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.