lay the mouse on the board, ventral side up.
2. Use forceps to lift the abdominal skin from the body, snip the raised skin to make an incision.
3. Insert the blunt side of your scissors under the skin, pulling upwards, cut the skin as far upwards
(anteriorly) as the mouth and down (posteriorly) to the urinary opening.
4. Pull the skin away from the body wall and lay it back, cut the skin towards the legs.
5. Repeat steps 2, 3 and 4 to open the body wall, but only for the abdomen.

Male mouse Female mouse

external nares external nares
incisors incisors

MEDIAN

LATERAL rib line nusrib

line
rinary
openingnipple of
mammary gland
start cut here

penis urinary opening

genital opening
scrotal sac
anus
CAUDAL
P5 - 4 Practical 5: Comparative Reproduction BIOL10002 : 2020

Your mouse should look like this when you

have completed these steps.
To reveal the reproductive system you
must first remove the digestive tract:
Step 1: Cut the oesophagus just below the
diaphragm.
Step 2: Cut the colon about 3 mm from
where it exits the body cavity.
Step 3: Remove the alimentary canal and
spleen, but do not damage the
underlying organs and vessels.
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 5

Reproductive Tract – Male Mouse

If you have a male mouse follow this procedure to observe and draw the male reproductive system.
Then observe the reproductive system of the female mouse from other members of your group and
draw that on the following page.

Step 1: Identify the bladder. In male mice this is often filled with urine. The two vas deferens
(vasa deferentia) enter the urethra, near the dorsal base of the bladder. This will appear
to you to be ‘behind’ the bladder. Pull the bladder posteriorly (towards you) to reveal the
vasa.
Step 2: Expose the testes by gently squeezing the scrotal sacs to push each testis back into the
body cavity.
Step 3: Trace the path of each thin, vas deferens back towards the testis. The vas deferens ends
at the epididymis, a large, pale body next to each testis, comprising a single, tightly coiled
tube continuous with the vas deferens.
Step 4: Identify the accessory glands associated with the reproductive system of the male.
• The seminal vesicles and associated coagulating glands are obvious. They are a pair of
large, knobbly, cream-coloured glands looking rather like horns, located anterior and dorsal
to the bladder. They connect the urethra close to where the vasa deferentia enter. The fluid
from these contributes to the ejaculate.
Other glands associated with the reproductive system of the male can be harder to find.
• The much smaller, pink prostate gland is lobed and is located posterior to the base of the
bladder. It is closely associated with the urethra and opens into it. You may see this gland if
you pull the bladder forward. The prostate surrounds the urethra at the base of the bladder.
• The smaller bulbourethral gland, also known as Cowper’s gland, is at the base of the penis
but it is small and difficult to observe.

After copulation the secretions of the

seminal vesicles, the prostate glands,
and the bulbourethral glands form the
copulatory plug to keep semen within
the vagina.
• A pair of preputial glands is located
under the skin. It supplies secretions
to the prepuce which surrounds the
penis.

On page P5-7, make a labelled drawing of

the male mouse reproductive tract. Ensure
the following have been labelled:

• testis • prostate
• epididymis • the urethra
• vas deferens • ureters
• seminal • kidneys
vesicle
P5 - 6 Practical 5: Comparative Reproduction BIOL10002 : 2020

Reproductive Tract – Female Mouse

If you have a female mouse follow this procedure to observe and draw the female reproductive
system. Then, observe the reproductive system of a male mouse and draw it on the previous page.

Step 1: It is easy to find the female reproductive tract once you locate the bladder, a small pale
usually empty sac. The bladder, and the urethra which leaves it, lie ventral to the vagina.
(To you this will appear behind the bladder).

Step 2: The vagina connects to the uterus, which in

the mouse, as in many other mammals, is
bicornuate (divided into two horns). One
‘horn’ lies on the right side of the abdomen,
the other on the left side. Follow the uterine
horns towards the animal’s head. Their ends
merge with a mass of fatty tissue, very close
to the dark red kidney.

Step 3: Gently prise the fatty tissue away from the

ends of the uterus. The fat surrounds each
ovary and oviduct.

Step 4: Identify the ovaries, these are two very small

pink or red, roundish organs lying near to
each much larger kidney.

Step 5: Next to each ovary is the paler, very tightly coiled oviduct. It is often difficult to distinguish
the ovary from the oviduct.
Use your dissecting microscope to distinguish these. It is even more difficult to see that the ovaries
are enveloped by a thin transparent sheet of tissue, which is the opening of the oviducts.

On the following page, draw and label your observations of the female mouse reproductive tract.
Ensure the following have been labelled:
• ovary
• oviduct
• uterus (has 2 uterine horns)
• vagina
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 7
P5 - 8 Practical 5: Comparative Reproduction BIOL10002 : 2020

Drawing of male and female mouse reproductive systems

BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 9
After observing the dissection of both sexes answer the following questions:

Question 1: In which organ does gametogenesis occur in a male mouse? In a female mouse?

Question 2: List the organs of the reproductive tract starting with the testis (in correct sequence)
through which sperm must move to be released.

Question 3: What is the purpose of each of the glands associated with the male reproductive
tract?

Question 4: When eggs are released during ovulation, into which organ(s) do they move?

Question 5: Where does fertilisation occur in the mouse?

Question 6: Are there any differences in the external reproductive structures of male and female
mice??

Do not dispose of your mice!

You will need to keep your dissected mice to compare to the toad in Activity 3.
P5 - 10 Practical 5: Comparative Reproduction BIOL10002 : 2020

Activity 2: Observation of the Mouse Ovary

Work as a group with the other students at your bench for this activity.

Do this after your dissection has been assessed.

Procedure:
For each female mouse in your group, place the mouse on a piece of paper towel under a
dissecting microscope:
Step 1: Use a pair of forceps and the sharp pointed scissors to carefully clear the connective
tissue and fat away from the area around each ovary.
Step 2: Identify the ovaries and oviducts.

Question 7: If you find a corpus luteum on the mouse ovary what does that tell you about the
reproductive history of your mouse?

Question 8: If you do find a corpus luteum, have a close look at the uterus of the mouse from
which the ovary came. What would you expect to find?

Question 9: Why would it be common to see multiple active corpora lutea on the ovary of a mouse
but not on a human or a cow?

Question 10: Would you expect to find a similar structure to the corpus luteum on the ovary of a
toad? Explain your answer.
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 11

Activity 3: Reproductive System of the Toad

In this activity we will examine the toad (Bufo marinus) as an example of a vertebrate animal that
uses external fertilisation. There may be other examples of external fertilisers you can observe. In
addition to your dissection, you should observe the preserved specimens of male and female toads
and take note of the differences in the reproductive systems of the toad compared to the mouse.
The skin is cornified but does not possess scales (distinct from reptiles) and is loosely attached to
the body. In Bufo, there are many glands in the skin. On the dorsal side of the pectoral region
behind the head is an aggregation of glands, the parotoid glands (paler patches near shoulders).
These glands produce a creamy secretion which is poisonous, so take care not to let it come into
contact with your eyes and mouth as it will cause stinging and discomfort.
IMPORTANT: You will need to wear gloves and safety glasses for this dissection. Avoid
touching the glands with bare skin if possible. Keep gloved hands away from your eyes, nose,
mouth, books or stationary while you dissect. Gloves and toads should be disposed of appropriately
once all activities have been completed.
*Note: It is imperative you have watched the TechTips on Cane Toad Dissection before coming into
Prac 5.

Dissection of the Toad

Equipment
• 1 toad per pair (ensure that your table has at • dissecting scissors (blunt ended)
least one male and one female toad)
• dissecting probes (blunt ended)
• lamp
• wax dissecting dish • dissecting forceps

Step 1: Place toad onto its dorsal side so its ventral side faces up in a wax dissecting dish. Before
the dissection, briefly note the external features of the body, which is divided into a head, trunk and
limbs. There is no neck (primitive feature) and no tail (specialized feature). The forelimbs are at
right angles to the body and the pectoral muscles are strongly developed to support the body's
weight. The hind limbs are long and specialized for hopping. The feet are only slightly webbed
since Bufo is more terrestrial than aquatic.
Step 2: Observe the external features of your toad and try to determine
its sex. Are there any differences between males and females? And how
does the external reproductive structure/structures compare to mice?

Step 3: Use the same procedure to open the toad as you did for the
mouse. Lift the skin in the mid-ventral line with forceps and make a small
incision to the skin about half way down the abdomen. There is a layer of
skin and a transparent abdominal wall which you will need to cut
through using your blunt-ended dissecting scissors. Continue the cut
anteriorly to the throat and posteriorly to the cloaca, and then cut laterally
towards each limb. Be careful not to damage underlying organs. Use the
diagram as a guide and speak to your demonstrator if you require any
additional assistance.
Ventral view of a toad.
Make your incisions as
indicated by the dotted
lines.

Look for the vocal sac, which is visible under the skin of the throat. It appears as a loose flabby
membrane, often flecked with black. The vocal sac is absent in female toads.
P5 - 12 Practical 5: Comparative Reproduction BIOL10002 : 2020

Free the skin from the body wall with closed scissors and peel back the skin. Cut through the
abdominal body wall a little to one side of the midline to avoid the ventral abdominal vein, which lies
on the inside of the body wall in the midline, makes a convenient guideline. When making these
incisions through the body wall, be sure to keep the lower blade of the scissors parallel to the body
surface and cut slightly upwards. This technique helps lessen the chance of damaging internal
organs. Extend this cut anteriorly as far as the pectoral girdle, going around the xiphisternal
cartilage, which is attached to the girdle posteriorly. Also extend this cut posteriorly as far as
possible. Next, cut laterally along the posterior border of the pectoral girdle, carefully severing the
xiphisternal cartilage and the abdominal vein. Make similar lateral cuts at the posterior end of the
abdomen.

Step 4: Cover the animal with water before going any further. This floats the organs apart. and
prevents them from drying out. Change the water when it becomes cloudy.

The liver is a large, dark brown organ three-lobes. The stomach is a thick-walled, tubular organ on
the left side of the animal. Anteriorly, its’ continuity with the oesophagus may be seen by turning
the stomach to the right side. The stomach leads to the small intestine. The large intestine is a
short wide tube, which merges into the cloaca. The lungs are situated dorsal and anterior to the
liver and stomach and if inflated look like bubble wrap.

Ventral to the large intestine is the large, bilobed thin walled urinary bladder. It is attached to the
large intestine and also to the body wall. It appears as a large flabby membrane when empty.
You will need to move the viscera aside to locate the kidneys, which are elongate, red-brown
organs on the mid-dorsal body wall.

Step 5: Observe both a male and female toad (you will have to share with another pair as you did
for the mouse) and complete the activities below.

Only dispose of your dissected mouse and toad AFTER all comparison and observational
activities have been completed, into the appropriate bin as instructed by your
demonstrator.
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 13

Reproductive Tract – Male Toad

The toad, unlike the mouse, relies on the availability of free water for external sexual reproduction.
They do not need to create a medium in which to release the sperm, as the sperm are able to use
the free-water to swim to the egg. The reproductive cycle is strongly seasonal, indicating that
environmental conditions play a large role in stimulating the hormonal cues required for
gametogenesis in the toad. Spermatogenesis is seasonal in this species.
The testis is closely associated with the anterior part of the kidneys and is located ventrally to it.
They lie adjacent to the kidneys and are cream-coloured. From each testis a number of very fine
efferent ducts pass to the tubules of the anterior part of the corresponding kidney.
The mature sperm is carried from the testis to the kidney and then down a duct to seminal vesicles
for storage. During mating it is released into the environment through the cloaca.
Observe the male toad and identify the following:
• fat bodies • kidneys
• testis • cloaca
• efferent ducts (connecting testis with kidney) • bladder
• seminal vesicle • urogenital duct
On your dissection, locate the structures above. You can use the demonstration material to help
you. Then label the seven structures on the drawing of the male toad urogenital* system (*urinary
and reproductive combined).

digestive tract removed

Figure 1: Male toad urogenital system (Image source: http://biodidac.bio.uottawa.ca)

Question 11: What structures are found in the male mouse that are not found in male toads?
How do each of these structures aid in fertilisation?

Question 12: Are there any observable differences between the external reproductive structures in
male and female cane toads?
P5 - 14 Practical 5: Comparative Reproduction BIOL10002 : 2020

Reproductive Tract – Female Toad

The reproductive tract of the female toad will look considerably different depending on whether she
is carrying eggs (the eggs of B. marinus are black).
The toad ovary is a multi-lobed structure occupying a large space in the female abdomen.
Eggs (oocytes) are ovulated from the two ovaries and enter the lumen of the oviduct where
several layers of jelly are placed around them. The jelly protects eggs from mechanical injury and
infection, and aids fertilisation. These eggs (about 2000 to 20,000 depending on the species) are
stored in the ovisac for 1 or 2 days before release through the cloaca. Release of the eggs is
triggered by the male toad mating with the female by clasping her while mounted on her back (an
arrangement called amplexus). The male will then release his stored sperm onto the egg mass.
Observe the female toad and identify the following:
• ovary
• oviduct
• ovisac
• cloaca
• kidney
• bladder
• ureters

On your dissection, locate the structures above. You can use the demonstration material to help
you. Then label the seven structures on the drawing of the female toad reproductive system.

digestive tract removed

Figure 2: Female toad urogenital system (Image source: http://biodidac.bio.uottawa.ca)

Question 13: Why is it necessary for the ovaries in female toads to be so much larger than in
female mice?
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 15

Activity 4: Histology of Ovaries

Ovaries are the sites of oogenesis in both the mouse and the toad. In this activity you will observe
the structure of ovaries in each of these animals.

Typical mouse ovary tissue will contain oocytes in several stages of development. An oocyte within
its surrounding layer of cells is collectively called a follicle.
A primary follicle has one layer of cuboidal cells around the oocyte, whereas a secondary follicle
has two or more layers. The more advanced antral follicles have a fluid-filled cavity. In a mature or
late antral follicle, the maturing oocyte, now commonly called an egg (ovum), and some associated
cells are completely surrounded by fluid. At this stage, oocytes are very large compared to the
other cells.

Procedure
1. Place Slide H15, a section through mouse
ovary, under your compound microscope.
2. Observe the oocytes in several stages of
development. If possible find a late antral Developing
follicle, and identify a mature oocyte (egg). follicle

Question 14: Calculate the diameter of a single

egg in a late antral follicle.
Developing
follicle
a

Question 15: Suggest a reason for the large

size of mouse ovum compared Ovum
to other cells in the ovary.

u
Mature follicle
!
Figure 3: Transverse section of mouse ovary.
(Image source: Dr Mary Familari, School of BioSciences)
P5 - 16 Practical 5: Comparative Reproduction BIOL10002 : 2020
Toad ovary tissue will also contain oocytes in different stages of development. They are not
surrounded by a conspicuous follicle in the toad; typically the connective tissue between the
oocytes, the theca interna, appears as a thin layer.

3. Place Slide H41a, a section through toad ovary, under your compound microscope.
4. Observe the oocytes in several stages of development.
!

Nucleus of
mature egg

1. Developing oocytes
2. Mature eggs

Figure 4: Transverse section of toad ovary. (Image source: Dr Mary Familari, School of BioSciences)

Question 16: Find a mature egg in the toad ovary and calculate its diameter. Suggest a reason for
the difference in size between the mouse and toad egg.

Question 17: Much of the dark staining material in the cytoplasm of the toad ovum is energy-rich
‘yolk’. Suggest a function for this yolk.

Question 18: Would you expect to see as much ‘yolk’ in the ovum of a mouse? Give a reason for
your answer.
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 17

Activity 5: Histology of Testes

Production of sperm, the male gamete, occurs in the testes. Within each testis there are many
coiled seminiferous tubules inside which the sperm are formed. The initial meiotic divisions occur in
spermatogonia, cells found around the edge of each seminiferous tubule. The resultant
spermatocytes move towards the lumen of the tubule as they develop, and when they become
sperm they are released into this lumen. In mammals, maturing sperm move through the lumen of
the seminiferous tubules until they reach the epididymis. The epididymis is a long coiled tube
where the sperm largely complete their maturation and are stored until ejaculation.

Procedure:
Step 1: Place Slide H16 (mouse testes) or
Slide H40 (toad testes) under your
compound microscope.
Step 2: Compare your slide to your partner’s
and answer the questions below.
*You will need to compare both slides*
Question 19: What do you notice about the
internal structure of these organs in the
mouse compared to the toad?

Figure 5: Transverse section through a single mammalian

seminiferous tubule. (Image source: Biology Laboratory)

Question 20: Describe how the structure of the mature sperm close to the lumen of the tubule
differs from that of the spermatocytes near the wall of the tubule?

Question 21: Why is it not necessary for a sperm to contain as much cytoplasm as an ovum?

Question 22: In both the toad and the mouse, huge numbers of sperm are produced and billions
can be released in a single ejaculation. Propose an explanation as to why this is
necessary.
P5 - 18 Practical 5: Comparative Reproduction BIOL10002 : 2020

Summary Table
Comparison of the characteristics of the reproductive tracts of species utilising internal and external
fertilisation.

Internal fertilisers External fertilisers

Structural
(gross
anatomy)

Histological
(microscopic)

Functional
BIOL10002 : 2020 Practical 5: Comparative Reproduction P5 – 19