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UNIVERSITI TUNKU ABDUL RAHMAN

CENTRE FOR FOUNDATION STUDIES

BIOLOGY II
LABORATORY MANUAL
UNIVERSITI TUNKU ABDUL RAHMAN
CENTRE FOR FOUNDATION STUDIES
LABORATORY SAFETY RULES

The following rules must be obeyed by all students in the science laboratory of the faculty. Wilful
or repeated inadvertent noncompliance may result in dismissal or suspension from the
laboratories.

I. No entry without permission:


i) Outsiders are not allowed to enter the laboratory without permission.
ii) Visitor must request for a lab coat from the laboratory officer before enter to the laboratory.
iii) No student is allowed to enter the laboratory unless permission has been given by laboratory officer or
lecturer.
II. At work in the laboratory:
i) No experiment may be attempted without the knowledge and permission of lecturer or lab officer.
ii) Laboratory coat must be worn at all times in the laboratory.
iii) Students must wear covered shoes in the laboratory. Students wearing open toes shoes such as
slippers or sandals are not allowed to work in the laboratory.
iv) Safety glasses must be worn when necessary.
v) Mobile phones are to be switched off at all times in the laboratory.
vi) Do not smoke, drink, eat, bite nails or pencils, or apply cosmetics in the laboratory.
vii) Do not pipette chemicals with mouth.
viii) Do not taste any chemicals, including diluted solutions. If any acid or alkali accidentally enters your
eyes or mouth, wash immediately with plenty of water. Inform your lecturer or laboratory staff, and
seek medical attention if necessary.
ix) Any accident must report to the lecturer or lab officer immediately.
x) Paper should never be used to light up the Bunsen burners.
xi) Used match sticks, filter papers, and other solid waste must never be thrown into the sinks. They must
be thrown into the dustbins provided. Lighted match sticks and smoldering materials must be
extinguished with tap water before thrown into the dustbins.
xii) Students must take responsibility for apparatus and equipment under their charge in the laboratory.
xiii) Any glassware breakages, apparatus lost and equipment damages or malfunctioning must be reported
to the laboratory officer.
III. Before leaving the laboratory:
i) Ensure all the equipment and working benches used are thoroughly cleaned and dried.
ii) Wash hands and arms with soap and water before leaving the laboratory.
iii) All stools must be kept under the benches.
iv) No student is allowed to take away any chemicals, equipment or other property of the laboratory
without permission.
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Table of Contents:

Practical Experiment Description Page


Practical 1 Introduction 1
Anatomy & physiological  Notes on biological drawings
studies I  Microscope usage (revision)

Practical 2 Nervous System 8


Anatomy & physiological
studies II

Practical 3 Circulatory System I 16


Anatomy & physiological  The mammalian heart
studies III  The use of sphygmomanometer and stethoscope

Practical 4 Circulatory System II 21


Anatomy & physiological  Haematology
studies IV
Practical 5 Histology of Mammalian Organs I (Endocrine System) 25
Anatomy & physiological  Microscopic investigation of the pancreas
studies V  Microscopic investigation of the liver

Practical 6 Histology of Mammalian Organs II (Urinary system) 36


Anatomy & physiological  Microscopic investigation of the kidney
studies VI

Practical 7 Histology of Mammalian Organs III (Digestive System) 45


Anatomy & physiological  Microscopic investigation of the digestive system
studies VII

Practical 8 Dissection of Mouse 48


Anatomy & physiological  Dissection of the mammalian alimentary canal
studies VIII  Dissection of the mammalian reproductive system
 Dissection of the mammalian excretory system

Practical 9 Human Reproduction & Development I 62


Anatomy & physiological  Microscopic examination of mammalian reproductive system
studies IX (male)

Practical 10 Human Reproduction & Development II 69


Anatomy & physiological  Microscopic examination of mammalian reproductive system
studies X (female)
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Important rules on lab assignments


 If you were absent for a particular lab session, please inform your instructor to make other
arrangement for lab report. Your lab report is not valid if you were absent for that particular
lab session.
 All biological drawings must be the original work of a student. No mark would be awarded to
work which is not original.
 As such, it would be a good practice to attach a photo of the captured microscopic view
together with your drawing.
 All full reports are to be submitted seven days after the commencement of lab session. Late
submission would be penalised.
 All partial reports (drawings) are to be submitted on the spot during the lab session
 All partial reports (photographs) are to be submitted in the following week.

Writing lab reports


For exercises involving purely making biological drawings (partial report), your lab report should
contain the following sections:
 Objective
 Drawings

For exercises not involving purely making biological drawings (full report), your lab report should
contain the following sections:
 Objective
 Apparatus & materials
 Procedure
 Observations and/or results
 Discussion
 Conclusion
 References

Your lab report should be preceded by a cover page which contains the following:
 Unit code
 Unit description
 Name
 Partner’s name
 Group
 Date
 Title of lab report
 Lecturer’s name

Example:
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Practical 1 Introduction

Objective:
To understand how to produce biological drawings and revise the usage of microscope.

Exercise 1 Notes on Biological Drawings

Drawings are an aid to precise observations and for this reason they are an important part of laboratory
work. In the practical examination, the examiner will have only your written recordings and drawings to
assess you. Therefore full recordings and neatly labelled drawings are of great importance.

Keep the following points in mind when making drawings:

1. Use a sharp, pointed HB/2B pencil.

2. Drawings should be as large as possible and made to fit into the space available.

3. Attention must be given to the general shape and proportion of the specimen. First, consider what
you want to show. Then plan your drawing so that various parts are in proportion and fit on the page.
Small marks indicating the length and breadth of the drawing are of great help in planning, and a faint
outline can be rapidly drawn to show the relative positions of the parts.

4. The final outline should be drawn with clean firm lines (not sketchy broken lines). Details should be
put in clearly with a sharp pencil. If important details are too small to be shown in proportion, they can
be shown in an enlarged drawing on the side.

5. Drawings should be accurate records of your observations. The biologist is not expected to be an
artist, but to become, in some degree, a draughtsman. Clear and accurate line drawings are
needed.

6. Shading and colouring should be avoided. It should be possible to make the drawing perfectly
clear by the judicious use of thick and thin pencil lines and careful cross-hatching. Get into the habit
of making your drawings large and clear.

7. As important as the drawing is the labelling. This should be done neatly in pencil and the letters
printed. Each label should be connected to the appropriate part of the drawing by a clear guideline
without arrowheads. Do not label too close to the drawings, and never write on the drawing itself.
Always make sure that each drawing is fully labelled before you leave it. Guidelines to the labels
must be drawn with pencil and ruler and never crossing one another.

8. Each drawing must always have a title. The title should specify whether it is a transverse section,
longitudinal section, whole mount, etc.

9. It is sometimes appropriate, particularly when drawing live specimens, to make succinct notes to the
labels. These are called annotated drawings, which are particularly valuable as they combine a
record of structure with functional observations. Annotations must be beneath the labels.

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Example of a detailed drawing:

Title: Detailed drawing of trachea


Magnification power: 10x. 10x

Example of an annotated drawing:

Title: Detailed drawing of a Hydrilla leaf cell


Magnification power: 10x. 40x

Chloroplast
(site of photosynthesis; stained
brownish with iodine solution)
Cytoplasm
(granulated;
found at
periphery of
cell; stained
light yellow with
iodine solution) Cell wall
(made of cellulose,
stained yellow)

Nucleus
(doubled membrane organelle embedded
in the cytoplasm; control center of the cell;
stained orange brown with iodine solution.)

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General instructions for students:


 Drawing is to be done on your own A4 sheets of paper.
 Students may need to do few drawings in one session. Plan your time and allocate enough time for
each drawing.
 In order not to lose marks unnecessarily, please ensure that you comply with the instructions on
writing lab reports and notes on biological drawing.

Checklist for biological drawings:


Remarks
Items
(from notes on biological drawing)
The title should specify whether it is a transverse
1. Appropriate & comprehensive title section, longitudinal section, whole mount, etc.
2. Magnification e.g. 10x.40x
(ocular lens power x objective lens power)
3. Correct labelling At least THREE correct labels for each drawing.

Clear guideline without arrowheads, drawn with pencil


4. Guidelines and ruler and never crossing one another.
…annotated drawings, which are particularly valuable
5. Annotations if requested as they combine a record of structure with functional
observations.
Memorized textbook drawings or diagrams, bearing
6. Drawing is as what is seen under microscope little likeness to the specimens or observations will not
(originality)
be awarded marks.

Warning:
Memorized textbook drawings or diagrams, bearing little likeness to the specimens or
observations will not be awarded marks.

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Exercise 2 Review of FHSB1214 Biology I on Microscope Usage

Note to students: Before any microscope work (viewing of histological slides) commences, please
ensure you have gone through this introductory session.

Introduction:
The microscope is a basic tool of the biologist. It is a valuable precision optical instrument easily
damaged by careless usage. It is very important for the student to become familiar with the
parts of the microscope and the procedures in the handling of it. Treat your microscope well and it
will serve you well.

Setting up the microscope:


The microscope when not in use is usually kept in a case. Remove it by grasping the handle
arm while placing one hand under the base. Set it down gently on the laboratory table and at
a reasonable distance from the table edge. Always keep the microscope upright in the vertical
position and never touch any of the lens surfaces with the fingers since it will deposit a thin
film of oil on the glass.

Parts of the microscope:

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Component Function
Arm For lifting and carrying the microscope.
Base To provide stability.
Body tube To house the lenses.
Eyepiece This is a set of lenses that rests loosely at the top end of the body tube. It is
or ocular lenses obvious that if the microscope is tilted while being carried, the lens may fall out
and be ruined.

The magnification of the eyepiece (given as 10X) is printed on the metal part of
the ocular.

Revolving Located at the lower end of the body tube, it carries 3 objectives of different
nosepiece lengths. Rotating this part changes the magnification of the objectives.

Objective lenses They are of different magnifications with the following visible properties:

Objectives Magnification Length Lens opening


• Scanning lens (red ring) 4x Shortest Widest
• Low power lens (yellow ring) 10x Short Wide
• High power lens (blue ring) 40x Longest Narrowest

Focusing These comprise two knobs located on either side of the microscope which are
adjustments used to change the distance between the object being viewed and the objective
lens. Changing the distance determines the focus.

For the object to be viewed in focus under high magnification, the lens must be
much closer to the object than when it is under low magnification.

• Coarse adjustment Made by the large knob beside the body tube for
focusing under low power magnification.
• Fine adjustment Made by the small knob, which is for focusing under
high power magnification and accurate focusing.

Precautions when using the focusing adjustments:

• Turn both adjustment knobs at the same time.


• Do not overturn the adjustment knobs (i.e. do not force them to go beyond their
limits)
• Do not use the coarse adjustment knobs when focussing under the 40x
objective lens.

Stage This is the platform for slides and specimens to be viewed under the microscope.
Mechanical stage This movable portion of the stage is attached to the specimen holder and allows
the slide to be moved in different directions to facilitate viewing.
Specimen holder This holds the glass slide in place.
Vertical feed knob Rotating this moves the glass slide in the vertical direction.
Horizontal feed This moves the glass slide in the horizontal direction.
knob
Condenser Located just beneath the stage of the microscope, it incorporates a lens which
collects light on the stage to bear on the object.
Iris diaphragm A rotating disk under the stage. This diaphragm is used to vary the amount light
that is projected upward into the slide.
Built-in light This is situated below the iris-diaphragm to provide light for illuminating the
source object. It can be switched on or off.
Brightness This provides adjustment to the illumination brightness.
adjustment knob
Main switch This ensures that power is turned on or off.

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Preliminaries before use:


1. Turn the nosepiece until the scanning objective is in-line with the eyepiece. You should hear a
soft click or else feel a distinct falling into place as the objective moves into position. If not, the
field of view is totally dark or an illuminated crescent instead of a complete circle.

2. Turn the diaphragm to its largest opening.

3. Look into the eyepiece and make a final adjustment to the light adjustment knob so that the
field of view (i.e., the lit circle which you see) is evenly illuminated. Any glare sh ould be
removed by adjusting the diaphragm.

4. Should either of the lenses appear dirty, wipe it gently with a piece of special lens paper. Use
a circular motion with very light finger pressure. You should never use any other type of
paper or cloth. Discard the lens paper after use.

5. The microscope is now ready for use.

6. If you’re using a binocular compound light microscope like the diagram above, position it so that
the stage faces you.

7. Connect the microscope to the power supply and turn on the built-in light.

8. Ensure that the microscope stage is at its lowest position. This will prevent breaking of slides and
lenses by mistake when adjusting the objectives by moving the stage with the coarse adjustment
knob.

Using a higher power objective

1. Most microscopes have parfocal objectives. This means that if one switches from viewing a
specimen in sharp focus under a lower power objective to a higher one, the object should
automatically come approximately into focus. Only some slight further focussing with the fine
adjustment knob is required to see the specimen clearly. Therefore, if you’re using the higher
power objectives, do not use the coarse knob to refine focus or you’ll risk breaking the
slide and lenses.

2. You are now ready to switch from the scanning objective to a higher power objective after
obtaining a sharp focus of the object.

3. If required, adjust the fine adjustment knob to see the specimen clearly. Great care must be taken
when using higher power objectives. DO NOT focus the high power objectives with the coarse
adjustment knob.

4. If you’re going to switch to the next higher power objective, look from the side of the microscope
and move the revolving nosepiece slowly till that higher power objective clicks into position. Be
careful that it does not touch the slide (normally it shouldn’t unless the specimen is too thick and
also covered by a thin cover slip).

5. Take care that the lower end of the high power objective does not touch the cover slip. If this
happens, you must repeat the whole procedure focusing again, starting with the scanning
objective.

Trouble-shooting
Below are some common problems associated with not being able to find and focus on an object
under high-power magnification.

 Is the objective lens in position?


 Is the cover slip on the slide facing upwards?
 Is the object in the centre of the stage?
 Are the lenses clean and free from dirt and moisture?
 Is the condenser adjusted and focused?
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Oil immersion lens:


If your microscope comes with a 100 x objective, please DO NOT use it. Used the improper way, it will
break.

If you require a particularly high magnification, immersion oil may be used. Fluid with the same
refractive index as the objective lens is placed between a special objective lens and the cover slip so
that it touches both. The fluid permits a larger cone of light rays to enter the objective from the
specimen, and this increases the resolving power obtainable.

Microscope care:
Like all laboratory instruments, the microscope needs proper care for best service. Observe the
following:
1. Turn the resolving nosepiece until the scanning objective is in position.
2. Ensure that the stage surface is clean and dry.
3. Return the microscope in an upright position to its storage case.

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Practical 2 Nervous System

Objective:
To study the microscopic structure of mammalian nervous system.

Introduction:
The human nervous system is by far the most complex system in the body histologically and
physiologically and is formed by a network of many billion neurons, all assisted by many more
supporting cells (glial cells). Nerve tissue is distributed throughout the body as an integrated
communications network. Anatomists divide the nervous system into the following:

 central nervous system (CNS), consisting of the brain and spinal cord
 peripheral nervous system (PNS), composed of the nerves that conduct impulses to and from
CNS (sensory and motor nerves respectively) and ganglia which are small groups of nerve
cell bodies outside the CNS

Exercise 1 Microscopic Investigation of the Mammal Peripheral Nerve c.s.

Observation:
1. Examine a cross section of peripheral nerve. Nerves are surrounded by a thick connective tissue
sheath called epineurium. Within the epineurium are bundles of nerve fibers called fascicles.

2. Examine that each fascicle is surrounded by a layer of connective tissue called perineurium.
Nerve fibers consist of groups of axons. Each axon is individually surrounded by a layer of
connective tissue called the endoneurium.

3. Observe at least THREE of the following labels:


 Epineurium
 Axon
 Schwann cell nucleus
 Endoneurium
 Perineurium
 Fascicle

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Figure 1 Overview of the central and peripheral nervous system

Figure 2 Peripheral nerve, cross section


[http://anesthesiology.pubs.asahq.org/Article.aspx?articleid=1931243]

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Figure 3 Peripheral nerve, cross section


BNF: bundle of nerve fibers; BV: blood vessels; AT:
adipose tissue; Epn: epineurium; Pn: perineurium;
A: axon; C: capillary; NI: Schwann cell
[Ross &Pawlina, 6e]

Note that the myelin sheaths were dissolved in the


slide preparation, leaving the round empty spaces
shown around the axons.

Figure 4 Myelinated axon (ma) and nonmyelinated


axon (nma)
Scn: Schwann cell; m: myelin sheath; pn:
perineurium
[http://eugraph.com/histology/nervous/nervecs.html]

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Exercise 2 Microscopic Investigation of the Spinal Cord c.s.

Observation:
1. With the scanning objective, identify the centrally located butterfly or H-shaped arrangements of
the gray matter. Identify the dorsal (posterior) and ventral (anterior) horns of the gray matter.
Identify the white matter and the central canal. The white matter is composed mostly of
myelinated axons that have been sectioned transversely.

2. Observe at least THREE of the following labels:


 Gray-matter
 White-matter
 Central canal
 Anterior median fissure
 Posterior median sulcus

Mammal/Human Spinal Cord, c.s

Figure 5A Human Spinal Cord, c.s [Mader 6e, Fig. 8.7.c]

Figure 5B Human Spinal Cord, c.s [McKinley 2e, Fig.16.3]

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal Peripheral Nerve, c.s

Magnification power: 10x. 10x

Endoneurium
Fascicle Perineurium

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Name: Seng Kuan Chaing


---SAMPLE---
Title of slide: Mammal Peripheral Nerve, c.s.

Magnification power: 10x.60x

Myelin sheath

Myelinated
axon

Schwann cell
cytoplasm

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Name: Seng Kuan Chaing


---SAMPLE---
Title: Human Nerve, sec. H&E
Magnification power: 10x. 10x

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Title: Mammal Spinal Cord


---SAMPLE---
Magnification: 10x. 4x

Posterior median sulcus

White matter

Central canal

Gray matter

Anterior median fissure

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Practical 3 Circulatory System I

The Mammalian Heart

Objective:
To study the structure of the mammalian heart

Apparatus& material:
Dissecting tray, dissecting kit, glass rod, goat’s heart

Procedure:
1. Distinguish between the dorsal and ventral sides of the heart. Identify both atria and both
ventricles.

2. Identify the four blood vessels, aorta and pulmonary artery which transport blood away from the
heart, and the vena cava and pulmonary vein which transport blood to the heart.

3. The interior of the left ventricle was exposed by cutting. The semilunar/aortic valve which guarded
the opening into the aorta at the left top end of the ventricle, and the bicuspid/atrioventricular
valve which guarded the atrioventricular opening were observed.

4. The relative sizes and relative thickness of the wall in the 4 chambers were observed.

Exercise 1
1. Pulmonary artery and aorta arise from the ___________________ and __________________
ventricles respectively; vena cava opens into the ___________________________; pulmonary
vein opens into the ___________________________; the __________________ arteries carry
oxygen and food to the wall muscles of the heart.

2. When water was run into the vena cava and pulmonary vein, it emerged from the
___________________ and ___________________ respectively.

3. The bicuspid valve has ___________ flaps and the tricuspid valve has ____________ flaps which
are supported by non-elastic cords, the ___________________________ attached to the
papillary muscle. The cords support the atrioventricular valves and prevent the valves from
turning inside out into the ____________________ when the ventricles contract.

4. In life, the atrioventricular valves (the left _______________________valve and the right
___________________ valve) prevent the _______________of the blood from the ventricles into
the respective atria.

5. The two ___________________ have much thinner walls than the two ________________ as
they only have to force blood into the _______________ and this does not require much force.
The chamber with the thickest wall is the ______________________. Its wall is about 3 times as
thick as that of the _________________, and this is because the _______________ pumps blood
round the whole body while the _________________ pumps blood to the lung which is a short
distance from the heart.

6. The flow of blood through the heart in a mammal is as follows:


Body → vena cava → ____________ atrium → ____________ ventricle →
___________________ → lungs → _____________________ → ______________ atrium →
___________ ventricle →________________ → body

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7. List three structural differences between artery and vein. Explain how diet may affect the arteries
supplying the heart, resulting in coronary heart disease.

Exercise 2
State the name and condition (open or close) of valves involved in boxes 1, 2, 3 and 4.
Label the curves in boxes 5, 6, 7 and 8.

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The Use of Sphygmomanometer and Stethoscope

Objective:
To learn to use the sphygmomanometer to measure blood pressure and listen to the heart sounds

Note: The lab needs to be quiet in order for any sounds to be heard via the stethoscope.

Exercise 1 Listening of Heart Sound

Procedure:
1. Listen to your heart sounds using the stethoscope. See figure below:

*LLSB = lower left sternal border

Figure 1 Position of stethoscope on the chest

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Exercise 2 Measuring of Blood Pressure

Figure 2 Measuring blood pressure using sphygmomanometer and stethoscope

Procedure:
1. Inflate the sphygmomanometer (blood pressure cuff) to a little above 180 mm Hg. This collapses
the major arteries to the arm (that's why it is uncomfortable).

2. Slowly release air by gently turning the air valve, and watch the pressure drop. When you first
hear a sound, that will be the systolic blood pressure. The sound you hear is the blood now
flowing in the artery of the arm. This means that the systolic pressure is now greater than the
pressure in the blood pressure cuff.

3. As you continue to watch the pressure drop, when you no longer hear any sounds; the last sound
will be the diastolic blood pressure.

4. Record your diastolic and systolic readings.

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Exercise 3 Calculation of BMI

Body mass index (BMI) is a measure of body fat based on height and weight that applies to adult men
and women.BMI compares your weight to your height, and is calculated by dividing your weight in
kilograms by your height in metres squared. It gives you an idea of whether you’re underweight,
having a healthy weight, overweight, or obese for your height. By calculating your BMI, you can get an
indication of whether your weight may be affecting your health.
2
Body mass index (BMI) = weight (kg) ÷ height (m )

Figure 3 Body mass index (BMI) chart for adults

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Practical 4 Circulatory System II

Haematology

Objective:
To understand the preparation of blood samples.
To determine blood group and observe different types of human blood cells.

Exercise 1 Preparation of Blood Slides

Procedure:
1. Prepare 2 new slides.

2. Insert a new sterilized lancet into the lancet device.

3. Sterilize your finger tip by swabbing them with 70% alcohol swab. Using the lancet device, prick
your finger tip.

4. Place a drop of blood on one of the slide and 3 drops of blood on another slide.

5. Use a cleaned cotton ball to clean your finger tip. (use alcohol swab if necessary).

6. Make a blood smear on the slide as shown below.

7. Allow the smear to dry.

Figure 1 Preparation of Blood Slides

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Exercise 2 Determination of Blood Group

Introduction:
Your body has around 4 – 6 litres of blood, which consists of red blood cells, white blood cells, and
platelets in a liquid called plasma. The plasma contains salt and various proteins, the red blood cells
transport oxygen and remove carbon dioxide and other waste products, white blood cells fight
infection, and platelets help the blood to clot.

There are differences in human blood known as blood groups. There are four main blood groups: A, B,
AB and O. These groups are then either positive or negative meaning that your blood group can be
one of eight variations.

Blood Transfusion
If you have a blood transfusion (where blood is taken from one person and put into another), you
would need to know your blood group, or have a blood group test. This is because some blood groups
cannot be mixed with each other.

For example, if you are blood group A, you cannot take blood from a person with B blood as the anti-
B antibodies in your blood will fight the B antigens in the donor’s blood. However, any antibodies in
the donor’s blood will be quickly diluted. Therefore, antibodies in the donors blood won’t try to fight the
A antigens in your own blood. That’s why O blood, with no antigens, can be given to anybody.

If you have a blood transfusion, your blood will always be mixed with a sample of the donor’s blood to
check for clumping, even if your blood groups are compatible. This is because there are many other
types of antigens in the blood, which on rare occasions will not be compatible.

A blood group test will always be done on a pregnant woman. If she is rhesus negative but the child
has inherited rhesus positive blood from the father, it could cause complications if it isn’t treated.

Procedure:
1. Use the antiserum provided; determine the type of blood group you have.

2. Provide the theoretical explanation to the occurrence of ABO blood grouping with reference to the
various antigen-antibody interactions.

Figure 2 Results of Blood Group Tests

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Exercise 3 Observation of Red and White Blood Cells

Introduction:
In mammals, blood consists of red blood cells, white blood cells, and platelets suspended in the fluid
medium, the plasma. Red blood cells (erythrocytes) are among the simplest of cells. They lack a
nucleus. Different types of white blood cells are observed in the human blood (see figures in the next
two pages). They are variable in size and shape but most of them are larger than the red blood cells.
Each cell has a nucleus.

Procedures:
1. In the preparation of blood smear, follow steps 1 to 7 of the previous Exercise 1.

2. For white blood cells, stain the smear by adding a few drops of Leishman’s stain till the whole
slide is flooded (No staining required for red blood cells). Leave for about 2 minutes.

3. Flood the slide with distilled water for 8 minutes (so that darker stains may be obtained).

4. Wash gently with running pipe water.

5. Blot gently with a clean tissue paper (DO NOT WIPE).

6. When dry, try to identify the red blood cells and various types of white blood cells.

7. Make a biological drawing of the blood cells according to what you see under the 40x objective
lens or take picture of the slide.

Note: the details of white blood cells (in the following pages) may only be distinctly visible under high
power microscopes.

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Classification of leukocytes

Figure 4 Key to the different types of white blood cell found in human blood as seen under the light
microscope after staining (McKinley 2e).

Note:The classification of leucocytes into agranular and granular is traditional and inaccurate. This is because with the advent
of more powerful microscopes could granular bodies be observed in ‘agranular’ leukocytes. Martini (2006, 7 th Ed)

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Lymphocyte Eosinophil

Basophil Neutrophil

Monocyte

Figure 5 Types of white blood cells stained by Leishman stain. (Saladin 2e & 4e)

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Practical 5 Histology of Mammalian Organs I (Endocrine System)

Objective:
To study the microscopic structure of pancreas and liver. By the end of this practical, you must be
able to describe the functional unit of each organ and their microscopic anatomy.

Exercise 1 Microscopic Investigation of the Pancreas

Introduction:
The pancreas has two distinct functions:
1. It secretes digestive enzymes into the pancreatic ducts down which they flow to the duodenum.
So it functions as an exocrine gland.
2. It secretes the hormone insulin and glucagon into the bloodstream. So it functions as an
endocrine gland.

Observation:
1. Examine a section of the mammalian pancreas under the microscope. The bulk of it is made up of
groups of enzyme secreting cells called acini. Here and there among the acini, are islets of
Langerhans which secrete hormones.

2. Examine a single islet of Langerhans.

3. Identify at least THREE of the following labels:


 Exocrine acinus
 Islet of Langerhans
 Pancreatic duct
 Stroma
 Blood vessel
 Adipose tissue

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Figure 1 A section from the liver and the pancreas is illustrated, with emphasis on the details of the
liver lobule and the duct system of the exocrine pancreas.
[Eroschenko, V.P. (2005). diFiore’s Atlas of Histology with Functional Correlations (11 e)]

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Figure 2 Illustration and microscopic view of pancreas


[Structures may only be distinctly visible under high power microscopes; Saladin 2e]

Figure 3 Microscopic structure of the pancreas. The islets of Langerhans can best be found with a
low magnification because then lesser stainability outlines them as roundish light areas
within the exocrine glandular tissue [Saladin 2e]

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[Marieb 3e]

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Exercise 2 Microscopic Investigation of the Liver

Introduction:
The liver has many functions. The two main ones:
1. It secretes bile into the bile duct down which it flows, through a gall bladder, to the duodenum.
2. It regulates the amounts of blood sugar, lipids and amino acids by removing them from the
bloodstream or adding them to it, as appropriate.

Observation:
1. Examine a section of the mammalian liver under the microscope. It consists of numerous closely
packed lobules. Try to locate the lobules and other structures.

2. Examine the centre of a lobule. Observe the branch of the hepatic vein in the centre. Sinusoids
(blood capillaries) radiate out from this vein. Between the sinusoids are rows of liver cells
(hepatocytes) and very narrow while bile channels (canaliculi).

4. Identify at least THREE of the following labels:


 Central vein
 Hepatocytes
 Interlobular septum
 Hepatic lobule and sinusoids
 portal triad bile duct, hepatic portal vein and hepatic artery

Figure 4 Hepatic lobules [McKinley 2e]

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Figure 5 Structure of liver lobule [Saladin 2e]

In a section these lobules appear as small, more or less roundish or irregular polygonal units
consisting of (1) hepatic cells arranged in plates or cords radiating around radiating around a central
blood vessel (central vein) and (2) the interconnecting liver sinusoids between.

The interlobular connective tissue is apparent mainly at points where three or more lobules meet to
form the portal area or canal. These regularly contain the interlobular bile duct together with branches
of the portal vein and the hepatic artery. The duct and two types of vessels are known as the “portal
triad” which lies in and constitutes the main constitutes of the portal canal.

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Figure 6 Portal triad [McKinley 2e]

Figure 7 Portal triad [Saladin 2e]

Figure 8 Transverse section of liver lobule. The border of one lobule separating it from an adjacent
one is called the interlobular septum

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal Liver, sec. (cross section)

Magnification power: 10x.4x

Interlobular septum

Central Vein

Hepatic Lobule

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---SAMPLE---
Name: Seng Kuan Chaing
Title: Mammal Liver, sec. (cross section)
Magnification power: 10x.10x

Stroma

Lymphatic
vessel Interlobular septum

Central vein
Branch of
hepatic
artery

Branch of hepatic
portal vein

Branch of bile duct

Hepatocytes
Hepatic Lobule

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal liver, sec. (section)

Magnification power: 10x. 60x

Branch of hepatic
portal vein

Hepatic
sinusoid

Branch of hepatic artery Branch of bile duct Hepatocytes

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Practical 6 Histology of Mammalian Organs II (Urinary System)

Microscope Investigation of the Kidney

Objective:
To study the microscopic structure of kidney. By the end of this practical, you must be able to describe
the functional unit of the organ and their microscopic anatomy.

Introduction:
The kidney is the mammal’s principal organ of excretion and osmoregulation. The basic unit of kidney
is the nephron. Nephron is responsible for removing waste from the body. Each kidney is composed
of over one million nephrons. A nephron consists of three parts: a renal corpuscle, a renal tubule, and
the associated capillary network.

Observation:
1. Examine the section of the kidney. Note the cortex and the medulla.

2. Examine the cortex to identify the renal corpuscle. The cortex also contains proximal and distal
convoluted tubules, collecting ducts and blood capillaries.

3. Examine the medulla. Try to identify the loops of Henle (descending and the ascending limbs),
collecting ducts and capillaries (vasa recta).

4. You should be able to identify these structures under different magnifications:


a. 4x: renal corpuscle (Bowman’s capsule + glomerulus), renal pelvis, medulla and cortex,
any other structure
b. 10x: Bowman’s capsular space, glomerulus, cortex, medulla, any other structure
c. 40x: Bowman’s capsule, glomerulus, simple cuboidal cell, any other structure (e.g.
tubules)

Figure 1 Overview of the kidney structure [Saladin 2e]

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Figure 2 Nephron structure

Figure 3 Renal corpuscle [Saladin 2e]

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Figure 4 Renal cortex [Marieb 3e]

Figure 5 Renal corpuscle [Marieb 3e]

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Figure 6 Renal corpuscle [Saladin 2e]

[McKinley 2e)]

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Figure 7 Histology of renal medulla


Thick limbs of nephron loops are lined by simple
cuboidal cells while thin limbs are lined by simple
squamous cells. [McKinley 2e]
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[Marieb 3e]

Figure 8 Renal medulla


Collecting duct is lined by simple cuboidal
cells with clear cytoplasm, has large lumen
and distinct cell-to-cell border. [Marieb 3e]

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal Kidney median, c.s.

Magnification Power: 10x. 10x

Renal
corpuscles

Medullary
rays

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal Kidney median, c.s.

Magnification Power: 10x. 60x

Glomerular capsule:
Visceral layer
Glomerulus

Glomerular capsule:
Parietal layer

Glomerular capsule:
Capsular space
Renal
corpuscle

Proximal
convoluted
tubule

Proximal convoluted
tubule

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---SAMPLE---
Name: Seng Kuan Chaing

Title: Mammal Kidney median, c.s.

Magnification Power: 10x.60x

Loop of Henle
(thick limb)

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Practical 7 Histology of Mammalian Organs III (Digestive System)

Microscope Investigation of the Digestive System

Objective:
To study the microscopic structures of mammalian digestive system.

Introduction:
The layers making up the wall of the intestine are shown diagrammatically in the following figure.

Figure 1 Histological organisation of the digestive tract.

[http://droualb.faculty.mjc.edu/Lecture%20Notes/Unit%206/Spring%2006%20Digestive%20system%2
0with%20figures.htm]

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The tissue in this tract can be divided into four layers:

Mucosa
The mucosa consists of an epithelial lining; an underlying lamina propria of loose connective tissue
rich in blood vessels, lymphatics, lymphocytes, smooth muscle cells, and often containing small
glands; and a thin layer of smooth muscle called the muscularis mucosae separating mucosa from
submucosa and allowing local movements of the mucosa. Intestinal villi are mucosal outgrowths and
project into the lumen. Villi are covered by a simple columnar epithelial cells of absorptive cells
(enterocytes) and goblet cells. Each enterocyte has dense microvilli which are only able to be seen
using electron microscope.

Submucosa
A denser connective tissue layer, with larger blood vessels, lymphatics, nerves, and can contain
mucous secreting glands.

Muscularis externa (smooth muscle layer)


There are usually two layers; the inner layer is circular, and the outer layer is longitudinal. These
layers of smooth muscle are used for peristalsis (rhythmic waves of contraction), to move food down
through the gut.

Serosa/adventitia
Where the digestive tract is free to move within the abdominal cavity, it is surrounded by a serous
membrane called the serosa. At places where the tract is firmly attached to surrounding structures,
the attaching connective tissue is called adventitia.

[http://www.proteinatlas.org/learn/dictionary/normal/small+intestine+1]

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[Fox 11e]

Observation:
1. Find a region of the section where one of the projections has been cut along its entire length. Use
the high power of your microscope to find a region along this projection where you can see clearly
the structure of the two different types of cells which make up the surface layer of the projection.
The less abundant cells, which are stained blue, are mucus-secreting cells (goblet cells).

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Practical 8 Dissection of Mouse

Objective:
The dissection of animals is important for many reasons. It helps in the learning about the internal
structures of animals. It also allows students to learn how organs and tissues are interrelated.
Another purpose of dissection is to allow the comparison of organisms in terms of their organs and
relative complexities.

Exercise 1 Alimentary Canal

Procedure:
1. Pin the rat to dissecting board, ventral surface upwards and the head pointing away from you.
2. Make a mid-ventral incision through the skin (not through the wall) and cut forward as far as the
lower jaw and backwards to the anus.
3. Cut either side of the urinogenital openings as shown in Figure 1.
4. Free the skin from the underlying body wall, using your fingers or the handle of a scalpel, then pin
back the skin.
5. With scissors cut through the body wall so as to expose the contents of the abdomen.
6. Identify the liver and the stomach.
7. Without cutting the mesentery, push the liver forward and spread out the small intestine to your
left.
8. Identify its two main parts: the duodenum and ileum.
9. Identify the pancreas in the loop of the duodenum and, IF possible, identify the bile duct and the
pancreatic ducts.

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Figure 3A Liver,
visceral aspect, indicating the cut levels for rats and mice.

Figure 3B Rat liver, visceral aspect. Figure 3C Mouse liver, visceral aspect, with
gall bladder.

Figure 4A Pancreas. Figure 4B Pancreas.

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Figure 5A Intestine in situ with mesentery and mesenteric lymph nodes.

Figure 5B Intestine (mesentery is Figure 5C Intestine in situ.


removed)

1: duodenum
2: jejunum
3: ileum
4: cecum
5: colon
6: rectum

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---SAMPLE---

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Exercise 2 Reproductive System

Procedure:
1. In each sex, there are two gonads (testes in the males and ovaries in the females). Tubes lead from
the gonads to the midline where they join a single tube to the exterior. In the males the tube is shared
by the urinary system.

2. Before starting the dissection, identify the sex of the rat. In the female, identify the nipples and in the
males the scrotal sacs.

3. Also identify the urinary and genital openings on the ventral side. Note that in the female there are
separate urinary and genital openings – the former being located just anterior to the latter – whereas
in the male there is just one urinogenital opening, at the end of the penis.

Male (Figure 6)
1. With the scissors, cut open the scrotal sacs.

2. With the forceps, grasp hold of the testis or the fat attached to it and pull it gently forwards.

3. Remove the muscle overlying the pelvic girdle.

4. With the large scissors, cut through the pelvic girdle on either side of the midline and remove the
pubis.

5. Keep the ends of the scissors as horizontal as possible. This will expose that urethra which leads into
the penis.

6. Identify the epididymis and vas deferens leading from the testis. Follow the vas deferens to the
midline and make sure the vas deferens of the other side opens into the anterior end of the urethra.

Figure 6A Mouse Reproductive System (Male)

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Figure 6B Mouse Reproductive System (Male)

In situ localization and fixation of urinary bladder

Instillation procedure step 1 Instillation procedure step 2 Instillation procedure step 3

Figure 7 Instillation procedure: in situ localization, ventral aspect, after opening of the abdominal cavity.

P: prostate
R: rectum
S: seminal vesicle
U: urinary bladder

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Figure 8 Testis, recommended transverse section


(vertical, green), optional section (horizontal, blue).

Figure 9 Seminal vesicle and coagulating gland.

Figure 10A (Rat) preputial glands (P), testis (T), penis Figure 10B (Mouse) preputial glands (P), testis
(Pe). (T), penis (Pe).

Figure 11A Prostate, rat, ventral aspect. Lateral lobes


not visible, dorsal lobe: only caudal part visible. Figure 11B Prostate, rat, dorsal aspect.

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Female (Figure 12)


1. Remove the muscle overlying the pelvic girdle.

2. With a large scissors cut through the pelvic girdle on either side of the midline and remove the pubis.
Be careful to keep the ends of the scissors as horizontal as possible.

3. This will expose the narrow urethra beneath which is the larger vagina. Identify the small ovaries and
– with the help of a hand lens – the short, coiled oviducts.

4. The oviducts on each side lead to a long V – shaped uterus, the two horns of which unite in the
midline to form the vagina.

5. The bladder receives the ureters from the kidney, and opens into the anterior end of the urethra.

6. A pair of clitoral (i.e., female: preputial) glands can be seen at the posterior end of the urethra. Note
that (in the female) the urethra (urinary duct) and the vagina (genital duct) are separate.

7. If the rat is pregnant the much expanded uterus will be seen to contain a variable number of fetuses,
each with an umbilical cord and placenta.

Figure 12 Mouse Reproductive System (Female)

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Figure 13A Uterus and vagina (V: vagina, C: cervix, B: body, H: uterine horn, Od: oviduct, Ov: ovary

Figure 13B Ovary and oviduct, if ovaries are not Figure 13C Ovary (Ov), oviduct (Od) and
weighed. uterine horn (H), if ovaries are weighed.

Figure 13D (Rat) clitoral glands (C), vulva (V), vena Figure 13E (Mouse) clitoral glands (C)
femoralis (VF), abdominal muscle (M). and vulva (V).

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Exercise 3 Excretory System

Procedure:
1. Remove the digestive system, by cutting the end of the esophagus (or near there) and the rectum,
where it disappears into the urinogenital organs.
2. Cut through the mesentery so as to lift out the whole gut, spleen and pancreas.
3. Be careful not to cut any major blood vessels beneath the gut.
4. Leave the liver.
5. The rat can now be used to study the excretory system.
6. Once the gut has been removed, identify the bean shaped kidneys (a pair).
7. Identify the ureter and the urinary bladder.

Figure 13 Mouse Excretory system

Figure 14 Mouse Excretory system

Figure 15A Kidney and ureter

Figure 15B Kidney and ureter (U) in situ.

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Exercise 4 Circulatory and Respiratory Systems

1. Remove most of the rib cage as you can till you can see the circulatory and respiratory organs.
2. Make a small cut through trachea and insert Pasteur pipette tip. Pump air into the lungs through the
cut and observe the inflated lungs.

Figure 16A Lung, ventral aspect, oral study. Figure 16B Lung, ventral aspect, oral study.

Figure 16C Heart

Ao: aorta,
Cv: conoventricular vein,
La: left auricle,
Lv: left ventricle,
Ra: right auricle,
Rv: right ventricle

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---SAMPLE---
Name: Yu Hui Fang, Chong Yuet Tian, Ooi Min Huey, Tan Jing Kuan, Teoh Yew Shen, Hoh KarHeng

Title: Dissection of mouse thoracic cavity (circulatory and respiratory system)

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Washing up
1. Remove the pins from the mouse and place them in a separate beaker provided which has been filled
with antiseptic (Dettol, Savlon).
2. Wrap the mouse up with newspaper and disposein the large plastic bag provided.
3. Rinse your utencils by soaking them in the beaker containing antiseptic.
4. Meanwhile, rinse your wax tablets under running water first, followed by just a little antiseptic.

Laboratory report
1. Submit a partial report (per group) in the following week.
2. Use a (handphone) camera to capture photos and do the labelling and annotation subsequently.

You should submit TWO photos (complete with labelling and annotations) of the following:

o Alimentary Canal
You should be able to identify the following:
o Pancreas
o Liver
o Stomach
o Duodenum/ small intestine
o Ileum/ small intestine
o Caecum
o Large intestine/ colon
o Rectum

o Excretory System/ Reproductive System/ Circulatory & Respiratory systems


You should be able to identify the following:
o Vulva
o Ovary
o Uterine horn
o Penis
o Testes
o Scrotum
o Seminal vesicle
o Kidney
o Ureter
o Urinary Bladder
o Lungs
o Heart
o Trachea

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Practical 9 Human Reproduction & Development I

Objective:
To study the various stages of spermatogenesis.

Introduction:
The testis and the ovary contain developing sperm and eggs respectively. In each case, diploid
primordial germ cells, associated with the germinal epithelium, divide mitotically to form the
spermatogonia and the oogonia. These grow and undergo meiosis to form the spermatozoa and the
ova respectively.

Observation:
Identify at least THREE of the following labels.

Magnification 10x.10x
 Seminiferous tubule
 Tubule lumen
 Interstitial cells / Leydig cells
 Basement membrane
 Blood vessel

Magnification 10x.40x
 Spermatogonia
 Primary spermatocyte
 Secondary spermatocyte
 Spermatids
 Sperm cells

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Human sperm, smear

Figure 1 The structure of human sperm

Figure 2 Human sperm, smear


[http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-95022012000400045]

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Human testis

Figure 3 Gross anatomy of the testis, with a section cut away to reveal the internal structures and
histology

Figure 4 Seminiferous tubule, cross section

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Figure 5A Seminiferous tubules

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Figure 5B Seminiferous tubules (80X)

Figure 5C Seminiferous tubules (160X)

Figure 5D Seminiferous tubules (160X)

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Figure 5E Seminiferous tubules, cross section

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Practical 10 Human Reproduction & Development II

Objective:
To study the various stages of oogenesis.

Introduction:
The testis and the ovary contain developing sperm and eggs respectively. In each case, diploid
primordial germ cells, associated with the germinal epithelium, divide mitotically to form the
spermatogonia and the oogonia. These grow and undergo meiosis to form the spermatozoa and the
ova respectively.

Observation:
Identify at least THREE of the following labels.

Magnification 10x.10x
 Primodial follicles
 Primary follicles
 Secondary follicles
 Graafian follicles
 Corpus luteum
 Corpus albican

Magnification 10x.40x
 Follicle cells
 Antrum
 Primary oocyte
 Secondary oocyte
 Zona pellucid
 Corona radiate
 Theca interna

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Human Ovary Active Phase, sec

Figure 1A Human ovary active phase

Figure 1B Human ovary active phase

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Mammal Graafian Follicles, sec

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Figure 2 Ovarian follicles and structure that develop in the ovary

Figure 3

Primodial follicle and primary follicle

Figure 4 Primary follicle and secondary follicle

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Figure 4A Primodial follicles and primary follicle (with oocyte)

Figure 4B Primodial follicles (with oocyte) and primary follicle (with oocyte)

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Figure 5 Primary follicles (with oocyte) and atretic follicle

Figure 6 Secondary follicles (with oocyte)

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Figure 7 Graafian follicle

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Figure 8 Corpus luteum

Figure 9 Corpus albican

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