LABORATORY EXERCISES FOR
VERTEBRATE EMBRYOLOGY
MARY
LEE SPARLING, PhD
PROFESSOR
OF BIOLOGY,
CALIFORNIA
STATE UNIVERSITY, NORTHRIDGE COPYRIGHT, 1986, 1991,1998
,2001
INTRODUCTION
This
EMBRYOLOGY lab is designed to do several things:
1.
Give you time to look at embryos to see how they are organized and how they
change
with
time. It is very valuable to be able to construct an embryo from the sections
you see,
or to
figure out where the particular section was made in the embryo. That will
develop
your
3-D image projection ability.
2.
acquaint you with biological experimentation as a non-exact science,
demonstrating
how
important it is to vary only one parameter at a time, allow you to find out
about
variation
in individual response, to discover that it is the experimenter's perception of
the
result
which can be inexact (every student will have a slightly different idea of how
the
experiment
went); 3. reinforce your knowledge of the experimental method, force you to
start
out each experiment with an hypothesis to test, make you aware of the
importance of
control
experiments for comparison. Our most common experimental variables will be
temperature,
calcium and magnesium ion concentration, cytoskeletal disruptors, substrate
concentration,
anesthetics which alter the membrane.
4.
allow you to learn some of the methods used in biological research in cell
biology.
This
will be a great advantage to those going into graduate work, or teaching,
or.professional school. This lab combines training in old morphological methods
with
chemical
methods. There will be labs which do not produce the expected results of the
hypothesis.
The common error of students is to think that these labs failed. It is just as
important
to your education in biological research to figure out why some experiments do
not
"work out" the way they were expected, as it is to get beautiful
results. Most of
research
is spent getting the experimental conditions to the point where data can be
collected
and meaningful results collected. Research is not cookbook chemistry, and I
have
not attemped to write a cookbook. I have used some experiments which require
practice
before beautiful results can be obtained. However, I have tried to adapt them
to
classroom
application. There are often time constraints which make this difficult.
Sometimes,
we try a different species due to availability. Sometimes experimental
animals
die right before the class, sometimes mistakes in solution making can occur,
since
graduate students often are also doing this for the first time, and sometimes
the lab
temperature
is very warm or very cold which can change rates of egg development, or
cell
motility. No lab should be considered wasted, since you will learn to handle
volumetric
measurement, microscopic proficiency, quantitation, methods for handling
gametes.
If you are consistently having problems completing labs when others are not,
chances
are you need to improve your prelab preparation or your data recording
technique.
You
can help insure good lab technique by the following: 1. take care of your
dissection
kit,
buy a glass marking pen, a ruler, a cheap stopwatch, and keep them in your
backpack.
2.
take good notes during the experiment about any variation of technique and
record
your
results accurately. You can save yourself enormous amounts of lab time by
setting
up
tables ahead of time for filling in the results.
3.
read about the experiment in your text and any references in the lab manual
before you
come
to the lab. You will get much more out of the experiment. You should write some
of
the possible conclusions to such experiments before you do them. For example, if
you
are
going to vary the ion concentration in a certain test, find out what that ion
does in
cells,
in relation to that cell so that you will have a possible explanation of your
results.
You
will not be able to figure out what your experiments mean unless you do this.
You
may
want to bring your texts to class as resource books, or your biochemistry or
genetics
text
may also help. Since you always have to turn in your lab results at the end of
the lab,
you
must have some of the work done before the lab.
4.
don't rely on the instructor to tell you what this experiment is supposed to
mean. I will
be
happy to answer questions, but I expect you to do some work before you come. It
is
not
possible to coordinate the lecture with the lab, so sometimes you will do
something in
lab
we have not yet discussed in lecture. Therefore, you will have to read in your
text to
find
out where it fits in the overall embryology. That reading will just make it
easier
when
we come to that part in lecture.
5.
always use clean glassware. Most of the time you can see dirt in glassware. You
will
not
have a problem if you always leave it clean at the end of the lab, to dry for
the next.time. We may have to share drawers with the other section of
embryology lab, so leave
them
clean glassware, and they will return the favor. Always take clean pipettes
from
stock
for the day, then put them back at the front of the lab to be washed at the end
of the
day.
Glassware should be immersed in hot soapy water and brushed individually, then
rinsed
eight times in running tap water and three times in distilled water. Invert to
dry in
your
drawer. Disposable pipette tips and cover glasses need not be washed.
6.
keep your lab exercises after they have been graded. Your lab book will be
handed in
at
the end of the semester for review and grade. The lab assistant will grade each
experiment,
but the instructor will make the final analysis. Do work with your lab
partners,
but write up your experiments by yourself, since they will be compared at the
end.
That is where the prelab work will show up.
SOME LAB TIPS
PIPETTING.
For good control of the pipette use your index finger on the tip of the
pipette,
not your thumb. Pull fluid up into the pipette until slightly above the place
where
you
want the volume to be. Release your suction bulb at the same time as you place
you
finger
on the tip. If the tip of the pipette is wet, you will not be able to easily
release a
part
of the volume to get it to the volume you wish to deliver, so don't have very
wet
fingers.
Probably the most accurate way to pipette is by the blowout method, where you
only
take up the amount you wish to release, each time you place a volume in a tube,
then
blow
out the fluid with your bulb after it flows to a stop. Always use the size
pipette near
the
volume you want (use a 1 ml for .1-1.0 ml; use a 5ml for 1.01 to 5; use a 10 ml
for
5.01
to 10ml; use a graduated cylinder for more than 10ml.) Never use two times of 5
ml
for
ten ml delivery. Each time you pipette, you magnify the error. Don't use large
pipettes
for
small amounts to deliver, because you cannot read the large diameter as
accurately as
the
smaller diameter pipettes. For amounts less than 1 ml use the automatic
pipettes with
disposable
tips. During the same lab, you can use the same tips, if you label them by
marking
them with a marker to prevent contamination of the reagents.
DILUTIONS.
To make dilutions, divide the desired concentration by the present
concentration,
then use that value to determine the desired volume of concentrated
reagent
to dilute with water to the final volume. Example: reagent prepared is 1 M.
Reagent
concentration desired in your reaction mixture is 6 mM or .006M. Divide .006
by 1=
.006. You need 25 ml of reaction mixture, so multiply 25 by .006= .15 ml you
need
to
add before bringing the mixture to a final volume of 25 ml. Often in making
reaction
mixtures
you have to add several reagents, so add up all the reagent volumes added,
subtract
from the 25 ml, then make up the difference by adding water. Bringing a
calculator
to lab is essential. IT IS IMPORTANT NOT TO DILUTE REAGENTS TO
THE
FINAL CONCENTRATION DESIRED BEFORE ADDING THEM TO THE
REACTION
MIXTURE BECAUSE THAT WOULD DILUTE THEM FURTHER..pH. The pH of reactions is very
important and must be controlled during experiments.
Make
sure that all reagents are the proper pH, and then add buffer at the controlled
pH
desired.
Usually, .1-.01 M buffers are used. They are usually supplied as 1M, and
dilution
is
done as above. Dilution does not change the pH. Learn to use the pH meter, and
ask the
instructor
to show you how to test your reaction mix.
SPECTROPHOTOMETERS.
Always check to be sure the optical filter system is correct
for
the wavelength you wish to use. Then turn it on 20 min before you need to use
it, so it
can
stabilize. Use only special tubes, and insert them so the markings are at the
front.
Make
sure you know how to zero the instrument, and have a control tube for doing
that.
WATER
BATHS. Fill the water bath with water the approximate temperature you want,
then
adjust it with ice and a thermometer. Then turn on the heating element and
adjust it
so
the light just comes on at that temperature, and goes off when you turn it a
little bit
lower.
Then watch it for a few minutes, to be sure it
REAGENTS.
Since the whole class has to use the reagents, it is important that they not
be
contaminated by dirty pipettes. Take the amount of reagent you have figured out
that
you
need for the whole day, placing it in an ehrlenmeyer flask, well marked. Since
there
are
20 students and ten pairs of partners, never take more than 1/10 of the total
volume.
ABSENCES.
There will be one absence allowed, or the lowest grade will be eliminated.
There
can be no makeup labs. Habitual tardiness, or long coffee breaks, or allowing
your
lab
partner to do the work, is always noted by the instructor and will be reflected
in your
final
grade. Grading will be subjective. An A grade will be for those who learn the
slides,
do
the work, write the reports, including drawing correct conclusions about the
experiments.
This will include getting results on unknown samples given by the
instructor.
To be able to do all of this in 3 hours takes organization, done largely before
coming
to lab. The instructor will do spot checks to see if you have done your prelab
work.
It is possible for everyone in lab to get an A, if everyone does the work well.
SLIDES.
You will have several slide boxes checked out to you during the semester.
Check
out the slides when you get the box, and make sure all the needed slides are
there,
and
not cracked unless noted by the instructor. You will be held responsible for
those
slides.
It is a misdemeanor to remove the slides from the campus, since they are worth
quite
a lot of money. If you break them or lose them, you own them. Over the years, I
have
noticed that those who stay for the full lab period and ask questions, do the
best, so I
plan
to have spot three minute papers during labs to test for preparation and
attendance.
SETTING GOALS FOR THE SEMESTER Adapted from
exercises
by William McKeachie Teaching Tips and lab manual for Learning to Learn.
If
you are going to get the maximum learning experience from this semester for the
amount
of work you do, you need to have completed the prerequisites, have a certain
level
of maturity that comes from being in upper division, your student goals must be
clear,
and compatible with my instructional goals. Think about your longterm goals.(become
a doctor, become a dentist, become a scientist, etc). Think about how your goal
will
be aided by taking this course. What are your long term goals for this course?
Possible
goals are: to get a certain grade; to get to know an instructor who can write you
a
letter
of recommendation; to fulfill a requirement; to prepare for the MCAT; to
prepare
for
medical or professional school by learning about anatomy and development. Some
may
have the goal of doing as little work as possible. Some may want to find out
how
they
each developed from a zygote and how they got to be different from others in
color,
sensory
ability, hairiness, sensuality, sex, immunity, size.
My
goal is to help you to learn the major concepts of developmental biology, to
learn
how
to design and carry out experiments in developmental biology, to learn how to
think
in 3D
when observing slides in 2D, to learn about relationships between mammals and
other
species regarding development, to improve your communication skills, your
thinking
and reasoning and questioning power, and ability to work in groups. My goals
are
going to require participation in class involving the reading to be done ahead
of class,
and
the experiments done during class.
Now
think of your long term goals for this course, and how you expect to achieve
them
through
setting short term goals. For example, you could develop short term goals such
as:
read all assignments ahead of class, do some extra reading in the reserve book
room,
carefully
prepare the lab tables to be filled in, or the purpose of the experiment before
coming
to class. You could answer all review questions and then discuss them with your
group.
You could strive to participate in discussions more, to become more organized
in
essay
writing. These goals will definitely be compatable with my goals. You need to
make
out a schedule to include work hours, class hours and study hours. Then you
will
know
how much time you have left for socializing. Set your priorities early so that
you
don't
end up studying all just before tests. You will get little out of the course if
you do
that,
and part of your grade will be on class participation, and your state of
preparation on
a
daily basis.
Another
of my goals is to try to help those who have special problems related to poor
previous
performance or preparation, problems related to the multi-cultural nature of
the
body
of students, poor writing skills, inability to participate in class
discussions, poor
study
habits. If you know that you have any of these problems, please call them to my
attention
as early as possible so I can give you special attention.
Fill
out an ideal schedule for each week of class, including class, work, study time
(remember
that 1-2 hour periods of study 4-8 times a week are probably better than 8 hrs
solid
study), exercise, socializing, phoning, errands. You need to commit a certain
amount
of time per week to study for this class. The minimum should be 2 hr/ hr of
lecture
and 2hr/ lab. That would be 8 hr/wk. Then you need to set when those hours will
be each
week. If you see that after you block out the hours for class and work you
don't
have
any time for study each week, you need to do something about your schedule,
because
you certainly will not fulfill any of your goals, and none of mine. Take this
with
you
and for the first week, fill out what you actually do with your time, being
strictly
honest.
This may help you become more realistic about your study habits. At the end
of.the week compare it with your ideal. Bring both to class next week. Your
group may be
able
to help make suggestions of how to make the fit better. This may involve a
shift in
priorities.
The
instructor will not look at your results unless you so desire.
IDENTIFYING
STUDENT'S IMPLICIT THEORIES OF LEARNING AND
TEACHING
The
papers resulting from the exercises on these pages will not be handed in, and
will not
be
graded.
Think
about how you learn. Write a three minute paper on things which help you learn,
the
most. Just start writing and write whatever comes into your head. Continue on
back of
page.
Now
write about how knowledge of developmental biology concepts and experimental
techniques
is going to lead to your empowerment as a scientist or science
professional..Now we are going to break up into groups to discuss your answers.
Each group will
select
a recorder who will compile a list of all answers. Then your group will try to
reach
concensus
on the best answer for each question which will then be reported back by a
reporter
whom you select. If there is no consensus, then you will report that, and why.
Did
your answers include going to professor’s office hours to discuss your needs? Please
don’t
just come to class expecting me to give you your education. You have to dig it
up
for yourself.