LABORATORYMANUAL

SBC3043 DEVELOPMENTALBIOLOGY

SEMESTER
S 1,S
EMESTER2, SESSION 2018/2019
ESSION2017/2018

DEPARTMENTOFBIOLOGY
FACULTYOFSCIENCE&MATHEMATICS
UNIVERSITI PENDIDIKANSULTANIDRIS

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 CONTENTS

LABORATORY TITLE PAGE

- Introduction: Laboratory Syllabus 1

1 Laboratory Exercise 1: Reproduction of Flowering Plants: 2

Observation of flowers

2 Laboratory Exercise 2: Reproduction of Flowering Plants: 4

Microsporogenesis /Microgametophyte

3 Laboratory Exercise 3: Reproduction of Flowering Plants: 7

Megasporogenesis /Megagametophyte
4-5 Laboratory Exercise 1: Mammalian reproductive System 9
and Development

5-6 Laboratory Exercise 2: Embryological Development 12

Laboratory Syllabus

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 SBC 3043 Developmental Biology

Introduction

In the lab portion of Developmental Biology students will learn and practice various
experimental approaches for investigating early embryonic development using different
model organisms. The lab course will emphasize experimental design, methodology, and
writing scientific reports. By the end of the term, Developmental Biology students will be
able to plan and execute experiments with living organisms, gather and interpret the
results, and present the outcome of these experiments in clear and concise scientific
reports. Exercises conducted during Developmental Biology Lab will relate to material
covered during lectures and material presented in lab. Lab grades will be based on
production of reports and the lab notebook.

Student Responsibilities.

You are responsible for leaving your work area clean and for rinsing out your own
glassware and dissecting equipment. You are responsible for maintaining and returning
your microscopes and dissecting equipment in good working condition. Shared equipment
(microscopes, dissecting kits) should always be left in perfect condition, so that the next
person can use it without having to clean it or to fix it. If you have questions about how to
use a piece of equipment, do not hesitate to ask the tutor or lab assistants. If you find
problems with tools or equipment, or if supplies or equipment are damaged in the course of
use, please report the problem to the instructor.

Absolutely no eating or drinking is allowed in the lab.

Lab report

You will write complete lab report.

Report will be prepared according to standard scientific style and format with Title,
Introduction, Methods / Procedures, Observation/ Results, Discussion and References.

Reflection

This reflection is a part of component relate to laboratory practical work. At the end of the
laboratory session, students will write a reflection. It should be submitted /attached with the
laboratory report.

Instructions for preparing your reflection:

1. Students’ will work individually to prepare a reflection. Those who are not participating
will not be given any mark.
2. Please refer to Laboratory Reflection Questions provided.
Lab Reflection Questions

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 1. Lab Exercise: __________________________

Note: [The reflective writing related to the laboratory exercise]

2. What is the purpose of this lab experiment?

3. What did you learn in this lab experiment?

4. What are the challenges you have in this practical?

5. How have your ideas changed as a result of this lab?

6. How do you apply the concepts learned in this practical?

7. What skills did you learn and/or practice in this lab?

8. Explain what student have learned and got something from doing this experiment?

Scoring Rubric
Details will be explained by the lecturer/tutor and laboratory during the course.
Late submissions your written reports will result in a reduction in marks available for the
course work.
Lab report will be given 10% and reflective writing is 5%.

Scoring Rubric For Laboratory Report

Poor Fair Good

Criteria What I’m looking for (1) (2) (3)
1. Title • A specific title on first line of report
 Included knowledge already known
about subject from other sources
2.Introduction  Explained purpose of experiment
 Demonstrated understanding of lab &
significance to biology
 Not a true materials and methods
3. Methods / section
Procedures • Flow chart diagram style preferred
• Accurate and complete of steps (step-
by-step list of how the experiment was
performed)
 Well thought out and organized

 Results and data are clearly recorded,

organized so it is easy for the reader to
4. Results see trends. All appropriate labels are
included all tables // figures //graphs)
are clearly labeled & organized
 All tables // figures are titled

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  A description of observations are
reported
 Data interpreted
5. Discussion  Supported by evidence
 Section is well thought out and
organized
6. Mechanics  Grammatical, spelling or punctuation
errors.
 At least 2 was cited in report Poor Good
7. References  Proper format (sources in italic or (1) (2)
underlined)

Total Score (20)

Scoring Rubric for Reflection

Criteria Proficient (3 m) Basic (2 m) Unsatisfactory (1 m)

1.Related
Topic
The reflection included all
required (answers of each
question) as well as
2. Required
additional information // All
element
required (answers of each
question) are included on
the reflection.
Most information of answers
3. Subject is clear, appropriate and
knowledge/ correct. // Most information
Information clearly relates to the main
topic.
The reflection explains the
students’ own thinking and
4. Reflective learning processes.
thinking
5. Making The reflection articulate
Connection multiple connection any
connection between
Answers are related to Answers are not
the topic/lab exercise related to the lab
(Good-1) exercise.
(Poor-4)
1-3 (some) required Several required
(answers of question) (answers of each
was missing on the question) were
reflection. missing
Some information of Information of
answers is confusing, answers is confusing,
incorrect or flawed. // incorrect or flawed. //
Some information clearly Information has little
relates to the main topic. or nothing to do with
the topic.
The reflection attempts The reflection does
to demonstrate thinking not address the
about learning but students’ thinking
unclear about the and/or learning
personal learning
processes.
The reflection articulate The reflection does
connections between not articulate to
learning experience and other learning

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 learning experience and content courses, life experience future
content courses, life experience and /or future goals
experience and /or future goals
goals.
Not submitted
Submitted on time (2 Submitted, but delay
Submission reflection writing -
mark) (1 mark)
(0 mark)
Total Mark= 15

TOTAL MARK = 20 + 15 = 35 = 15%

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 Laboratory 1 : Reproduction of Flowering Plants

Learning Objectives:

1. Identify the parts of flower

2. Draw a flower and label the parts

Flowers. The basic structural unit of sexual reproduction in angiosperms is the flower.
Although flowers come in a spectacular array of sizes, shapes, and colors, they are, in
essence, believed to have developed from shoots that have become modified for
reproductive purposes.

Flowers typically have fours kinds of foliar appendages usually arranged in a series of
whorls: sepals, petals, stamens, and carpels, arranged in this order from the bottom to the
top of the floral axis. Although the typical flower displays all of these types of appendages,
sometimes the parts are modified, reduced in size, fused with other parts, or they fail to
develop or abort. These developmental changes contribute to the complexity of flowers.
The two outermost sets, sepals, and petals, are sterile. That is, they do not produce
spores.

Sepals are typically green, and petals are often brightly colored. However, both sepals and
petals can be green or showy. Collectively, the sepals are known as the calyx and the
petals as a unit, the corolla. Together the calyx and corolla comprise the perianth. The term
“perianth” is especially useful in flowers which do not have a distinct whorl of petals and
sepals i.e. only one set of appendage is present instead of the usual two.

Next along the floral axis, inside of the perianth, are the stamens. Stamens are considered
to be microsporophyll. A stamen typically consists of a four-chambered anther, in which
pollen is produced, and a supporting stalk called the filament. Each anther chamber is
equivalent to a microsporangium.

The zygote undergoes mitosis and cytokinesis to reproduce a two-cells embryo. Numerous
subsequent divisions produce an increasingly large and complex embryo.

Materials:

Fresh flowers
Forceps
Razor blade
Dissecting microscope
Mature pollen of Impateins ; prepared slide

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Experimental procedure:

a. Observe the parts of flowers:

1. Obtain 3 different flowers per group.

2. With each flower and examine its form with the dissecting microscope.
3. Discard the green bract that surrounds the base of each flower near the stalk. Note the
brightly colored structure referred as corolla and individual components of the calyx are
called petals and the three outermost petals are calyx made up of sepals
4. Use the razor blade to carefully cut off, the colored sepals and petals at the point where they
join the green ovary of the flower. The remaining structures in the centers of the flower are
the true reproductive organs.
5. Identify and count the number of sepals, petals, stamens, and pistils. Use a dissecting
microscope to help you see stamens and pistils.
6. Use tweezers to remove the sepals and petals. Look at the stamen and pistil using the
dissecting microscope. Draw and label the stamen.
7. Identify the male and female structure of the flower. Note the female carpel.
8. Use razor blade to cut a very thin cross section from the center of the ovary. Place the
section flat on the slide and examine under the microscope. Note: the whitish ovules
(immature seeds) within the chamber of the ovary (locule).
9. Draw (floral diagram) and label the flower parts:

i) Female structure
ii) Male structure
iii) Other structures

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 Laboratory Exercise 2: Reproduction of Flowering Plants:
Microsporogenesis / Microgametophyte

Overview

The pollen, or male gametophyte (gamete-producing plant) and the ovule, or female
gametophyte, represents the sexual generation in the life-cycle of the flowering plant. The
pollen may be transferred to the stigma of the same flower or to the stigma of another
flower of the same species. In the former case the process is called self-pollination, in the
latter case, cross-pollination.

On the stigma, the pollen germinates, and produces the male gametes, which are
transported to the ovule in a pollen tube. One of these male gametes fuses with the
female gamete within the ovule. This fusion, called fertilization, marks the beginning of the
development of the embryo plant.

The tissues of the ovule surrounding the embryo form the seed, while the tissues of the
ovary, sometimes with some of the surrounding structures, form the fruit. The fruit is the
ripened ovary (ovaries) with the enclosed seeds, if any, and in some cases may include
other parts of the parent flower. The characteristics of the fruit are thus closely related to
the characteristic of the flower (or flowers), and especially the ovary (or ovaries) from which
it was produced.

Objectives

1. Identify the location of microsporangia, microspore (pollen grains), megasporangia

(ovules), and gametophytes in angiosperms.
2. Describe the cell cycle of flowering plants
3. Observing the pollen germination

Materials:

Mature pollen of Impateins ; prepared slide

Mature Lily anther c.s; prepared slide
Lily x.s. anther, tapetum, spores
Lily anther tetrads of microspores
Pollen sac
Pollen in anther Pollen
Lily Pollen grains

A. Observation: Microsporogenesis / Microgametophyte

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 1. Examine a prepared slide of a lily anther cross section under microscope. First, look
at the lower power. Identify the pollen sacs (microsporangia) where microspores
arise.
2. Use high power to examine the cells in the interior of the pollen sacs. The cells may
be mature pollen grains or microspores that are in stage of pollen development. The
walls of the anther split open the pollen is fully developed.
3. Identify and draw the structure of microspore, and stages of pollen development and
mature pollen grains. Include your drawing sketches of microspores and mature
pollen grains and pollen tubes.

B. Observation: Pollen Germination

Instruction:

i) Collect a range of flowers.

ii) Make up the solutions of sucrose and mineral salts pollen culture medium.

iii) Observe and sketch your field of view at time = 0, after 30 to 40 minutes, after one
hour and after 24 hours

Procedure:

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10
 Laboratory Exercise 3: Reproduction of Plants:
Megasporogenesis / Megagametophyte

Materials:

Mature Lily ovary; prepared slide :

Lily x.s. ovary with young ovules
Lily embryo sac- 4 nucleate
Lily embryo sac- 8 nucleate
Lily - nuclei dividing final division
Lily - mature embryo sac

Capsella embryo developing :

Capsella embryo globular
Capsella embryo heart
Capsella embryo torpedo
Capsella embryo (mature seed)

Observation: Megasporogenesis / Megagametophyte

1. Examine a prepared slide of lily ovary cross section using lower power.
Identify three chamber within ovary contain ovules. You may be able to see the
embryo sac (female gametophyte) as an oval arrangement of larger cells in the
center of the ovule.

2. Identify the stages of embryo sac (female gametophyte) development other

than the mature stage with eight nuclei simply due to chance. Each embryo sac of
an ovule contains an egg cell that may be fertilized by a sperm nucleus if a pollen
tube should reach it.

3. Draw the lily ovary in cross section. Include in your drawing of a mature
embryo sac and indicate which nuclei are involved in double fertilization.

Lily x.s. ovary with young ovules

Lily embryo sac- 4 nucleate
Lily embryo sac- 8 nucleate
Lily - nuclei dividing final division
Lily - mature embryo sac

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Observation: Embryo Development

1. Observe the series of demonstrations slides illustrating the development of the

embryo in the female gametophyte of Capsella.

2. The slide shows globular stage; spherical mass of cells, heart-shaped stage, and
torpedo shaped stage.

Capsella embryo developing

Capsella embryo globular
Capsella embryo heart
Capsella embryo torpedo
Capsella embryo (mature seed)

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 Laboratory Exercise 4 & 5: Mammalian Reproductive System and
Development

Objectives

The specific objectives for the lab are the following:

1) To study the major structures of the male and female reproductive system using
both models and diagrams
2) To understand the major functions of major male and female reproductive system
structures.
3) To examine basic histology of select male and female reproductive structures
4) To study spermatogenesis and understand how sperm are produced the male
5) To study oogenesis and understand events leading to ovulation and production
of an ovum

Preparation

The following resources are available to prepare you for lab.

1) Review relevant photomicrographs in the lab manual’s histology atlas

2) Review of mitosis in relation to spermatogenesis and oogenesis.

Materials:

The following materials will be available in the laboratory:

models of male and female reproductive structures

prepared slides of human sperm
section of mammalian testes
section of mammalian ovary

Procedure:

A. The male reproductive structures

1. Using your textbook and the available model, identify the following reproductive
structure:

Testes; scrotal sac, epididymis, vas deferens, seminal vesicle, Prostate gland
Bulbourethral, and gland penis

2. State the function of each structure identified above.

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B. Female reproductive structures

1. Using your textbook and the available models, identify the following reproductive
structures vulva vagina cervix uterus, ovary fallopian tubes broad ligament urethra

2. State the functions of each structure identified above

D. Human Sperm

Focus the specimen using the low power objective. Switch to high power and increase the
amount of light by opening the diaphragm. Sketch the appearance of human sperm under
high power magnification.

E. X-section of mammalian testes

1. Using the scanning power of the microscope, focus on a seminiferous tubule.

2. Switch to high power. Identify the spermatogonia. These cells are destined to become
spermatozoa through a process called spermatogenesis.
3. Look closely at the lumen of the tubule. The dark, fiber-like structures that seem to fill the
lumen are the flagella of spermatozoa. These sperm will eventually go out of the testes
and into the vas deferens on its way out of the body.
4. Sketch a seminiferous tubule and label the spermatogonia, spermatozoa.
5. Return to the low power objective and find two adjacent seminiferous tubules.
6. Switch back to the high power and focus on the cells between the adjacent seminiferous
tubules. These are the interstitial cells. Under the influence of FSH and LH, these cells
secrete the male sex hormone testosterone.
7. Sketch and the interstitial cell and the adjacent seminiferous tubules.

F. X-section of mammalian ovary

1. Using the scanning power of the microscope, identify the growing follicles.
2. Identify the corpus luteum. What is the function of this structure?
3. Sketch the field of view showing the maturing follicle.
4. Locate a follicle that is close to the surface of the ovary, getting ready to be ovulated.
5. Slide/ Model of Ovary:

a) Examine a mature follicle and locate the mature ovum within the follicle. With the
help of Microscopic ovary anatomy (from references book/internet exploring, etc),
examine a prepared slide of an ovary. Under low power you will see a large number
of small, primary follicles near the outer edge. A primary follicle contains a primary
oocyte.

b) Find the secondary follicle (use high power). Note the secondary oocyte (egg)
surrounded by numerous cells; the Graafian follicle with contain a mature secondary
oocyte. How many secondary follicle and Graafian follicle can you find on your slide?

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G. Model of mammalian ovary

1. Using your textbook, identify the following structures in the model of a mammalian ovary
maturing follicles antrum ovarian artery
mature follicle ovarian vein corpus luteurn
2. Sketch the model and label the structures identified above.
3. State the function of the structures identified above.

Laboratory Review: Gametogenesis

1. What is gametogenesis? In general, how many chromosomes are in a. gamete?

2. What is spermatogenesis? How many chromosomes does a human sperm have?

3. What is oogenesis? How many chromosomes does a human egg have?

4. Following fertilization, how many chromosomes does the zygote, the first cell
of the new individual, have?

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 Laboratory Exercise 5 & 6: Embryological development

Overview

All sexually reproducing, multicellular organisms begin life as single-celled zygotes, and
progress through stages of growth, differentiation, and morphogenesis. Growth is the
irreversible increase in size of an organism. Differentiation is the structural and functional
specialization of groups of cells. Morphogenesis is the development of pattern, shape, and
form. Embryology is the study of these processes in the early development of an organism.

Objectives

1. Describe the stages of embryological development common (processes of cleavage,

blastulation, gastrulation, and organogenesis
2. Define terms describing the formation of ectoderm, endoderm, mesoderm, and body
cavities.
3. Describe variations in strategies of development among the sea-star, frog, and chick.

Introduction

Early stages of embryology are similar in advanced invertebrates and vertebrates, and
include formation of body cavities and multiple cell layers. During these stages the
development of embryos must overcome fundamental problems. For example,
development always requires energy (i.e., food), protection of the embryo, and a waste
disposal system. However, the specific morphologies and structures involved in
development vary significantly among groups of animals. In other words, different
organisms have different strategies to cope with the same problems.

During this lab exercise you will compare features and developmental strategies of the
three earliest stages of development of the sea-star, frog, and chick.

These stages are:

1. Fertilization - Male and female gametes fuse to form a zygote.

2. Cleavage - Zygote divides into a larger and larger number of smaller and smaller cells
that eventually form a hollow sphere of many cells. Cleavage ends when
groups of cells begin to differentiate.

3. Gastrula - Cells of the sphere formed during cleavage continue to divide and move
inward to form three germ layers.

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Materials

prepared slides of sectioned frog embryos

prepared slides of sea urchin eggs
models of sea urchin zygotic development
models of frog embryonic development
model of mammalian ovary
model of frog eggs in different stages of development

Laboratory Procedure

1. Before coming to lab, go to the following websites, print the appropriate pictures
from each, label them, and tape them into your notebook with citations.

Sea star development:

http://www.jburroughs.org/science/resources/SeaStarembryology/starfishembryology.html
http://www.uoguelph.ca/zoology/devobio/210labs/cleavage1.html
http://www.luc.edu/depts/biology/dev.htm

Frog development
http://www.cascadia.ctc.edu/FacultyWeb/instructors/ccollin/frog_development.htm

Chick development:
http://web.grcc.edu/biosci/pictdata/104contents.htm
http://www.uoguelph.ca/zoology/devobio/splab4/

2. Examine a slide of a sea-star development: egg two-celled blastula, four-celled blastula,

early, mid, and later gastrula.

3. Sketch a sea star egg, two-celled blastula, four-celled blastula, and a gastrula.

4. Examine prepared slides of a frog egg and zygote. Sketch and label each.

5. Examine slides of early and late cleavage and a slide of a frog blastula cross section.

6. Observe the demonstration specimen of a living 33- and 72-hour chick embryo.

7. Examine 24-, 33-, and 72-hour chick embryos, and follow the formation of the brain,
heart, and eye.

8. Discuss the stages of development of the sea-star, frog, and chick.

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Laboratory Review

1. Explain the process of gastrulation in each of the organisms observed. What are the
major differences between them?

2. Why do human embryos have a trophoblast while bird embryos do not?

3. How are development in birds and humans similar?

4. Why do frog eggs have proportionately less yolk than bird eggs?

5. Why is human cleavage holoblastic?

6. At what point in its development does a human embryo implant in the uterus?

7. What is the function of each of the extraembryonic membranes?

8. Explain the difference between holoblastic and meroblastic cleavage.

9. Compare and contrast a blastopore and a primitive streak.

10. What structures do each of the 3 germ layers develop into in an adult?

11. What is yolk? How does it influence cleavage?

12. State the function/significance of each of the following structures, and state during
which stage of development (and in which organism) they appear: primitive streak,
blastomeres,gray crescent, epiblast, archenteron, germinal disc, and chorion.

13. Explain the differences between a zygote, a blastula, and a gastrula.

14. Sketch a frog zygote and label the animal and vegetal poles, and the gray crescent.
Label where yolk is found, and explain how these divisions will affect the axes of
development of the adult.

15. Explain the difference between a telolecithal and a isolecithal egg.

16. At what stage in the development does the embryo change shape? How does this
change occur?

17. As the embryo becomes a tadpole, the tail becomes a prominent structure. How does
the adult frog lose its tail?

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