NAME ______
LAB SECTION ______LAB TIME ______
SEAT NUMBER ______
ADDRESS ______
______
______
PHONE NUMBER ______
VERTEBRATE DEVELOPMENT, BIOL 4410
LABORATORY HANDOUTS
FALL 2016 (revised 2/2/16)
INSTRUCTOR - DR. STEPHEN C. KEMPF
DEPARTMENT OF BIOLOGICAL SCIENCES
AUBURN UNIVERSITY
1
TABLE OF CONTENTS
LECTURE, LAB READING, SLIDE #s, AND EXAM SCHEDULE ------3
COURSE POLICIES ------10
LABORATORY REQUIREMENTS/INFORMATION ------11
LAB NOTEBOOK REQUIREMENTS ------12
LAB NOTEBOOK – FREQUENTLY ASKED QUESTIONS ------14
POSSIBLY USEFUL STUDY HINTS ------16
LAB EXAM AND QUIZ SAMPLE QUESTIONS ------17
HANDOUT 1A, LABORATORY ORIENTATION ------20
HANDOUT 1B, USE OF THE COMPOUND MICROSCOPE ------23
HANDOUT 2A, ROUTINE METHODS ------29
HANDOUT 2B, MITOSIS, MEIOSIS, AND GAMETOGENESIS ------35
HANDOUT 2C, BASIC MICROSCOPY METHODS ------37
LABORATORY ID LISTS – OVERVIEW ------42
HANDOUT 3A, REPRODUCTIVE ORGANS: SPERMATOGENESIS ------43
HANDOUT 3B, REPRODUCTIVE ORGANS: OOGENESIS ------44
HANDOUT 4A, STARFISH DEVELOPMENT ------45
HANDOUT 4B, EARLY FROG DEVELOPMENT ------46
HANDOUT 5, 4 - 7 MM FROG TADPOLE ------48
HANDOUT 6, 10 MM FROG TADPOLE ------51
HANDOUT 7A, CRANIAL NERVES AND GANGLIA ------54
HANDOUT 7B, 18 AND 24 HOUR CHICK ------58
HANDOUT 8, 33 HOUR CHICK ------60
HANDOUT 9, 48 HOUR CHICK ------62
HANDOUT 10, 72 HOUR CHICK ------65
HANDOUT 11, 96 HOUR CHICK ------72
HANDOUT 12, 6 MM PIG ------78
HANDOUT 13, 10 MM PIG ------83
HANDOUT 14, TOOTH DEVELOPMENT ------90
2
VERTEBRATE DEVELOPMENT - BIOL 4410 LECTURE and LAB FALL 2016 - LECTURE AND LAB TOPICS, STUDY ASSIGNMENTS C - Carlson (6th edition), S - Schoenwolfe (7th edition), D - Digital Lab Manual (If you have a different edition of the text, the required page numbers may be different.) ______------Aug 17 W Class orientation, drops and adds, lab switches Introduction, Developmental biology as a science Gametogenesis I: Gametes, where do they come from. C: pp. 1-56, pp. 57-74
W/Th NO LAB TODAY! ______------Aug 19 F Finish Introduction, Developmental biology as a science Gametogenesis I: Gametes, where do they come from. C: pp. 1-56, pp. 57-74 ______------Aug 22 M Gametogenesis I: Gametes, where do they come from C: pp. 57-74
M/T ATTENDANCE AT THIS LAB IS REQUIRED!!!! Lab: Equipment assignments. Use of microscope. D: Introductory materials, Approaches to learning Routine methods of Microtechnique Microscopy: Use of the Microscope ______------Aug 24 W Gametogenesis II: Spermatogenesis C: pp. 75-93
W/Th ATTENDANCE AT THIS LAB IS REQUIRED!!!! Lab: Histological sections, a 2-dimensional view of 3-dimensions. Reproductive organs. Tray #1 & 2. D: Developmental Events and Mechanisms, General Background Information, Gametogenesis, Fertilization M: pp. 1-15, 74-77, 126-129 ______------Aug 26 F Gametogenesis II: Spermatogenesis . C: pp. 75-93 ______------
3 Aug 29 M Gametogenesis II: Finish Spermatogenesis. Start Oogenesis C: pp. 75-93 C: pp. 94-120
M/T Lab: Starfish development. Tray #3. Quiz 1 D: Starfish Development, Descriptive Text M: pp. 50-56 FIRST LAB QUIZ TODAY! ______------Aug 31 W Gametogenesis III: Oogenesis C: pp. 94-120
W/T h Lab: Early frog development. Tray #4. Quiz 2 D: Amphibian Development, Early Frog Development, Descriptive Text M: pp. 78, 81-96 ______------Sept 2 F Gametogenesis III: Oogenesis C: pp. 94-120 ______------Sept 5 M LABOR DAY HOLIDAY 15th day of classes tomorrow ______------Sept 7 W Finish Fertilization, C: pp. 121-142
W/Th Lab: 4mm Frog tadpole. Tray #5 Quiz 3 D: Amphibian Development, 4mm Frog Tadpole, Descriptive Text for Wholemount and Transverse sections M: 97-105 ______------Sept 9 F Fertilization C: pp. 121-142 ______------Sept 12 M Cleavage. C: pp. 143-150, 151 - 188
M/T Lab: Frog development, 4-7 mm, Tray #5. Quiz 4 D: Amphibian Development, 7mm Frog Tadpole, Descriptive Text for Wholemount and Transverse sections M: pp. 97-105, 106-116 ______------
4 Sept 14 W Cleavage. C: pp. 143-150, 151 - 188 .
W/Th Lab: Frog development, 4-7 mm, Tray #5. Quiz 5 D: Amphibian Development, 7mm Frog Tadpole, Descriptive Text for Wholemount and Transverse sections M: pp. 97-105, 106-116 ______------Sept 16 F Cleavage. C: pp. 143-150, 151 - 188 ______------Sept 19 M Gastrulation. C: pp. 189-226
M/T Lab: Frog development, 10mm. Tray #7. Quiz 6 D: Developmental Events and Mechanisms, Cleavage D: Amphibian Development, 10mm Frog Tadpole, Descriptive Text for Wholemount and Transverse sections M: pp. 117-123 ______------Sept 21 W FIRST LECTURE EXAM (through Monday's lecture)
W/Th Lab: Frog development, 10mm. Tray #7. Quiz 7 D: Developmental Events and Mechanisms, Cleavage D: Amphibian Development, 10mm Frog Tadpole, Descriptive Text for Wholemount and Transverse sections M: pp. 117-123 ______------Sept 23 F Gastrulation. C: pp. 189-226 ______------Sept 26 M Gastrulation. C: pp. 189-226
M/T Lab: Chicken development, 18 hr, 24 hr. (4 somite) Tray #8. Quiz 8 D: Developmental Events and Mechanisms, Gastrulation D: Avian Development, Major Events in Early Avian Development Descriptive Text D: Avian Development, 18 hr and 24 hr chick embryo Descriptive Text for 18 hr and 24 hr embryos, Wholemounts and Transverse sections M: pp. 131-134, M: pp. 134-144 ______------
5 Sept 28 W Neurulation and Induction Read information in Lecture Handout
W/Th Lab: FIRST LAB EXAM (through 24 hr chick). ______------Sept 30 F Neurulation and Induction Read information in Lecture Handout ______------Oct 3 M Embryonic adaptations (membranes). C: pp. 255-273 (birds & mammals), 274-290 (primate, human)
M/T Lab: Chicken development 33 hr. (12-13 somite) Tray #9. Quiz 9 D: Developmental Events and Mechanisms, Neurulation D: Avian Development, 33 hr Chick Embryo Descriptive Text for Wholemount and Transverse Sections M: pp. 145-152
Your lab notebook completed through the 10mm frog is due at beginning of your lab! ______------Oct 5 W Embryonic adaptations (membranes). C: pp. 255-273 (birds & mammals), 274-290 (primate, human)
W/Th Lab: Chicken development 48 hr. Tray #10. Quiz 10 D: Avian development, 48 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 153-170
MID-SEMESTER IS TODAY, YOU MUST HAVE PERMISSION FROM THE DEAN'S OFFICE TO DROP AFTER TODAY! ______------Oct 7 F Finish Emrbyonic adaptations, C: pp. 255-273 (birds & mammals), 274-290 (primate, human) C: pp. 311-324 ______------Oct 10 M Differentiation C: pp. 311-324
M/T Lab: Chicken development 48 hr. Tray #10. Quiz 11 D: Avian development, 48 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 153-170)
______------
6 Oct 12 W Differentiation C: pp. 311-324
W/T h Lab: Chicken development 72 hr. Tray #11. Quiz 12 D: Developmental Events and Mechanisms, Morphogenesis D: Avian development, 72 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 171-189 ______------Oct 14 F FALL BREAK! ______------Oct 17 M Early human (mammalian) development. C: pp. 274-310
M/T Lab: Chicken development 72 hr. Tray #11. Quiz 13 D: Avian development, 72 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 171-189 ______------Oct 19 W Organogenesis (Intro + Ectoderm). Nervous system. C: pp. 227-239, 427-484
W/Th Lab: Chicken development 96 hr. Tray #12. Quiz 14 D: Avian development, 96 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 190-195) ______------Oct 21 F Organogenesis (ectoderm 2). Nervous system I. C: pp. 227-239, 427-484 ______------Oct 24 M Organogenesis (ectoderm 3). Nervous system II. C: pp. 227-239, 427-484
M/T Lab: Chicken development 96 hr. Tray #12. Quiz 15 D: Avian development, 96 hr Chicken Embryo Descriptive Text for Wholemount and transverse sections M: pp. 190-195 ______------Oct 26 W SECOND LECTURE EXAM (through Monday's lecture)
W/Th Lab: 6mm pig. Tray #13. Quiz 16 Descriptive text for transverse sections M: pp. 198-237 ______------7 Oct 28 F Organogenesis (Mesoderm 1). Musculo-skeletal system I C: 311-353 ______------OCT 31 M Organogenesis (Mesoderm 1). Musculo-skeletal system I C: 311-353
M/T Lab: 6mm pig. Tray #13. Quiz 17 Descriptive text for transverse sections M: pp. 198-237 ______------Nov 2 W Organogenesis (mesoderm 2). Circulatory system I - Origin C: pp. 607-644 C: pp. 619-644
W/Th Lab: 6mm pig. Tray #13. Quiz 18 Descriptive text for transverse sections M: pp. 198-237 ______------Nov 4 F Organogenesis (mesoderm 2). Circulatory system II - Arteries C: pp. 607-644 C: pp. 619-644 ______------Nov 7 M Organogenesis (mesoderm 2). Circulatory system II - Arteries/veins C: pp. 607-644 C: pp. 619-644
M/T Lab: 10mm pig. Tray #14 & #15-1. Quiz 19 M: pp. 201-237 ______------Nov 9 W Organogenesis (mesoderm 2). Circulatory system II - Veins C: pp. 607-644 C: pp. 619-644
W/Th 10mm pig. Tray #14 & #15-1. Quiz 20 M: pp. 201-237 ______------Nov 11 F Organogenesis (mesoderm 3). Urogenital system II C: pp. 569-606 ______------
8 Nov 14 M Organogenesis (mesoderm 3). Urogenital system I C: pp. 569-606
M/T Tooth Development, Tray #15-2, Quiz 21 ______------Nov 16 W Organogenesis (mesoderm 3). Urogenital system III C: pp. 569-606
W/Th LAB NOTEBOOKS DUE immediately before the exam! LAB FINAL EXAM - comprehensive with emphasis on chick & pig. Lab Clean-up. Turn in microscopes and slides. ______------Nov 18 F Organogenesis (endoderm). Face, Visceral arches, lips, tongue, teeth C: pp. 513-526, 537-546 ______------Nov 21-25 Thanksgiving Vacation ______------Nov 28 M Organogenesis (endoderm). Face, Visceral arches, lips, tongue, teeth C: pp. 513-526, 537-546
M/T GO OVER LAB FINAL EXAM. QUIZ AVERAGES WILL BE RETURNED ______------Nov. 30 W Organogenesis (endoderm). Face, Visceral arches, lips, tongue, teeth C: pp. 513-526, 537-546
W/Th No lab today! (-;{ ______------Dec 2 F Organogenesis (endoderm). Face, Visceral arches, lips, tongue, teeth LAST DAY OF CLASSES C: pp. 513-526, 537-546 ______------Dec FINAL LECTURE EXAM (through last lecture, COMPREHENSIVE) at 8:00 A.M. - 9:30 A.M. in lecture rooms (SCC 115/122). ______------
9 COURSE POLICIES:
Disabilities: If you have a disability that requires special consideration, please talk to me during the first two weeks of classes so that arrangements to accommodate your needs can be made.
Equipment Responsibilities: Students will be responsible for keys and laboratory equipment assigned to them. Failure to check in keys and all lab equipment at the end of the semester in good condition will result in an Incomplete course grade until the matter is resolved.
Attendance: Attendance is required at the first 2 lab sessions. Attendance is required for lecture and lab exams/quizzes. Absence will be condoned only if an acceptable, verifiable, written excuse is provided. Absence without an acceptable excuse will result in a grade of zero (0) for the exam missed. Make-up examinations will be given as soon as practical. No make-ups are given on lab quizzes (see below). No unannounced quizzes will be given.
Lab Quizzes: Starting with the second lab, a quiz will be given immediately before the introductory lecture to the lab. Quizzes will cover the material worked on in the previous lab. There will be no make-up quizzes. If you can provide an acceptable, verifiable, written excuse for missing a quiz, that quiz will not be included in the final calculation of your cumulative quiz grade. Quizzes will not be given on lab exam days; however, quizzes WILL be given on lecture exam days.
Academic Honesty: Cheating is defined, and rules regarding the reporting of honesty cases are described, in the Tiger Cub. Cheating, including plagiarism of class work from the efforts of students who took Vertebrate Embryology in previous semesters, is a very serious offense and will be dealt with by the Auburn University Academic Honesty Committee. Please note that I do not allow students to keep my exams. Thus, there should be no copies of my exams available to you. If some copies of my exams have somehow escaped my “grip”, they have been obtained by illicit means and using such exams to study is also cheating.
Honesty and the digital age: The digital age brings with it new problems in regard to academic honesty. Items such as smartphone cameras, “spy” pens, lapel and eyeglass cameras, and other sorts of recording devices are now available. Use of such devices to acquire copies of exams or exam questions is a form of cheating. Similarly, using materials acquired by such devices to study for exams is also cheating. So, please do not use such devices to illicitly copy exam materials in this course and do not use materials acquired by such devices to study for exams. If you are found guilty of copying exams with a digital device, you will receive an automatic F in the course and it will be recommended to the Academic Honesty Committee that you be suspended.
NOTE! When graded exams are returned for your examination during class, all digital devices must be put away either in your backpack or pocket. If we see you have a cell phone or other digital device in your hand or on your desk/bench top while we are going over exams, 10 pts (one letter grade) will be subtracted from your exam score. No excuses will be accepted for this sort of infraction, so please be sure that all digital devices are in pocket or backpack prior to exams being returned.
10 LABORATORY REQUIREMENTS/INFORMATION:
!!ATTENDANCE AT THE FIRST TWO LABS IS REQUIRED. IF YOU FAIL TO ATTEND YOU WILL LOSE 1 POINT ON YOUR FINAL CLASS AVERAGE FOR EACH LAB MISSED!! Laboratory assignments in this Vertebrate Development course, as in most others, require an intensive study of histological sections of fixed tissues (gonadal and embryonic). In order to do well in the laboratory portion of this course it will be necessary to devote considerable time to the examination of your microscope slides. For most students, this will mean study time outside the scheduled laboratory periods if high grades are desired on the lab exams. To facilitate such study, access to the lab is controlled by a card-swipe lock, i.e. your student ID card can be used to open the lab door. The rules for lab use will be given to you at the first lab. Be there!!!!! A list of required identifications will be handed out for each slide, or set of slides, used in the course. You are to locate the listed “structures” on your slides and relate them to their position, function, and shape in the embryo or tissue where applicable. It is important to know, 1) what these structures are, 2) in some cases, what they will become or what they were, 3) what germ layer they are derived from 4) and what they do in the embryo and/or adult. Some of this information will be obvious, some will be presented in lecture or lab, and some will be found in your reading assignments.
LAB EXAMS: Two lab exams will be given during the semester. These exams will cover the material indicated in the class schedule. The second lab exam is comprehensive. Examples of Lab Exam and quiz questions are given starting on page 17 of this handout. CHECK THE SYLLABUS AND NOTE THE LAB EXAM DATES!! IF YOU PLANS ON THOSE DATES THAT WOULD CAUSE YOU TO MISS THE LAB EXAM, CHANGE THEM NOW! IF YOU HAVE SOME EVENT DURING THE DAYS DIRECTLY PRECEEDING A LAB EXAM, PLAN ACCORDINGLY! There are very few excuses that I will accept for missing a lab exam on its scheduled date.
LAB QUIZZES: There will be a short quiz given during each lab period starting with the second lab. Quizzes will cover the material worked on in the previous lab. Quizzes will continue to be given at every subsequent lab except those during which a lab exam is given. There will be no make-up quizzes. If you can provide an acceptable, verifiable, written excuse for missing a quiz, that quiz will not be included in the final calculation of your cumulative quiz grade. Please note that quizzes WILL be given on "lecture exam" days.
Each quiz will be worth 4 points and will consist of two identifications of embryonic structures/tissues from slides projected on the screen at the front of the class room and 2 short answer questions concerned with the structures/tissues you identified. At the end of the semester your quiz average (%) will be calculated as follows. Quiz Average Example: (For an example where 10 quizzes are given)
Total points received for all your quizzes - 34 Total points possible - 40 Quiz average = (34/40) X 100 = 85% Examples of Lab exam and quiz questions are given starting on page 17 of this handout
11 LAB NOTEBOOK:
Each student will prepare a lab notebook that summarizes their work in the laboratory portion of this course. Specific requirements for this notebook are given in the Laboratory Handout Packet available on the class web site. PLEASE NOTE: Lab notebooks must be turned in for grading on the two dates indicated in the syllabus schedule. On those days, the lab notebooks are due at the beginning of your lab period. In order to receive a grade other than "0" for a lab notebook turned in after this time, you must have an acceptable, written, verifiable excuse for your failure to turn the notebook in on time. Your final Lab Notebook grade will be the rounded average of the two grades you receive for the notebook.
PREPARATION OF LAB NOTEBOOK:
EACH STUDENT WILL PREPARE A LAB NOTEBOOK THAT SUMMARIZES THEIR WORK IN THE LABORATORY PORTION OF THE COURSE.
YOUR LAB NOTEBOOK SHOULD BE ARRANGED IN THE FOLLOWING SECTIONS AND CHAPTERS. EACH CHAPTER WILL REPRESENT ONE OF THE ORGAN SYSTEMS/EMBRYONIC STAGES YOU HAVE EXAMINED.
SECTIONS AND CHAPTERS:
TABLE OF CONTENTS
INTRODUCTORY MATERIAL I. REPRODUCTIVE ORGANS II. STARFISH DEVELOPMENT
FROG DEVELOPMENT III. EARLY FROG DEVELOPMENT IV. 4 mm FROG V. 7 mm FROG VI. 10 mm FROG
CHICKEN DEVELOPMENT VII. EARLY AND 18-24 hr CHICK VIII. 33 hr CHICK IX. 48 hr CHICK X. 72 hr CHICK XI. 96 hr CHICK
PIG DEVELOPMENT XII. 6 mm PIG XIII. 10 mm PIG
FOR THE EL PERFECTO NOTEBOOK A GOOD TABLE OF CONTENTS
EACH CHAPTER OF THE PERFECT LAB NOTEBOOK WILL CONTAIN THE FOLLOWING ITEMS IN THE FOLLOWING ORDER FOR EACH ORGANISM (i.e., Reproductive organs, Starfish, Frog, Chicken and Pig):
12 1. DETAILED LAB LECTURE NOTES FOR ALL LAB LECTURES THAT WERE CONCERNED WITH THAT ORGAN SYSTEM/EMBRYONIC STAGE.
2. ALL QUIZZES
3. ALL LABELED DRAWINGS OF SECTIONS SHOWING EVERY STRUCTURE YOU ARE SUPPOSED TO BE ABLE TO IDENTIFY FOR THAT ORGAN SYSTEM/EMBRYONIC STAGE.
4. A TABLE IDENTIFYING THE GERM LAYER ORIGIN OF EACH TISSUE/ORGAN/STRUCTURE TO BE IDENTIFIED, WHAT IT WILL FORM, AND WHAT ITS EVENTUAL FUNCTION WILL BE. (This is not required for Reproductive Organs or Starfish.)
DETERMINATION OF LAB NOTEBOOK GRADES:
See the Lab Notebook grading sheets available in the Lab Notebook section on the course web site.
THE LAB NOTEBOOK WILL REPRESENT 20% OF YOUR LAB GRADE.
13 LAB NOTEBOOK - FREQUENTLY ASKED QUESTIONS (Prepared by Kyle Barrett, 2004; updated by Maria Mays, 2012) Q. Does it matter how I organize my notebook? A. Yes. When you're putting the notebook together, please organize items as described in your lab packet. This means you'll have the notebook divided into sections for the notes, drawings, quizzes, and germ layer charts. Within those sections, you'll have your notebook organized based on the organism and stage of development. You DO NOT need to produce a germ layer chart for each stage. Only for each organism (i.e., the frog, chick, and pig).
Q. How should the notebook be bound? A. The contents of your notebook should go into a three-ring binder.
Q. Do my table of contents need to have page numbers? A. No. You do not have to number each page in the notebook and you do not need to have page numbers in the table of contents. Just list the order you have placed things in the notebook.
Q. How many drawings are required for a section? A. There is no required number of drawings. You need to have as many drawings as it takes for you to be able to label the structures listed in the ID lists in your course lab packet. Often, from a single drawing you can label a dozen structures or more. Sometimes, you may have to make a single sketch just to label one item. If this is the case, partial sketches are fine (i.e., you don't have to draw the entire embryo; just show us enough so we can tell where your drawing is coming from).
Q. Do I have to label every single term on the list for a particular organism and stage? A. No. Any time a term is new to an embryo you must label it on a drawing (new terms will appear in italics). Often times terms will continue to appear on the list after they have first developed. For example, you will see the term prosencephalon show up when you are studying the 24 hr chick. Because it is the first time you've seen the term for the chick, you'll want to label it. The term will continue to show up on future chick lists (48 hrs, 72 hrs, 96 hrs). You do not have to re-label the structure for these stages. For each type of embryo you will also be assigned 1 or 2 organ systems that you have to continue drawing (see the included full list of what you have to draw). Also, even if you have already labeled a term from a previous type of embryo (e.g. frog), you must draw and label it again if the same term applies to a new embryo (e.g. chicken). If in doubt about whether or not you should draw and label something, just ask. Q. But I can't draw very well, will you take off points for that? A. Nope. Just do the best you can. Even if you are not very artistic try to be neat (that helps us look at your pictures and give you all the points you deserve).
Q. Should I include my course packet in the notebook? A. No, you keep it - we already have a copy.
14 Q. What sort of paper/pencils should I use for my drawings? A. Plain white paper is preferred, but not required. Pencils are preferred over pen, but again, not required. If you use a pen that is very inky and bleeds through the paper (even very faintly), we ask that you draw only on one side of the paper.
Q. Mitosis, meiosis, and other processes, not structures, are on the list. How do I draw these? A. Any time a process shows up on the list (they rarely do) you should just sketch a diagram of it. For example, with meiosis, you could draw a circle representing the cell and lines within that circle representing chromosomes. Using these symbols and the appropriate labels you should be able to show what happens during the process of meiosis.
Q. In the section on the frog, the 4-7 mm stages are listed together. Will one set of drawings for these stages be OK? A. No. You have separate slides for the 4 and 7 mm frog and you should make separate drawings. However, see the question "Do I have to label every single term..." for more on this.
Q. What's a Germ Layer Chart? A. The Germ Layer Chart is a table that contains germ layer, fate, and function information on the structures on your ID lists in the course lab packet. You must make three of them, one each for the frog, chicken, and pig. The germ layer charts must be typed. Here's an example of what it should look like:
Structure/Tissue Germ Layer What if forms Function or System Archenteron Endoderm Embryonic Gut Digestion Myotome Mesoderm Skeletal Muscle Movement Otic vesicle Non-neural Ectoderm Inner Ear Sensory-Auditory Prosencephalon Neural Ectoderm Forebrain Central Nervous System Q. How will my notebook be graded? A. See the lab notebook grading sheets.
Q. If I encase the pages of my notebook in plastic cover sheets will my TAs be impressed with my initiative and give me a better score?
A. No. Please don't use plastic cover sheets for any portion of your notebook. They make it difficult to write notes/make corrections on your pages.
15 POSSIBLY USEFUL STUDY HINTS:
You may find it useful to bring Red, Yellow, Orange, Blue, and Green colored pencils to lab and lecture. I will be drawing colored figures on the board that indicate cells that will give rise to, or tissues that are derived from, the various germ layers. Blue - ectoderm, Red - mesoderm, Green - chordamesoderm (a special type of mesoderm), Yellow - endoderm, Orange - yolk.
A glossary is available at the end of your lab manual (Wright, 2005). I strongly recommend that you make maximum use of this study aids.
Other study aids available in both lab texts are the various figures in each chapter. These can be used in a number of ways,
1. As a comparative aid in studying your slides. 2. As an aid in reconstructing a 3-dimensional image of an embryo in your mind’s eye. 3. As an aid in relating various organs and structures to each other. 4. As a means of identifying specific organs and structures. 5. As a means of organizing organs and tissues into groups, i.e. those derived from ectoderm, mesoderm, or endoderm; or those associated with a specific organ system, for instance the digestive tract. In the case of organs and tissues derived from ectoderm, mesoderm, or endoderm, it will be helpful to draw and color in some of the figures with the appropriate colors corresponding to these germ layers.
BLUE - ectoderm RED - mesoderm YELLOW - endoderm
Notochord, which is derived from chordamesoderm, is colored GREEN to signify its special effects on development. Yolk, which is present in large amounts in frog and particularly chicken eggs, is colored ORANGE.
6. As a means of testing your knowledge by labeling specific organs, tissues, and structures in figs. where they are not labeled.
16 LAB EXAM AND QUIZ SAMPLE QUESTIONS:
The following are examples of the sort of questions that will be asked on the laboratory exams and quizzes.
LABORATORY EXAMS (Sec. I): On laboratory exams, the first group of questions will be concerned with structures I will point out on projected slides. e.g.
1.The lab instructor points to the Graafian follicle of a mammal and asks,
What structure is this?
or
Give two synonymous names for this structure. answer - Graafian follicle, tertiary ovarian follicle
2. The lab instructor points to the spermatogonial cells in a lobe of the grasshopper testis and asks,
What are these cells called? answer - spermatogonia or
What function do these cells perform? answer - give rise to primary spermatocytes or - they function as stem cells for the male germ cell line
3. The lab instructor points to the chordamesoderm of an early frog embryo and asks,
What embryonic structure will form from these cells? answer - notochord or
What characteristic embryonic structure in the frog is involved in the internalization of these cells?
answer - dorsal lip of the blastopore
17 LABORATORY EXAMS (Sec. II): The second group of questions on laboratory exams will involve identification of specific structures or processes using your microscope and slide set. You will locate the item indicated on the appropriate slide and put the very tip of the pointer directly on/over that item. Once you have done this, you will raise your hand a lab instructor will come to your place, check the identification, and mark it either right (+) or wrong (0). After each identification will be a question about the structure or process that requires a short, simple, written answer (usually one or two words). e.g.
Identify the following and answer the question after each identification. 1. primary spermatocyte
In terms of chromosome number, what is the ploidy of this cell immediately following the mitotic division of the spermatogonial cell that gave rise to it?
answer - diploid or
What type of cell immediately preceded the formation of this cell?
answer - type B spermatogonium
2. zona pellucida
Is this structure cellular or acellular?
answer - acellular or
What is this structure composed of?
answer - glycoproteins
3. liver diverticulum
Name an adult organ will form from the cells surrounding this diverticulum?
answer - liver or gall bladder or
What cavity within the 4 mm frog is the lumen of this structure continuous with?
Finally, when you are making identifications on your slides,
THE VERY TIP OF YOUR MICROSCOPE POINTER MUST BE DIRECTLY OVER (“ON”) THE STRUCTURE TO BE IDENTIFIED.
ALMOST DOESN’T COUNT!
18 LABORATORY QUIZZES: Quizzes will use slides that are projected on the screen in front of the classroom. Each quiz will consist of either two, 2-part questions or 4 one part questions and be worth a total of 4 points. For the 2-part questions, the lab instructor will first point to a tissue or structure on the screen and ask you to identify it. The second part will consist of a short answer question about some aspect of the tissue or structure you identified. One part questions will involve identifying a structure or answering some question about it.
1. a. The lab instructor points to a spermatid on a projection of a grasshopper testis slide and asks, "Give a specific name for this cell".
answer - spermatid
b. The lab instructor asks "What is the name of the process that results in the development of a spermatid into a mature spermatozoon?"
answer - spermiogenesis or spermateleosis or spermatozoon metamorphosis (these are 3 different names for the same process, i.e. synonyms)
2. a. The lab instructor points to the notochord on a projection of a frog tadpole transverse section and says "Give the specific name for this structure".
answer - notochord
b. The lab instructor says "What germ layer is this structure derived from?"
answer - mesoderm or chordamesoderm
(if the lab instructor had said "What specific germ layer is this structure derived from?", then only "chordamesoderm" would have received full credit.)
19 Lab Handout 1A VERTEBRATE DEVELOPMENT BIOL 4410
LAB ORIENTATION
LABORATORY ORIENTATION:
SEAT ASSIGNMENTS
Remain the same for entire semester once assignment is made. Seat assignment corresponds to equipment assignment.
EQUIPMENT ASSIGNMENT
You will be assigned one set of microscope slides of histological sectioned and stained embryos and tissues. A binocular compound microscope with electric light source will be assigned and kept in the locked cabinet adjacent to your seat. You are responsible for these items and they should be returned in good condition at the end of the course. Charges will be made for lost or damaged equipment.
EXTRACURRICULAR. LAB USE
Arrangements have been made so that it will be possible for you to use the 24/7, except on football weekends. This will be discussed further in lab.
Entrance to the SCC building after 5 PM or on weekends is accomplished by using your ID card in the "swipe" lock of the door closest to the class lab or in the middle of the building on the Chemistry building side. DO NOT PROP THE OUTSIDE OR LAB DOORS OPEN! IF THIS IS DONE THE CLASS WILL LOSE ITS 24/7 PRIVILAGES.
During lab use outside of regular class time, the last person to leave the lab is responsible for making sure the lab door is locked. Be sure to actually test the door by trying to turn the door knob and pulling on the door. Sometimes, for whatever reason, the lock mechanism fails to function.
If you leave the lab for any reason (going to the rest room, a cigarette break, to buy a coke, etc.) and there is no one else in the lab, then the door must be locked while you are gone. NO EXCEPTIONS.
Use of the lab outside regular lab time will continue only so long as everyone observes the rules set down above. IF ONE PERSON BECOMES LAX, THEN THIS SORT OF LAB USE WILL BE CURTAILED FOR EVERYONE. If you are the cause of this, I suspect your classmates will not be too happy with you.
20 LAB INTRODUCTION:
1. THE FIRST LAB WILL BE CONCERNED WITH INTRODUCTORY MATERIAL. IF YOU CANNOT ATTEND BECAUSE OF SCHEDULING DIFFICULTIES, YOU MUST ARRANGE TO SEE ONE OF THE TAs ABOUT MICROSCOPE USE BEFORE YOU USE YOUR MICROSCOPE. NO EXCEPTIONS!
2. CHAIRS UNDER BENCH AT END OF PERIOD.
3. MICROSCOPES AND SLIDES IN DRAWER OR CABINET AND LOCKED WHEN YOU ARE THROUGH USING THEM.
4. LAB USE DURING TIMES OTHER THAN SCHEDULED LAB PERIODS. THIS WILL ONLY WORK AS LONG AS EVERYONE RESPECTS RULES CONCERNING THIS SORT OF LAB USE. BE SURE DOOR TO LAB IS LOCKED IF YOU LEAVE ROOM AND NO ONE ELSE IS IN IT. DO NOT PROP THE OUTSIDE OR LAB DOORS OPEN! IF THIS IS DONE THE CLASS WILL LOSE ITS 24/7 PRIVILAGES.
5. IT IS TO YOUR ADVANTAGE TO ATTEND SCHEDULED LABS. THAT IS THE TIME SOMEONE WILL BE PRESENT TO ANSWER QUESTIONS CONCERNING YOUR SLIDES. IN ADDITION, YOU WILL WANT TO BE PRESENT FOR THE LAB QUIZZES GIVEN DURING EACH LAB PERIOD. YOUR SCORES ON THESE QUIZZES WILL DETERMINE 20% OF YOUR LAB GRADE. EVERYONE MUST BE IN LAB ON THE DATES OF SCHEDULED LAB EXAMS. NO EXCEPTIONS!
6. LAB QUIZZES WILL BE GIVEN DURING EVERY LAB PERIOD STARTING WITH THE SECOND LAB OF THE SEMESTER. THESE QUIZZES WILL DETERMINE 20% OF YOUR LAB GRADE. SEE p. 19 FOR A BRIEF DESCRIPTION OF QUIZZES. SEE p. 19 OF THIS HANDOUT FOR EXAMPLES OF THE SORTS OF QUESTIONS THAT WILL BE ASKED ON LAB QUIZZES.
7. LAB EXAMS ARE PRACTICAL AND OBJECTIVE. YOU WILL HAVE TO MAKE IDENTIFICATIONS ON YOUR SLIDES WHICH I WILL CHECK. IN ADDITION, THERE WILL BE A SHORT ANSWER QUESTION ABOUT THE STRUCTURE IDENTIFIED. YOU WILL ALSO HAVE TO IDENTIFY AND ANSWER QUESTIONS ABOUT TISSUES AND STRUCTURES I PROJECT ON THE SCREEN. DO NOT ASK TO BE EXCUSED FROM LAB EXAMS UNLESS YOU ARE TRULY SICK OR HAVE SOME SORT OF REAL EMERGENCY. IF YOU ARE SICK YOU WILL NEED TO PROVIDE A DOCTOR'S EXCUSE THAT SPECIFICALLY STATES YOU WERE TOO SICK TO ATTEND THE LAB EXAM. CLINIC EXCUSES THAT SIMPLY SAY YOU VISITED THE CLINIC WILL NOT BE ACCEPTED. IF YOU MISS AN EXAM WITHOUT AN ACCEPTABLE EXCUSE YOU WILL RECEIVE A ZERO (0) FOR THAT EXAM. SEE pp. 17 - 18 OF THIS HANDOUT FOR EXAMPLES OF THE SORTS OF QUESTIONS ASKED ON LAB EXAMS.
8. FINALLY, YOU ARE RESPONSIBLE FOR YOUR MICROSCOPE AND SLIDES. SO BE CAREFUL WHEN USING THEM.
IMPORTANT THINGS TO REMEMBER WHEN USING THE EMBRYOLOGY LAB
1. When you remove your microscope from its cabinet or return it to the cabinet, use BOTH hands and take care not to bump the mechanical stage controls against the sides of the cabinet.
21 2. WHEN CLEANING LENSES, USE ONLY NEW, VIRGIN, LENS PAPER OR Q-TIPS! Once you have wiped a lens never re-use the Q-tip or lens paper. Throw it away and get out a new one. Lens paper and Q-tips are cheap, objectives, oculars and other lenses are very expensive.
3. WHENEVER YOU CHANGE SLIDES OR LOSE FOCUS ON THE SLIDE YOU ARE VIEWING, ALWAYS RE-START YOUR VIEWING BY FOCUSING WITH THE 4X OR 10X OBJECTIVE! Then move back to higher power objectives focusing with each one before moving to the next highest power.
4. IF YOU ARE THE LAST PERSON TO LEAVE THE LAB AT TIMES OTHER THAN REGULAR CLASS SESSIONS, BE SURE THE DOOR IS CLOSED AND LOCKED WHEN YOU LEAVE!
22 Lab Handout 1B VERTEBRATE DEVELOPMENT BIOL 4410
MICROSCOPY
USE OF THE COMPOUND MICROSCOPE
HOW TO HANDLE A MICROSCOPE:
A compound microscope should be treated as a VERY, VERY, VERY fragile piece of equipment.
1. Adjustments should be made gently and with finesse.
2. ALWAYS use BOTH HANDS when picking the microscope up and moving it.
3. When focusing on a slide, ALWAYS start with either the 4X or 10X objective. Once you have the object in focus, then switch to the next higher power objective. Re-focus on the image and then switch to the next highest power. Etc. NEVER advance more than one objective before focusing.
4. Use ONLY the fine focus control when focusing for higher power objectives (20X, 40X, 100X). The coarse focus control is too coarse for focusing with these objectives. Objectives are fragile and must not be rammed into slides.
5. If an objective or ocular needs to be cleaned use the Q-tips and breath moisture or the methanol available on the front desk in the lab. THE FOLLOWING NOTES ON CLEANING ARE VERY IMPORTANT! a. USE ONLY NEW, UN-USED Q-TIPS FOR CLEANING. Even Q-tips that have only been used once may have dirt on them that could scratch a lens or contaminate the methanol and cause scratches when the now dirty methanol is used for cleaning in the future. b. WHEN LENS PAPER IS USED TO CLEAN OBJECTIVES OR OCULARS, USE A NEW, CLEAN SECTION OF THE PAPER EACH TIME YOU WIPE THE LENS SURFACE. c. NEVER SAVE USED LENS PAPER OR Q-TIPS. THEY SHOULD NEVER BE RE-USED ON ANY OF THE LENSES. d. IF YOU RUN INTO A PARTICULARLY TENACIOUS BIT OF DIRT ON A LENS, SEE THE CLASS INSTRUCTOR ABOUT REMOVING IT RATHER THAN TRYING TO DO IT YOURSELF.WITH THESE THINGS IN MIND LET’S RUN THROUGH THE USE OF THE MICROSCOPE WITH AN EXAMPLE SLIDE.
INITIAL PROCEDURES:
1. REMOVE PLASTIC COVER AND USING BOTH HANDS, REMOVE YOUR MICROSCOPE FROM ITS CABINET AND PLACE IT ON THE LAB BENCH IN FRONT OF YOU. PUT PLASTIC COVER IN CABINET.
2. PLUG THE POWER CORD INTO THE BENCH SOCKET AND TURN ON THE LIGHT.
3. MAKE SURE THAT THE 10X OBJECTIVE IS IN POSITION OVER THE VIEWING AREA. THE OBJECTIVE SHOULD BE POSITIONED ABOUT 1/4” - 3/8” ABOVE THE STAGE. 23
4. PLACE A SLIDE ON THE MICROSCOPE STAGE SUCH THAT THE PORTION OF THE SLIDE YOU WANT TO VIEW IS UNDER THE OBJECTIVE.
5. FOCUS ON SPECIMEN, FIRST USING THE COARSE AND THEN THE FINE FOCUS CONTROLS. YOU MAY HAVE TO MOVE THE SLIDE AROUND ON THE STAGE OF THE MICROSCOPE TO BRING THE SPECIMEN INTO THE VIEWING AREA.
ADJUST THE POSITION OF THE OCULARS (the interocular distance) SO THAT A SINGLE IMAGE CAN BE SEEN WHEN LOOKING THROUGH BOTH OCULARS AT THE SAME TIME.
If your eyes are too close set or far apart for the intraocular distance to be adjusted properly, you will have to use your microscope as a monocular instrument (i.e. look through one eyepiece with one eye). If you do this, it is important to keep both eyes open in order to avoid eyestrain. With a little practice, you should be able to train yourself to “see” only what is being viewed with the microscope, and ignore whatever the other eye is seeing. If you can’t do this, a trick that works is to buy a cheap pair of sunglasses, knock out the dark lenses and put a piece of cardboard in the lenses over the eye that you don’t look through the microscope with. This will allow you to “see” only what the eye looking through the microscope ocular sees. In any case, practice keeping both eyes open while looking through the microscope. Eyestrain can give you headaches.
6. VISION DIFFERS BETWEEN PEOPLE AND ALSO BETWEEN EYES. IT IS LIKELY THAT WHILE THE IMAGE YOU ARE VIEWING MAY BE IN FOCUS FOR ONE OF YOUR EYES, IT IS NOT IN FOCUS FOR THE OTHER. THE LEFT OCULAR ON YOUR MICROSCOPE HAS ADJUSTABLE FOCUS TO ACCOUNT FOR THIS. TO ADJUST THE OCULAR FOCUS, START BY LOOKING THROUGH THE NON- ADJUSTABLE RIGHT OCULAR AND COVERING YOUR LEFT EYE WITH THE INDEX CARD THAT IS IN THE TOP DRAWER AT YOUR SEAT. DON’T CLOSE THE EYE YOU ARE COVERING! NOW, CAREFULLY FOCUS ON THE IMAGE USING THE FINE FOCUS CONTROL ON THE MICROSCOPE. ONCE THE IMAGE IS IN FOCUS, COVER YOUR RIGHT EYE WITH THE INDEX CARD AND LOOK THROUGH THE LEFT OCULAR WITH YOUR LEFT EYE. ADJUST THE FOCUS OF THE LEFT OCULAR BY TURNING ITS FOCUSING RING TO THE LEFT OR THE RIGHT UNTIL THE IMAGE IS IN GOOD FOCUS. ONCE THESE STEPS ARE COMPLETED YOU WILL HAVE MATCHING FOCUS IN BOTH OCULARS. SINCE SOMEONE ELSE IN ANOTHER LAB SECTION WILL BE USING YOUR MICROSCOPE IT MAY BE NECESSARY TO PERFORM THIS ADJUSTMENT EACH TIME YOU USE THE MICROSCOPE.. AGAIN, THIS WILL HELP ELIMINATE EYESTRAIN.
ONCE THE SPECIMEN IS IN FOCUS, IT IS TIME TO ADJUST THE CONDENSER DIAPHRAGM APERTURE. THIS IS DONE BY ROTATING THE PLASTIC RING ON THE THE CONDENSER ASSEMBLY THAT IS UNDERNEATH THE STAGE. YOU WILL NOTICE THAT THERE IS A WHITE DOT ON THE RING AND BELOW THAT A WHITE LINE THAT AT ONE END HAS A DIAGRAM OF A CIRCLE WITH A HEXAGON IN IT AND AT THE OTHER END, JUST A CIRCLE..
7. CONDENSER DIAPHRAGM ADJUSTMENT. a. WHILE LOOKING THROUGH THE OCULAR OF YOUR MICROSCOPE, ROTATE THE RING CLOCKWISE (TO THER LEFT) ALL THE WAY TO WHERE IT STOPS ROTATING. AT THIS POINT THE APERTURE IS AT ITS SMALLEST SIZE AND ALLOWS THE LEAST AMOUNT OF LIGHT TO PASS THROUGH. 24 b. NEXT, SLOWLY TURN THE RING COUNTER-CLOCKWISE (TO THE RIGHT) UNTIL YOU REACH THE POINT THAT THE VIEWING FIELD IS AS BRIGHT AS IT WILL GET. THE APERTURE SHOULD BE ADJUSTED TO THIS POINT AND NOT BEYOND IT. YOU NOW HAVE ADJUSTED YOUR CONDENSER DIAPHRAGM FOR MAXIMUM RESOLUTION AT A REASONABLE CONTRAST. IF YOU HAVE COMPLETED THIS ADJUSTMENT CORRECTLY, THE GRADUATED PLASTIC RING ON THE CONDENSER A SHORT DISTANCE FROOM THE HEXAGON IN THE CIRCLE..
CONTRAST CAN BE INCREASED BY CLOSING DOWN THE CONDENSER DIAPHRAGM TO ALLOW LESS LIGHT THROUGH; HOWEVER, THIS ALSO CAUSES A DECREASE IN RESOLUTION. AN INCREASE IN CONTRAST MAY BE HELPFUL IN VIEWING SOME SLIDES.
THE APPROPRIATE ADJUSTMENT OF THE CONDENSER APERTURE MAY CHANGE DEPENDING ON WHICH SLIDE YOU’RE VIEWING AND WHAT OBJECTIVE YOU ARE USING. SO BE AWARE, THAT IT MAY BE HELPFUL IN SOME CASES TO CHANGE THE ADJUSTMENT OF THE CONDENSER DIAPHRAGM.
8. AFTER THE SPECIMEN IS IN FOCUS AND THE CONDENSER PROPERLY ADJUSTED, IT IS TIME TO BRING THE FIELD DIAPHRAGM INTO FOCUS AND CENTER IT.
9. FIRST, MAKE SURE THE TISSUE ON YOUR SLIDE IS IN GOOD FOCUS. THEN CLOSE THE FIELD DIAPHRAGM TO IT’S SMALLEST OPENING BY ROTATING THE PLASTIC KNURLED RING ON THE FIELD DIAPHRAGM ASSEMBLY COUNTER-CLOCKWISE UNTIL IT STOPS.
10. NOW, BY TURNING THE CHROME-PLATED ADJUSTMENT SCREWS ON THE LEFT AND RIGHT SIDES OF THE BASE OF THE CONDENSOR ASSEMBLY, LOOK THROUGH THE OCULARS AND CENTER THE DIAPHRAGM IN THE VIEWING FIELD. CENTERING IS CRITICAL IF YOU ARE TO OBTAIN THE BEST IMAGE OF THE MATERIAL YOU ARE LOOKING AT. AFTER YOU HAVE CENTERED THE SMALLEST OPENING OF THE FIELD DIAPHRAGM, OPEN THE DIAPHRAGM UNTIL THE OPENING ALMOST FILLS THE FIELD. NOW YOU CAN RE-CENTER THE DIAPHRAGM OPENING FOR EVEN BETTER ALIGNMENT.
11. ONCE YOU HAVE CENTERED THE FIELD DIAPHRAGM, OPEN THE DIAPHRAGM UNTIL THE OPENING JUST FILLS THE FIELD OF VIEW. NO FURTHER!
12. IDEALLY, EACH TIME YOU CHANGE OBJECTIVES YOU SHOULD RE-FOCUS, RE-SIZE AND RE-CENTER THE FIELD DIAPHRAGM IF YOU WISH TO OBTAIN THE BEST IMAGE POSSIBLE.
WHAT YOU HAVE JUST DONE IS SET YOUR MICROSCOPE UP FOR “PROPER KOHLER ILLUMINATION”. THIS WILL GIVE YOU THE BEST RESOLUTION POSSIBLE WITH YOUR MICROSCOPE.
13. ONCE YOU HAVE THE IMAGE OF THE TISSUE YOU ARE VIEWING IN FOCUS WITH A GIVEN OBJECTIVE YOU MAY ADVANCE THE OBJECTIVE TURRET TO THE NEXT
25 HIGHER MAGNIFICATION AND RE-FOCUS AND READJUST YOUR MICROSCOPE AS DESCRIBED ABOVE.
------THE FOLLOWING INSTRUCTION MUST ALWAYS BE ADHERED TO!!!
WHENEVER YOU ADVANCE TO THE NEXT HIGHER MAGNIFICATION OBJECTIVE, YOU MUST RE-FOCUS THE IMAGE BEFORE ADVANCING TO AN EVEN HIGHER MAGNIFICATION!!! ------14. NEVER, I REPEAT NEVER, USE THE COARSE FOCUS WITH THE HIGHER POWER OBJECTIVES (20X, 40X OR 100X objectives).
BEFORE REMOVING A SLIDE FROM THE STAGE!!!! WHEN YOU ARE FINISHED VIEWING, MOVE THE OBJECTIVE TURRET TO BRING THE 4X OR 10X OBJECTIVE INTO POSITION OVER THE VIEWING AREA. THEN REMOVE THE SLIDE FROM THE STAGE. REMOVING THE SLIDE FROM UNDER HIGHER POWER OBJECTIVES MAY CAUSE THE SLIDE SURFACE TO DRAG OVER THE OBJECTIVE LENS SINCE THE LENS IN THESE OBJECTIVES WILL BE VERY CLOSE TO THE SLIDE. THIS COULD RESULT IN DAMAGE TO THE LENS.
OIL IMMERSION (not used in this course)
OIL IMMERSION VIEWING IS USED TO 1) DIRECT THE GREATEST AMOUNT OF LIGHT THROUGH THE OBJECTIVE LENS and 2) TO ACHIEVE THE HIGHEST POSSIBLE RESOLUTION WHEN VIEWING THROUGH THE HIGHEST POWER OBJECTIVES.
FOR YOUR MICROSCOPE, THIS WOULD BE WHEN VIEWING SLIDES WITH THE 100X OBJECTIVE.
IN THIS COURSE, THE 4X, 10X, AND 40X OBJECTIVES WILL BE SUFFICIENT FOR ALL OF YOUR LAB WORK. HOWEVER, IN SOME INSTANCES, THE OIL IMMERSION OBJECTIVE (100X) MAY BE USEFUL. IN ORDER FOR THIS OBJECTIVE TO PROVIDE AN IMAGE THAT IS IN GOOD FOCUS, A DROP OF OIL MUST BE PLACED BETWEEN THE OBJECTIVE LENS AND THE SLIDE.
15. BEFORE PERFORMING OIL IMMERSION MICROSCOPY, YOU MUST FIRST GO THROUGH THE PROCEDURES NECESSARY TO OBTAIN GOOD FOCUS WITH THE 40X OBJECTIVE.
16. THAT ACCOMPLISHED, MOVE THE OBJECTIVE TURRET SUCH THAT THE SPACE BETWEEN THE 40X AND 100X OBJECTIVES IS OVER THE VIEWING AREA.
17. NOW, PLACE A SMALL DROP OF IMMERSION OIL ON THE SLIDE OVER THE POINT WHERE THE OBJECTIVE LENS WILL RESIDE WHEN YOU MOVE IT INTO POSITION OVER THE VIEWING AREA. THIS POINT IS WHERE YOU CAN SEE THE LIGHT BEAM PASSING THROUGH THE SLIDE. THE DROP OF OIL SHOULD BE VERY SMALL, NOT MUCH IS NEEDED AND THE LESS THERE IS, THE EASIER IT WILL BE TO CLEAN THINGS UP WHEN YOU ARE FINISHED.
18. NEXT, ROTATE THE OBJECTIVE TURRET TO BRING THE 100X OBJECTIVE OVER THE VIEWING AREA. DO THIS CAREFULLY, AND WATCH TO MAKE SURE THAT THE 26 OBJECTIVE LENS DOES NOT CONTACT THE SURFACE OF THE SLIDE. IF YOU FOCUSED PROPERLY WITH THE 40X OBJECTIVE IN STEP 10, THERE WILL BE NO PROBLEM.
19. ONCE THE 100X OBJECTIVE IS MOVED INTO POSITION, THE LENS WILL BE IMMERSED IN OIL. NOW, USING ONLY THE FINE FOCUS CONTROL, LOOK THROUGH THE OCULARS AND FOCUS ON THE SLIDE. THIS SHOULD REQUIRE ONLY A SMALL ADJUSTMENT OF THE FINE FOCUS KNOB.
WHEN YOU ARE DOING THIS FOCUSING, IT IS IMPORTANT TO BE VERY CAREFUL. IT WILL NOT TAKE MUCH ROTATION OF THE FINE FOCUS KNOB TO CAUSE THE 100X OBJECTIVE TO RAM THROUGH THE SLIDE, DAMAGING BOTH THE OBJECTIVE AND THE SLIDE.
BE CAREFUL!!!
20. ONCE THE TISSUE ON THE SLIDE IS IN FOCUS, IT MAY BE MOVED AROUND ON THE STAGE. AS LONG AS THE DISTANCE MOVED IS NOT TOO LARGE, THE OIL DROPLET WILL REMAIN BETWEEN THE 100X OBJECTIVE AND THE SLIDE. YOU WILL NOTICE THAT WHEN THE SLIDE IS MOVED, IT WILL BE NECESSARY TO RE-FOCUS THE 100X OBJECTIVE USING THE FINE FOCUS CONTROL. AT VERY HIGH MAGNIFICATIONS, VERY SMALL CHANGES IN THE DISTANCE BETWEEN THE SLIDE AND THE OBJECTIVE WILL CAUSE THE IMAGE TO GO OUT OF FOCUS. IRREGULARITIES ON THE SLIDE AND ON THE STAGE OF THE MICROSCOPE ARE LARGE ENOUGH TO CAUSE SUCH CHANGES IN FOCUS.
ONCE YOU ARE DONE VIEWING A SLIDE UNDER OIL IMMERSION, IT WILL BE NECESSARY TO CLEAN BOTH THE SLIDE AND THE OBJECTIVE.
21. TURN THE OBJECTIVE TURRET SUCH THAT THE SPACE BETWEEN THE 100X AND LOWEST POWER OBJECTIVE IS OVER THE VIEWING AREA.
22. REMOVE THE SLIDE FROM THE STAGE AND THOROUGHLY WIPE THE OIL FROM ITS SURFACE USING A PIECE OF LENS PAPER. MORE THAN ONE PIECE OF LENS PAPER MAY BE REQUIRED. WHILE SOME PRESSURE MUST BE USED IN ORDER TO CLEAN THE OIL FROM THE SLIDES SURFACE, BE CAREFUL NOT TO PRESS TOO HARD. IT IS POSSIBLE TO CAUSE THE COVERSLIP TO SLIDE OFF THE SLIDE IF TOO MUCH PRESSURE IS USED.
DO A GOOD JOB OF CLEANING THE SLIDE SO THAT YOU WILL NOT HAVE TO CLEAN IT BEFORE VIEWING IT IN THE FUTURE. DRIED OIL IS HARD TO REMOVE!
23. REPLACE THE SLIDE IN YOUR SLIDE BOX.
24. NEXT, THOROUGHLY WIPE THE 100X OBJECTIVE WITH A NEW, CLEAN PIECE OF LENS PAPER. AGAIN, IT MAY BE NECESSARY TO DO THIS MORE THAN ONCE, WITH MORE THAN ONE PIECE OF LENS PAPER.
25. WIPE UP ANY EXCESS OIL THAT IS ON THE MICROSCOPE STAGE.
26 PUTTING MICROSCOPE AWAY: 27 1. Make sure 4X objective is over viewing area 2. Raise stage to highest point 3. Raise condensor if necessary 4. Fold electrical cord between stage and condensor 5. Remove plastic cover from cabinet. 6. Put scope in cabinet with oculars facing back of cabinet. 7. Put Plastic cover back on scope. 8. Close cabinet door
CONGRATULATIONS!
YOU HAVE JUST FINISHED A SHORT COURSE IN MICROSCOPY. THE INSTRUCTIONS ABOVE ARE TAILORED TO THE MICROSCOPES USED IN THIS COURSE. THEY MAY NOT BE COMPLETELY ADEQUATE FOR MORE SOPHISTICATED MICROSCOPES. IF YOU HAVE THE OPPORTUNITY TO USE A MORE SOPHISTICATED MICROSCOPE IN THE FUTURE, BE SURE TO REVIEW THE INSTRUCTION MANUAL THAT COMES WITH THE MICROSCOPE BEFORE USING IT. FOLLOWING THIS COURSE OF ACTION WILL SAVE YOU TIME, THE HIGH COST OF REPAIRS, AND PROVIDE THE BEST VIEWING.
A FINAL REVIEW OF AN IMPORTANT POINT.
WHENEVER YOU CHANGE SLIDES, OR LOSE FOCUS ON SLIDES YOU ARE VIEWING, ALWAYS START YOUR VIEWING WITH THE 4X OR 10X OBJECTIVES. FOCUS ON SPECIMEN, AND THEN SWITCH TO HIGHER POWER OBJECTIVES.
NOT FOLLOWING THIS PROCEDURE WILL LEAD TO BROKEN SLIDES AND DAMAGED OBJECTIVES. BOTH ARE EXPENSIVE. PARTICULARLY THE OBJECTIVES WHICH, FOR YOUR MICROSCOPES, CAN COST AS MUCH AS $400.00 EACH. ON MORE SOPHISTICATED MICROSCOPES, THE COST OF OBJECTIVES CAN BE $2000.00 - $10,000.00 EACH DEPENDING ON THE TYPE OF OBJECTIVE..
IF YOU DAMAGE ANY OF YOUR OBJECTIVES, IT MAY BE WEEKS BEFORE WE CAN GET A REPLACEMENT. YOU MAY BE WITHOUT THE BENEFIT OF THAT OBJECTIVE ON YOUR MICROSCOPE FOR AS LONG AS IT TAKES.
28 Lab Handout 2A VERTEBRATE DEVELOPMENT BIOL 4410
ROUTINE METHODS
Units of measurement.
METRIC SYSTEM ONLY!
Common units of measurement encountered when working with histological sections are,
Meter (m) = 100 cm = 1000 mm = 106 µm = 109 nm = 1010 Å
Centimeter (cm) = 0.01 m = 100 mm = 10,000 µm = 10,000,000 nm - 100,000,000 Å
Millimeter (mm) = 0.001 m = 0.1 cm = 1000 µm = 1,000,000 nm = 10,000,000 Å
Micron = micrometer (µ or µm) = 0.000001 m = 0.0001 cm = 0.001 mm = 10-6 m = 1000 nm = 10,000 Å
Nanometer (nm) = millimicron (mµ ) = 0.000000001 m = 0.001 µm = 10-9 m = 10 Å
Angstrom = Å = 0.1 nm = 0.0000000001 m = 10-10 m
HISTOLOGICAL METHODS:
While the science of Histology involves use of both the light and electron microscopes, the term histological generally refers to the examination of tissues with the light microscope. i.e. If you talk about a histological study, you are generally referring to an investigation using the light microscope. An investigation using the electron microscope is generally referred to as an ultrastructural study.
BE AWARE, HOWEVER, THAT THIS IS NOT AN ABSOLUTE RULE. SOME INVESTIGATORS WILL USE THE TERM HISTOLOGICAL TO REFER TO INVESTIGATIONS USING THE ELECTRON MICROSCOPE.
In examining histological sections with the light microscope in this class we will be dealing with measurements on the order of the um. For instance, cells are generally on the order of 10 - 20 µm in diameter; however, some types are smaller, say 1 - 9 µm. IN FACT, THE SMALLEST STRUCTURES YOU CAN SEE (RESOLVE) WITH THE LIGHT MICROSCOPE ARE ABOUT 0.2 µm DIAMETER, LENGTH OR WIDTH.
EXAMPLES:
SOME CELLS ARE QUITE SMALL.
Red blood cells,
erythrocytes - 8 µm
29 Unicellular phytoplankton
Gymnodinium microadriaticum - 8 µm Pavlova lutheri - ~7 µm
Some bacteria - 1 µm
OTHER CELLS ARE MUCH LARGER:
Chicken egg - 7 cm
slug neuron - 500 µm
human egg - 140 µm
In working with histological sections for the light microscope, we are working with 3 dimensions; however, one of those dimensions, namely the thickness of the section, is generally quite small. The sections on your slides will be on the order of 1 - 20 µm in thickness depending on the technique used to prepare them and/or what sort of tissue the sections were taken from.
WHY SO THIN?
You may ask why sections are so thin, or why the thickness varies. I think the why part of the question is fairly self-evident. In most cases, light must pass through the section in order for us to observe the structure of the tissue the section is composed of. If the section is too thick it may be naturally opaque, or it may stain too heavily to allow sufficient light to pass through it.
The thicker the section, the less detail can be see since structures are superimposed over or under each other
There are exceptions to this general rule of thinness. In some investigations reflected light is used for observation of tissues. For instance, whole organisms (say an insect) or very thick sections might be viewed in this manner. In other cases, some types of fine detail cannot be adequately observed if a section is too thin.
A few pages hence, we will be talking about fixation, embedding and sectioning techniques. All of these can have an effect on how thin a section can be. Some types of fixation, embedding, or sectioning will not allow for very thick sections. Other types will not allow for very thin sections.
ULTRASTRUCTURAL METHODS
The term ultrastructural is used to refer to investigations of tissue structure that utilize the electron microscope. In ultrastructural investigations we are usually talking about measurements ranging from a few um down to fractions of a nanometer. IN FACT, THE SMALLEST OBJECTS THAT CAN BE SEEN WITH AN AVERAGE ELECTRON MICROSCOPE ARE ON THE ORDER OF 5-20 A IN DIAMETER OR LENGTH. Even finer resolution than this can be achieved.
30 Ultrastructural investigations often utilize very thin sections of tissue (60 - 130 nm usually) - Such microscopy is called transmission electron microscopy, the electron beam passes through section and forms an image on a fluorescent screen.
In another type of electron microscopy, thick sections or pieces of whole organisms can be examined with the electron microscope using a technique that is analogous (but definitely not homologous) to the reflected light technique described above. This type of electron microscopy is called scanning electron microscopy. The same principles concerned with why sections vary in thickness and why they must be thin that we discussed relative to light microscopy are also applicable in electron microscopy. Of course, in the case of electron microscopy we are talking about an electron beam rather than a light beam, but the properties are similar so we can treat light and electron beams in much the same way.
BACK TO SECTION THICKNESS!
Sections for both light and electron microscopy are, of course, 3-dimensional. They have length, width, and thickness. However, since they are very thin for most intents and purposes we often treat sections as 2-dimensional objects. THIS CAN BE DANGEROUS RELATIVE TO YOUR UNDERSTANDING OF WHAT YOU ARE LOOKING AT!
It’s important not to loose track of the fact that tissue sections are components of a 3-dimensional object. In order for you to gain a better understanding of how a sectioned organ or tissue is constructed, it is important to consider how a 3 dimensional object might look in sections taken at different angles (i.e. with the object in different orientations with respect to the knife blade that cuts the sections). Your understanding of the structure of cells, tissues, organs, and embryos will be totally dependent on whether or not you are able to relate the essentially 2-dimensional sections on a slide to the 3-dimensional object that they are parts of.
ONWARD TO PREPARATION OF TISSUES FOR MICROSCOPIC EXAMINATION.
In order to study tissue and cellular structure, tissues must be prepared for microscopic examination.
Two major categories
1. Methods involving direct observation of living cells
2. Methods involving observation of dead cells in which the cellular structure has been preserved in some manner.
For the purposes of this course, you will be mainly concerned with the second of these two categories. You will be looking at permanent preparations of embryos, organs, tissues and cells that have undergone processes called
1. Fixation, 2. embedding, 3. sectioning, and 4. staining.
To understand why this is the case, it might be best to first consider the characteristics of living tissues when prepared for microscopic examination.
While a number of kinds of information can be gleaned from the examination of living tissues, there are certain drawbacks that limit the amount and kind of information that may be obtained.
31 1. May be too thick. 2. opaque or translucent 3. live cells die and fall apart if sectioned, so there will often be dead tissue over and/or under the live tissue. This dead tissue can interfere with observations. 4. Low contrast 5. Methods to increase contrast limited to vital stains or special types of microscopy (phase, interference, or dark field microscopy). 6. Cannot be examined while alive with electron microscope. i.e. in living tissues you cannot examine ultrastructure, sub-cellular structure with the electron microscope.
It is important to realize that even with these inherent problems, examination of living cells or tissues can provide very important information for our understanding of structure and function.
However, detailed structure (that is cellular, sub-cellular, and often chemical structure) of cells and tissues is best observed in preserved, dead material.
So, you might say, all right, that’s easy, we’ll just kill the tissue, section it, and look at it. IT’S NOT THAT SIMPLE. THERE ARE A NUMBER OF IMPORTANT CONSIDERATIONS.
FIRST, TISSUES MUST BE FIXED:
FIXATION
1. You not only want to kill the tissue, you also want to preserve its structure. It is imperative that the original structure of the live tissue be preserved as closely as possible to it’s original condition. Thus, you want a fixative that will not disrupt the structure of the tissue.
Qualities of a good fixative. a. Fast penetration - fast fixation, so enzymes, membranes, etc. are fixed before they can either degrade or cause degradation of cellular structure. b. Similar temperature, pH, and osmolarity to cytoplasm of cells composing tissue - minimizes shrinkage and/or swelling of cellular components. c. Fixative should cause sufficient cross-linking of proteins, such that the tissue will maintain its integrity during embedding and sectioning. d. Minimal change in structure of molecules composing cells and extracellular matrix. This is particularly important in preparations that are to be used in histochemical or immunocytochemical studies.
Obviously, things have to be balanced out. You can’t optimize all these qualities of good fixation at once. So qualities of a fixative will be determined by what kind of tissue you’re fixing and what you want to do with it after it’s fixed.
32 The most common fixatives for light microscopy are
1. Formalin 2. Alcohols 3. Mercuric dichloride 4. Potassium dichromate 5. and various acids, e.g. picric acid
Often mixtures of 2 or more different fixatives, along with other reagents (such as buffers), are used for fixation of tissues.
The most common fixatives for electron microscopy are
1. Glutaraldehyde 2. Osmium tetroxide
In electron microscopy, pH and osmolarity are very important since ultrastructural examinations involve looking at the sub-structure of organelles such as mitochondria or at molecules. These are structures that are easily disrupted by inappropriate pH and osmolarity of the fixative.
EMBEDDING
Tissues are sometimes frozen to make them rigid enough for sectioning, but usually they are embedded in a supporting material. This material is in liquid form initially so that it can infiltrate the cells of the tissue. It is then hardened in some manner to form a rigid block that can be sectioned.
How embedding is accomplished depends on the medium that the tissue is embedded in.
Light microscopy
The classical medium for embedding tissues for light microscopy is wax. Wax is still used for the majority of histological procedures even today.
Wax is not water miscible, so, since fixatives usually contain water, tissues must undergo dehydration after fixation.
This is usually done by transferring the fixed tissue through an alcohol series, though acetone is sometimes used.
To dehydrate a fixed tissue it might be passed through a series of solutions in the following order.
After fixation,
Rinse in distilled water, 30% ethanol, 50%, 70%, 80%, 95%, 100%, 100%, 100%, toluene or xylene, toluene or xylene, hot (60o C) wax. Finally, the wax is cooled to form a hard block that can be sectioned.
Timing of treatment and temperature are very important. These must be adjusted to preserve tissue structure as closely as possible to the original living tissue.
33 Electron microscopy
Similar procedures are used for embedding tissue in plastic polymers such as Polybed 812, Epon, or Araldite. One difference is that propylene oxide is used after 100% alcohol rather than toluene or xylene. This allows for better preservation of the tissues ultrastructure.
General procedures are essentially the same as in wax embedding except that tissues are dehydrated through propylene oxide and then transferred to mixtures of propylene oxide and a plastic such as Epon, and finally to 100% plastic. A catalyst mixed with the plastic is responsible for its polymerization (hardening). Polymerization is usually accomplished at temperatures of about 60 o C, though with some plastic formulations ultraviolet light can be used to polymerize the plastic at low temperatures (e.g. - 20 o C).
Tissues embedded in plastic are generally much better preserved than those embedded in wax; however, plastics are more difficult to section and sections embedded in plastics such as Epon require special treatment if they are to be used in the majority of histochemical techniques. Often they can’t be used because the molecular structure of the embedded tissues has been changed by the polymerization process to the point that the stains used in histochemical procedures for the light microscope will no longer recognize the substance that they are supposed to stain.
In recent years, water miscible plastics such as Polyscience’s JB-4 medium have come into use. These circumvent the need for extensive dehydration procedures and are readily usable with most stains without special treatment of sections prior to staining. These plastics still present some problems with regard to the ease of sectioning. Hopefully these problems will be overcome in the near future.
Once the tissues are embedded, and the wax or plastic hardened, they are sectioned on a microtome. This is an instrument designed to cut thin sections from the face of a block of wax or plastic that contains the embedded tissue.
The block moves up and down on the arm of the microtome. A mechanical mechanism retracts the block away from a knife on the upstroke. The block is advanced one increment of distance on the downstroke and a section is cut on the knife. The end result is that you get a “ribbon” of sections. In the case of wax, sections are cut with a steel knife blade. Plastic sections are cut with a glass or diamond knife (to cut plastic the knife edge must be exceedingly sharp and very hard. Steel is very, very quickly dulled by plastic).
For light microscopy the sections are picked-up and transferred to a slide for mounting. They are usually floated on water and then the water is evaporated. Sections adhere to the slide, the wax is removed with a solvent such as xylene or toluene, the sections are rehydrated, stained, dehydrated, and a coverslip is mounted over the sections using a non-polar mounting medium such as cedarwood oil, or Permount. For electron microscopy, sections are cut with a glass or diamond knife, floated onto water as they are cut, then picked-up with a small, thin copper screen called a grid. The sections are allowed to dry and then are stained with heavy metals such as uranium and lead.
The final step in both light and electron microscopy is to examine the sections with the microscope.
34 Lab Handout 2B VERTEBRATE DEVELOPMENT BIOL 4410
MITOSIS, MEIOSIS, AND GAMETOGENESIS
HOW MEIOSIS AND MITOSIS DIFFER
MITOSIS MEIOSIS
1. Occurs in somatic cells and 1. Occurs only in gametocyte the stem cells of the germ stages of the germ cells. cell line.
2. One division resulting in 2. Two divisions 2 new cells resulting in 4 new cells.
3. Each of the 2 new cells 3. Each of the 4 new receives one complete pair cells receives only one of each homologous pair of chromosome of each chromosomes. homologous pair of chromosomes.
4. Each of the 2 new cells 4. Each of the 4 new contains a diploid number cells contains a (2n) of chromosomes and haploid number (1n) of diploid (2n) genetic chromosomes and content. haploid (1n) genetic content.
35
Lab Handout 2B VERTEBRATE DEVELOPMENT BIOL 4410
MITOSIS AND MEIOSIS IN A DIAGRAMATIC SENSE
Look at “PLOIDY, WHAT IS IT?” on the class web page.
36 Lab Handout 2C VERTEBRATE DEVELOPMENT BIOL 4410
BASIC MICROSCOPY METHODS
In examining slides of sectioned tissues with the light and electron microscopes, one should be aware that some of the structures observed may not be real, that is, they may be artifacts. Artifacts are the result of changes in tissues structure or the addition of new structures that are usually the result of fixation, dehydration, embedding, sectioning, staining, and/or section mounting techniques. Types of artifacts that are commonly encountered are listed below. Light microscope examples of these are available for viewing in the auto-tutorial slides available in the lab.
REVIEW OF BASIC ARTIFACTS.
1. Swelling of tissue components 2. Shrinkage of tissue components
Artifact types 1 and 2 are the result of poor fixation and/or dehydration techniques, i.e. osmolarity of fixative may be wrong, pH wrong, too short a fixation time, dehydration of tissue too rapid. Swelling and shrinkage can sometimes result in rupture of membranes. This sort of damage is particularly evident at the ultrastructural level.
3. wrinkles in section 4. tears in section 5. air bubbles 6. dust
Artifact types 3, 4, and 5 are usually the result of poor sectioning technique or poor technique during mounting of sections. In some cases, poor fixation and/or embedding can be responsible for tears or wrinkles in sections by modifying fixed tissue consistency such that the tissue cannot be sectioned without its tearing or wrinkling.
7. stain precipitate
This sort of artifact can result from use of old stain solutions, use of unfiltered stain solutions, mistakes made during preparation of the stain, or poor staining technique.
THE LIGHT MICROSCOPE:
We have gone over the use of your light microscopes during lab and you have a handout describing how to set-up your microscope for viewing such that “proper Kohler illumination” is established. In setting up “proper Kohler illumination” you are adjusting the path of the light such that a minimum of light reflection within occurs within the scope and the maximum amount of light passes through the center of the various lenses within the microscope. In addition, light is restricted to the central portion of the lenses. The reason for this is that their are more and more defects in the image the lens produces as you move away from its center.
The end result of your adjustments for “proper Kohler illumination” is that you achieve the maximum resolution of the image of the tissue you are viewing that is possible with your microscopes. This means that you will be able to see the maximum amount of structure within the tissue that can be seen with your microscopes. 37
The objective and ocular lenses are responsible for magnifying the image of the specimen being viewed.
Total magnification = Objective magnification X ocular magnification
So for 10X objective and 10X ocular,
Total magnification = 10 X 10 = 100X (this means that the image being viewed will appear to be 100 times its actual size).
For a 40X objective and 10X ocular,
Total magnification = 10 X 40 = 400X
Magnification is not of much value unless resolving power is high.
Resolution is a measure of the ability to distinguish 2 points as two points. That is, when viewing something through a microscope, how close together can two points be that you can still see some space between them?
** * *
We can’t say much more about resolution without a few words about numerical aperture (n.a. or NA). The value for numerical aperture measures to what extent the light that passes through a specimen is spread out over and collected by the objective lens. The light that passes through the specimen contains information about what the specimen looks like, that is, about its structure.
AS YOU READ THE FOLLOWING DISCUSSION ON NUMERICAL APERTURE AND RESOLUTION, IT MAY HELP TO REFER TO FIGURE 1-3 IN THE HISTOLOGY TEXT (PAGE 5) ON THE SHELF AT THE BACK OF THE LAB.
If we consider the cone of light that originates from the specimen and enters the objective lens, Numerical aperture can be defined as,
NA = n x sin µ ( x is the multiplication symbol) n = refractive index of substance between specimen and objective lens (usually air, n = 1.0; quartz, n = 1.5; glass, n= about 1.5; water, n = 1.3)
µ = 1/2 the aperture angle (also called the semiangle). The aperture angle is the angle described by the cone of light that enters the objective lens after passing through the specimen. This angle will depend on the curvature of the lens and also on how close the objective lens is to the specimen when it is in focus.
So, for an objective with an aperture angle of 120 o with air between specimen and objective lens,
NA = 1 x sin 60o = sin 60 o = 0.87
If oil with refractive index of 1.5 is used between objective lens and specimen,
NA = 1.5 x sin 60 o = 1.5 x (.87) = 1.31 38
Now, numerical aperture is important because it allows us to calculate the resolving power of the objective. Remember, that’s what we really were interested in determining initially.
R = 0.61 x ( λ / NA)
R = resolution of the objective λ = wavelength of light (average value for white light ~ 550 nm).
NA = numerical aperture
So, for the air situation,
R = 0.61 x (550nm/.87) = 386 nm = 0.000000386 m = 0.386 µm
For oil immersion,
R = 0.61 x (550nm/1.31) = 256 nm = 0.000000256 m = 0.26 µm
Thus, one can see that higher resolution is possible if the substance lying between the specimen and the objective lens has a refractive index as close as possible to that of the lens itself without exceeding the lens’ refractive index.
It is important to realize that while both the ocular and objective lenses are responsible for the final magnification on a compound microscope, ONLY the objective lens is responsible for resolution.
The discussion above should demonstrate the importance of resolution. By using the appropriate lenses I can create extremely high magnifications, say 5000X with the light microscope. However, magnification tells us nothing about resolution. If resolution of objective lens is 0.3 µm, no matter how much I magnify the specimen image, the resolution will remain the same. At 5000X, I will still only be able to resolve points a minimum of 0.3 µm apart. Points that are closer together may be visible, but the will be superimposed and blurred, appearing as one fuzzy point. So nothing has been gained by increased magnification. The amount of visible information available at 5000X is the same as at lower magnifications of 1500X.
Using the mathematical equations given above and the values for maximum numerical aperture attainable with the lenses of a light microscope it can be shown that the maximum useful magnification on a light microscope is between 1000X and 1500X. Higher magnification is possible, but resolution will not improve.
In addition to numerical aperture and an incorrect light path, there are 3 major lens defects that can affect the quality of the image in a compound microscope and result in decreased resolution. These are,
A. Chromatic aberration - caused by spherical a lens bringing different wavelengths of light into focus at different levels. Thus, you get multiple images superimposed on top of each other. This defect is corrected in achromatic objectives.
39 B. Spherical aberration - optical quality of image lessened due to the fact that the center of lens has slightly different qualities than the edges. Both spherical and chromatic aberration are corrected in apochromatic objectives.
C. Curvature of field - causes image to be in focus centrally, but out of focus peripherally or vice versa. This defect is corrected in planar objectives.
The type of objective, magnification, numerical aperture, and even the best coverslip thickness to use on your slides is listed on the side of an objective.
There are a number of special types of light microscopy that can enhance certain features of a specimen that is being examined. Some of these are listed below.
1. Phase contrast microscopy - takes advantage of phase differences in light beam that are caused by different refractive indexes of components within a tissue.
Consider air, n=1.0; water, n=1.3; glass, n=1.5. Light travels fastest through air and slowest through glass. Thus, if a light beam encounters three different spaces of equal thickness that are filled with air water, and glass, the beam will emerge first from the air filled space and last from the glass filled space. These light beams are said to be out of phase with each other.
In the phase contrast microscope, the condenser and objectives are specially made to detect the phase differences of light passing through different components within a tissue specimen. The construction of the condenser and objective lenses is such that these phase differences are made visible by increasing the contrast between light waves of different phase. As a result, components of cells that are normally of low contrast (clear or nearly clear), are given higher contrast and, thus, made visible.
2. Polarizing microscopy - A polarizing filter (called the polarizer) is placed below the condenser and allows only light vibrating in one plane to reach the condenser. A second polarizing filter (called the analyser) is placed between the objective and ocular. If these two filters are oriented such that their axes of light transmission are perpendicular, no light will pass through the analyser to the ocular. So nothing will be seen. One use of polarizing light microscopy is related to the fact that certain crystals found in or associated with some cells can bend light waves because of their refractive index. If some of the light waves that have passed through the polarizer are bent into different planes as they pass through crystalline parts of the specimen, then some of these light waves will be able to pass through the analyser even if it is oriented at 90o to the polarizer. This property of crystals to bend polarized light waves is called birefringency. It is important in identifying certain crystalline structures in or associated with cells.
3. Interference or Nemarski interference microscopy. - this is another method utilized to observe structures of different refractive index, but similar optical density. It is not the same as phase contrast microscopy. Nemarski interference microscopy requires 2 different light beams that are recombined after passing through the specimen. Differences in phase between the two beams are visualized as depth. The result is an image with depth (sort of 3-D). This type of microscopy is particularly useful for viewing living cells.
ELECTRON MICROSCOPY
Functioning of this instrument is dependent on the fact that an electron beam has many properties that are similar to a light beam. 40
In fact, a beam of electrons may be treated as either 1.) a beam of particles or 2.) as a wave (i.e. like a light wave). As it turns out, both properties are necessary in order for an electron microscope to work. The fact that the effective wavelength of an electron beam is very much smaller than that of the shortest visible light wave makes very high resolution possible with this instrument (i.e. 5 - 20 A)
Recall that, R = 0.61 x (λ/NA)
This means that very high useful magnification is possible since very small distances between two points can be resolved. The highest magnification commonly used with the electron microscope is 200,000X. However, higher useful magnifications are possible.
Suffice it to say, that for the purposes of this course, we can consider the electron microscope in relatively simple terms. An electron beam is produced by inducing a high voltage between a cathode (-) and an anode (+). Electromagnets are used to direct the path of this beam and also to act as magnetic lenses that are responsible for magnification of the image of the specimen. As the electron beam passes through the specimen, electrons are either unaffected, scattered, or absorbed by the tissues of the specimen and various stains (usually heavy metals) that have been applied to the tissues. The unaffected electrons and many of the scattered electrons pass through the specimen and then are focused by magnetic lenses on a fluorescent viewing screen. The number of electrons hitting various parts of this screen determine how brightly these parts fluoresce and thus form an image of the specimen on the screen that can be examined by the person using the scope. In addition, the focused electrons can be used to expose photographic film from which black and white pictures can be printed. The photographs produced are actually more useful in interpreting electron microscope images because they are permanent and of higher contrast than the fluorescent image.
41 Laboratory Identification (ID) Lists
Overview
In the vertebrate embryology laboratory we study reproductive and embryonic structures at different stages of development. Some of the terms we use are stage-specific (e.g., lens vesicle), whereas others remain consistent throughout development (e.g., head).
The Lab ID Lists contain the terms for structures and processes for which you are responsible. Obviously, you can’t point at a process, but you can point to the structure that performs that process. So yes, you need to relate form and function.
Just as an embryo begins development from apparent simplicity and progresses toward greater complexity, so does the list of terms you will learn in this course. If you have already peeked at the list for later stage embryos, we say, “Courage, Grasshopper! It can be done.” Don’t try to just memorize terms. Attach them to your visual images of structures. That means that you actually have to find them and look at (study, ponder, evaluate) them.
Repetition is the “engine” of memory, and as you progress through the course you will find that many terms will be repeated in subsequent lists. You will also find that some structures have more than one name. With each succeeding stage of development, you are responsible for those terms previously studied, in addition to those being introduced anew. For the embryos of various developmental stages (e.g., frog, chick, pig) the first time a term is added to the list, it is italicized, but not subsequently. As you progress to later stages, you will be able to identify the new terms easily by this style.
In the amniote (e.g., chick, pig) lists, the terms are grouped according to origin (e.g., extraembryonic or embryonic), or function (e.g., nervous system, digestive system, etc.). Some terms apply to tissues that involve both embryonic and extraembryonic structures (e.g., somatic and splanchnic). Observe that when a column head is followed by an ellipsis (e.g., Embryonic…) and a term in that column is preceded by an ellipsis (e.g., …mesoderm), you are responsible for identifying (e.g.,) “embryonic mesoderm.”
As you identify a structure on your slides, place a check mark in the space beside its name to keep track of your progress. Most of the amniote structures to identify are embryonic in nature, so the middle column is usually the longest, giving you room to make notes on either side of the entries. (Just one more service we provide.)
Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos identically labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your instructor to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
42 Laboratory Identification List
Testis & Spermatogenesis NOTE: Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Grasshopper Testis Vertebrate Testis (frog, rat, rabbit, monkey, human) ____ apical end of lobe ____ cyst ____ interstitial cells of Leydig ____ septum ____ seminiferous tubule ____ septum ____ Sertoli cell
Spermatogenesis Spermatogenesis ____ *mitosis ____ *mitosis ____ *meiosis (I & II) ____ *meiosis (I & II) ____ spermatogonium ____ spermatogonium
Meiosis I Meiosis I ____ primary spermatocyte ____ primary spermatocyte
Stages of First Meiotic Prophase Stages of First Meiotic Prophase ____ leptotene ____ leptotene ____ *zygotene ____ *zygotene ____ pachytene ____ pachytene ____ diplotene ____ diplotene ____ diakinesis ____ diakinesis ____ tetrad ____ tetrad
____ reductional division ____ *reductional division
Meiosis II Meiosis II ____ secondary spermatocyte ____ secondary spermatocyte ____ *dyad ____ *dyad ____ *equational division ____ *equational division ____ *spermiogenesis (=spermateleosis, ____ *spermiogenesis (=spermateleosis, =spermatozoan metamorphosis) =spermatozoan metamorphosis) ____ spermatid ____ spermatid ____ spermatozoon (=spermatozoan) ____ spermatozoon (=spermatozoan) ____ sperm head ____ sperm head ____ acrosome ____ acrosome ____ nucleus ____nucleus ____ midpiece ____ midpiece ____ tail (=flagellum) ____ tail (=flagellum) ____ *spermiation ____ *spermiation 43
Laboratory Identification List
Ovary & Oogenesis NOTE: Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Amphibian (frog) Avian (chick) Mammalian (cat) ____ animal pole ____ cortex ____ germinal epithelium ____ vegetal pole ____ medulla ____ tunica albuginea ____ *oogonium ____ *oogonium ____ cortex ____ medulla ____ corpus luteum ____egg nests ____ *oogonium
____ ovarian follicle ____ ovarian follicle ____ ovarian follicle ____ follicle cells ____ follicle cells ____ follicle cells ____ theca folliculi ____ theca folliculi ____ primordial follicle ____ theca externa ____ stratum granulosum ____ primary follicle ____ theca interna ____ secondary follicle ____ *vitelline membrane ____ tertiary follicle (=Graafian follicle) ____ theca folliculi ____ theca externa ____ theca interna ____ stratum granulosum ____ antrum ____ liquor folliculi ____ cumulus oophorus ____ *corona radiata ____ zona pellucida
____ oocyte (primary, ____ oocyte (primary, ____ oocyte (primary, *secondary) *secondary) *secondary) ____ germinal vesicle ____ germinal vesicle ____ nucleus ____ lampbrush chromosome ____ nucleus ____ germinal vesicle ____ nucleolus ____ yolk ____ nucleus ____ yolk
44 Laboratory Identification List
Starfish Development
For identifications that involve specific embryonic structures and/or tissues, it will be important to start determining which germ layer (ectoderm, mesoderm, endoderm) from which the tissue or structure is derived. Be sure you can identify the various embryonic/larval stages. You will not always be able to find the landmarks/structures necessary to identify the animal and vegetal poles; however, you should know what these landmarks/structures are and be able to use them to make these identifications.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Oocyte—Zygote Early Cleavage Blastula Gastrula Bipinnaria
____ fertilization ____ fertilization ____ fertilization ____ archenteron ____ blastocoel membrane membrane membrane ____ archenteric ____ stomodeum ____ perivitelline ____ perivitelline ____ perivitelline vesicle = mouth space space space ____ blastopore ____ oral field ____ germinal ____ blastocoel ____ blastocoel (=anus) ____ esophagus vesicle ____ blastomere ____ blastomere ____ blastocoel ____ stomach ____nucleus ____ coelomic ____ intestine ____*animal pole ____*animal pole ____*animal pole sacs (late ____ anus ____*vegetal pole ____*vegetal pole ____*vegetal pole gastrula) ____ coelomic ____ gastrocoel sac ____ ciliated (archenteric band cavity) ____*mesenchyme cells ____ oral lobe (late gastrula) ____ stomodeum = mouth (mid to late gastrula)
____ animal pole ____ vegetal pole
45 Laboratory Identification List
Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Early Frog Development (page 1 of 2)
Early Frog: Cleavage through Gastrulation
Early-Mid Cleavage Blastula Early Gastrula Yolk Plug ____ pigmented cortex ____ pigmented cortex ____ pigmented cortex ____ pigmented cortex ____ animal ____ animal ____ animal hemisphere hemisphere hemisphere ____ animal pole ____ animal pole ____ animal pole ____ vegetal pole ____ vegetal pole ____ vegetal pole ____ fertilization ____ fertilization ____ fertilization ____ fertilization membrane membrane membrane membrane ____ blastomere ____ blastomere ____ macromere ____ macromere ____ macromere ____ micromere ____ micromere ____ micromere ____ cleavage furrow ____ *gray crescent ____ nucleus ____ nucleus ____ blastocoel ____ blastocoel ____ blastopore ____ blastocoel ____ blastoderm ____ blastopore ____ *blastulation ____ dorsal lip of blastopore ____ dorsal lip of blastopore ____ * ventral lip of blastopore ____ archenteron (=gastrocoel) ____ yolk plug ____ *emboly ____ *epiboly ____ *involution ____ *fate map
Presumptive… ____ …neural ectoderm ____ …non-neural ectoderm ____ …mesoderm ____…chordamesoderm ____ …endoderm 46
Early Frog Development (page 2 of 2)
Early Frog: Neurula Stage
Neural Plate – Neural Fold Stage Neural Tube Stage
Nervous System Nervous System ____ neural plate ____ *neural crest ____ neural fold ____ neurocoel ____ neural groove ____ neural tube ____ *neural crest ____ *prosencephalon ____ neurocoel ____ *optic vesicle ____ *mesencephalon ____ *rhombencephalon ____ spinal cord
Digestive System Digestive System ____ *foregut ____ pharynx ____ *liver diverticulum ____ liver diverticulum ____ midgut ____ midgut ____ *hindgut
Miscellaneous Organs & Tissues Miscellaneous Organs & Tissues ____ ectoderm (neural, non-neural) ____ ectoderm (neural, non-neural) ____ epidermis ____ epidermis ____ *adhesive gland
____ *head mesenchyme ____ *head mesenchyme ____ mesoderm ____ mesoderm ____ notochord ____ notochord ____ somite mesoderm (=epimere) ____ somite mesoderm (=epimere) ____ intermediate mesoderm ____ intermediate mesoderm (=mesomere) (=mesomere) ____ lateral plate mesoderm ____ lateral plate mesoderm (=hypomere) (=hypomere) ____ hypochordal (subnotochordal) rod ____ hypochordal (subnotochordal) rod ____ endoderm ____ endoderm ____ yolk endoderm ____ yolk endoderm
47 Laboratory Identification List 4 – 7 mm Frog Embryo Page 48 of 3
Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
NOTE! NOT ALL OF THESE STRUCTURES/TISSUES ARE PRESENT IN BOTH THE 4mm AND 7mm STAGE. PART OF YOUR RESPONSIBILITY FOR THESE TWO STAGES IS TO FIGURE OUT WHAT CHANGES.
Nervous System Digestive System Respiratory System
Central Nervous System ____ Foregut ____ external gills ____ tuberculum posterious ____ oral plate ____ prosencephalon ____ stomodeum (=forebrain) ____ pharynx ____ prosocoel ____ liver diverticulum ____ telencephalon ____ thyroid rudiment ____ telocoel ____ proctodeum (=anus) ____ diencephalon ____ diocoel ____ Midgut ____ mesencephalon ____ yolk endoderm (=midbrain) ____ mesocoel (=aqueduct of ____ Hindgut Sylvius) ____ yolk endoderm ____ infundibulum (=neurohypophysis) ____ rhombencephalon (=hindbrain) ____ rhombocoel ____ spinal cord
48 4 – 7 mm Frog Embryo Page 2 of 3
Nervous System Digestive System Respiratory System
____ epiphysis (=pineal body) ____ sensory retina ____ presumptive pigmented retina ____ hypophysis (=pituitary body) ____ optic stalk ____ optic vesicle (maybe 4mm) ____ optic cup (7mm) ____ opticoel ____ *choroid (optic) fissure
Peripheral Nervous System ____ olfactory pit ____ otic vesicle ____ lens placode/vesicle
Circulatory System Urogenital System Pharyngeal Structures
Arterial (7mm) Urinary System ____ visceral (=pharyngeal, ____ aorta (dorsal, ventral) ____ pronephros =branchial) arch ____ aortic arch ____ pronephric tubules ____ visceral (=pharyngeal, ____ pronephric duct =branchial) cleft/groove Venous (=Wolffian duct, ____ visceral (=pharyngeal, ____ vitelline veins archinephric duct) =branchial) pouch ____ mesomere (= nephrotome) ____ mandibular arch Cardiac ____ hyoid arch ____ heart ____ hyomandibular pouch ____ pericardial coelom Genital System ____ hyomandibular cleft ____ pericardium (nothing obvious yet) ____ atrium (7mm) ____ ventricle (7mm) ____ conus arteriosus (=bulbus cordis,=bulbus arteriosus) (7mm) ____ myocardium ____ endocardium
49 4 – 7 mm Frog Embryo Page 3 of 3
Skeleto-muscular System Endocrine System Miscellaneous Structures
____ notochord ____ epiphysis (=pineal gland) ____ adhesive gland (=ventral ____ hypochordal ____ hypophysis (=pituitary sucker) (subnotochordal) rod gland) ____ head mesenchyme ____ epimere (=somite) ____ infundibulum of ____ epidermis ____ hypomere (=lateral plate diencephalon ____ coelom mesoderm) (=neurohypophysis, ____ dermatome =posterior pituitary) ____ myotome ____ Rathke’s pocket/pouch ____ sclerotome (=adenohypophysis, =anterior pituitary ____ thyroid
50 Laboratory Identification List 10 mm Frog Embryo Page 51 of 90 Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for. Ectodermal Derivatives
Central Nervous System Special Sense Organs ____ Prosencephalon ____ Eye ____ telencephalon ____ lens ____ telocoel ____ pigmented retina ____ olfactory nerve ____ sensory retina ____ diencephalon ____ cornea ____ diocoel ____ anterior chamber ____ epiphysis ____ vitreous chamber ____ infundibulum (=neurohypophysis) ____ optic cup ____ optic nerve ____ optic nerve (cranial nerve II) ____ optic cup ____ optic chiasma ____ Ear ____ optic recess (groove) ____ saccule (=sacculus) ____ torus transversus ____ *semicircular canal ____ utricle (=utriculus) ____ Mesencephalon ____ endolymphatic duct ____ mesocoel (=aqueduct of Sylvius) ____ otic vesicle
____ Rhombencephalon ____ Nose ____ rhombocoel ____ nasal cavity ____ metencephalon ____ olfactory nerve (cranial nerve I) ____ metacoel ____ nares ____ myelencephalon ____ internal nares ____ myelocoel ____ external nares ____ posterior choroid plexus (neural portion only) Miscellaneous Ectodermal Derivatives ____ stomodeum ____ Spinal Cord ____ proctodeum ____ dorsal root ____ tuberculum posterious ____ dorsal root (spinal) ganglion ____ *spiracle ____ ventral root 51 Laboratory Identification List 10 mm Frog Embryo Page 2 of 3 Ectodermal Derivatives
Central Nervous System Special Sense Organs
____ ependymal layer ____ gill (internal) ____ mantle layer ____ *gill raker ____ marginal layer ____ opercular cavity ____ spinal canal ____ *velar plate ____ tongue ____Pituitary Gland ____ adenohypophysis (from Rathke’s pocket) ____ neurohypophysis (infundibulum of diencephalon)
Endodermal Derivatives
Digestive, Respiratory & Endocrine Organs ____ pharynx ____ esophagus ____ stomach ____ intestine ____ liver ____ lung buds ____ thyroid ____ trachea ____ hyomandibular pouches (What do these tissues form in the adult?)
Mesodermal Derivatives
Circulatory System Skeleto-muscular System
Venous System Epimere (=somite) ____ posterior (=caudal) cardinal veins ____ dermatome ____ anterior (=cranial) cardinal veins ____ myotome ____ common cardinal veins (ducts of Cuvier) ____ sclerotome
Cardiac System (Heart) Mesomere (=nephrotome) ____ sinus venosus ____ mesonephros ____ atrium (=auricle) ____ mesonephric duct (=Wolffian duct; ____ ventricle formerly pronephric duct = archinephric ____ bulbus arteriosus (=conus arteriosus, duct) =bulbus cordis, =conotruncus) ____ truncus arteriosus [also part of arterial system] ____ pericardium 52 Laboratory Identification List 10 mm Frog Embryo Page 3 of 3 Mesodermal Derivatives
Circulatory System Skeleto-muscular System
____ pericardial cavity (coelom) Hypomere (=lateral plate mesoderm) ____ endocardium ____ splanchnic mesoderm ____ myocardium ____ somatic mesoderm ____ trabeculae carnae ____ coelom ____ mesentery Arterial System ____ pericardium ____ ventral aorta (=truncus arteriosus) ____ pericardial cavity ____ dorsal aorta ____ peritoneum ____ internal carotid artery ____ peritoneal cavity ____ lymph sinus
Mesodermal Derivatives (continued)
Skeleto-muscular System (continued) ____ mesenchyme ____ cranial cartilage ____ visceral arch #1 (mandibular arch) ____ visceral arch #2) (hyoid arch) ____ visceral arch #3 ____ visceral arch #4
53 Laboratory Identification List
Cranial Nerves and Ganglia (page 1 of 4)
In this course the twelve pairs of vertebrate cranial nerves are studied only in the chick and pig embryos, where they are seen to best advantage. (Several, however, are indicated in the 10 mm Frog labeled transverse sections.) Cranial ganglia are associated with cranial nerves V, VII, VIII, IX, X, and XI, although the latter (XI) is obscure and will not be found easily.
Cranial nerves and associated ganglia originate from neural ectoderm of brain and spinal cord, from neural crest cells, and from dorsolateral and epibranchial placodal ectoderm. Some cranial nerves are purely sensory (afferent), some purely motor (efferent), and some mixed. Sensory fibers are usually generated by cells from neural crest and/or dorsolateral placodal ectoderm, although epibranchial placodal ectoderm forms some sensory fibers associated with certain visceral (pharyngeal) pouches. Motor fibers are usually derived from neural ectoderm of the central nervous system (somatic efferent and first order autonomic efferent fibers), but may also be formed by neural crest cells (second order autonomic efferent fibers). Cranial nerves I (olfactory), II (optic), and VIII (auditory) are referred to as “special sensory” because of the great amount of sensory input they provide to the central nervous system.
The information in the following three tables should help you understand the basic relationships between cranial nerves, cranial ganglia, and their developmental origins.
Locate as many of the cranial nerves and their ganglia as possible. You are responsible for answering the following questions:
Which cranial nerves are sensory? Which are motor? Which are mixed (sensory and motor)? What does each of the cranial nerves innervate? Which cranial nerves are associated with ganglia? What are the names of those ganglia? What are the developmental origins of the cranial nerves and ganglia?
A traditional mnemonic (memory aid) for the order of the twelve cranial ganglia of vertebrates is:
“On Old Olympus’ Towering Top A Finn And German Vaulted And Hopped”
(There is no scientific meaning to this other than word order. We tend to remember ridiculous things easier than, e.g., the order of the vertebrate cranial nerves.)
From anterior to posterior, the twelve cranial nerves of vertebrates are:
Olfactory, Optic, Oculomotor, Trochlear, Trigeminal, Abducens, Facial, Acoustic, Glossopharyngeal, Vagus, (Spinal) Accessory, Hypoglossal
Compare the order of the first letter of each word in the mnemonic with that of the anterior to posterior sequence of cranial nerves.
54 Laboratory Identification List Cranial Nerves and Ganglia (page 2 of 4)
Table 1: The Vertebrate Cranial Nerves & Associated Ganglia
Mnemonic No. Cranial Function Brain Associated Ganglia Study in: Nerve Association On I Olfactory special telencephalon chick (72, sensory 96), pig Old II Optic special diencephalon chick (48, sensory 72, 96), pig Olympus’ III Oculomotor motor mesencephalon chick (72, 96), pig Towering IV Trochlear motor mesencephalon
Top V Trigeminal mixed myelencephalon Gasserian chick (48, (=semilunar) 72, 96), pig A VI Abducens motor myelencephalon
Finn VII Facial mixed myelencephalon geniculate chick (72, 96), pig And VIII Acoustic special myelencephalon vestibular & acoustic chick (72, sensory 96), pig German IX Glosso- mixed myelencephalon superior (proximal) & chick (72, pharyngeal inferior (distal or 96), pig petrosal) Vaulted X Vagus mixed myelencephalon jugular (proximal) & chick (72, nodose (distal) 96), pig Accessory And XI (Spinal motor myelencephalon Froriep’s (degenerate chick Accessory) occipital ganglia) (96), pig Hopped XII Hypoglossal motor myelencephalon chick (96), pig © S.R. Haley & S.C. Kempf, 7/25/11
Note: With respect to cranial ganglia, the terms superior (or proximal) and inferior (or distal) indicate position on the cranial nerve relative to the central nervous system (brain and spinal cord). e.g., The jugular ganglion on the Vagus nerve (X) is closer (proximal) to the central nervous system than is the distal nodose ganglion.
55 Laboratory Identification List Cranial Nerves and Ganglia (page 3 of 4)
Table 2: Origin & Distribution of Cranial Nerves *Legend S = Sensory A = Autonomic (motor) Large Case = major component SS = Special Sensory 1’ = First order autonomic small case = minor component M = Motor (somatic) 2” = Second order autonomic
No. Cranial *Function *Developmental Origin Distribution Study Nerve in: olfactory bulb of chick I Olfactory SS olfactory placode telencephalon (72, 96), pig II Optic SS sensory retina visual centers of chick (lateral diencephalon) brain (72, 96) chick III Oculomotor M, a ventrolateral mesencephalon (M, 1’a); intraocular & 4 (72, 96), neural crest (2” a) extraocular eye pig muscles IV Trochlear M ventrolateral mesencephalon 1 extraocular eye muscle visceral arch I chick V Trigeminal S, m neural crest & epibranchial placode (S); derivatives (48, 72, ventrolateral myelencephalon (m) 96), pig VI Abducens M ventrolateral myelencephalon 1 extraocular eye muscle neural crest & epibranchial placodes (S, visceral arch II chick VII Facial S, M, a 2”a); derivatives (72, 96), ventrolateral metencephalon (M, 1’a) pig Acoustic cochlea & chick VIII (Auditory) SS otic placode vestibular (72, 96), apparatus pig neural crest (S, superior ganglion; 2”a), visceral arch III chick IX Glosso- S, M, a epibranchial placode (S, petrosal derivatives (72, 96), pharyngeal ganglion); pig ventrolateral myelencephalon (M, 1’a) neural crest (S, jugular ganglion; 2”A), visceral arches IV chick X Vagus S, M, A epibranchial placode (S, nodose & VI derivatives; (72, 96), ganglion); heart, visceral pig ventrolateral myelencephalon (M, 1’A) organs Accessory visceral arch IV Chick XI (Spinal M, a myelencephalon & spinal cord (M, 1’a); derivatives, neck, (72), pig Accessory) neural crest (2”a) shoulder XII Hypoglossal M ventrolateral myelencephalon muscles of tongue Chick (96), pig
56 Laboratory Identification List Cranial Nerves and Ganglia (page 4 of 4)
Table 3: Origin of Cranial Ganglia Neurons whose cell bodies lie in cranial ganglia are sensory in function.
No. Cranial Function Associated Ganglion Origin Study in: Nerve Ganglion special chick (72, I Olfactory sensory none 96), pig special chick (48, II Optic sensory none 72, 96), pig chick (72, III Oculomotor motor none 96), pig IV Trochlear motor none chick (48, V Trigeminal mixed Gasserian neural crest & 72, 96), (=semilunar) epibranchial placodes pig
VI Abducens motor none chick (72, VII Facial mixed geniculate neural crest & 96), pig epibranchial placode
special vestibulo- chick (72, VIII Acoustic sensory acoustic otic placode 96), pig
IX Glosso- mixed proximal (superior) neural crest (proximal) & chick (72, & distal (petrosal) pharyngeal epibranchial placode (distal) 96), pig
X Vagus mixed proximal (jugular) neural crest (jugular) & chick (72, & distal (nodose) epibranchial placode 96), pig (nodose)
Froiep’s ganglia are chick (72, XI Accessory motor Froiep’s degenerate occipital spinal 96), pig ganglia (neural crest) XII Hypoglossal motor none chick (72, 96), pig
57 Laboratory Identification List NOTE: Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
18 and 24 hr Chick Embryo
Extraembryonic… Embryonic… Both ____ area pellucida ____ primitive streak ____ area opaca ____ primitive knot (=Hensen’s ____ yolk node) ____ primitive groove ____ primitive pit ____ primitive fold
24 hr Chick Embryo (page 1 of 2)
Extraembryonic… Embryonic… Both ____ area opaca vasculosa ____ epidermis The following must be identified as ____ area opaca vitellina ____ head “embryonic” or extraembryonic” ____ proamnion ____ head mesenchyme when you identify them on exams. ____ coelom (extraembryonic ____ subcephalic pocket ____ primitive streak coelom = exocoel) ____ subgerminal cavity ____ primitive knot (=Hensen’s ____ ectoderm (non-neural) ____ hypoblast node) ____ mesoderm ____ yolk sac ____ primitive groove ____ endoderm ____ primitive pit ____ epiblast ____ primitive fold ____ hypoblast
____ somatic mesoderm
____ somatopleure Nervous System ____ splanchnic mesoderm ____ neural ectoderm ____ splanchnopleure ____ neural folds ____ neural plate ____ neural tube ____ neural groove ____ neurocoel ____ prosencephalon ____ prosocoel 58 Laboratory Identification List
24 hr Chick Embryo (page 2 of 2)
Extraembryonic… Embryonic… Both
Digestive System ____ anterior(cranial) intestinal portal ____ foregut ____ midgut
Skeleto-muscular System ____ notochord ____ somite = epimere ____ lateral plate mesoderm (=hypomere)
59 Laboratory Identification List
33 hr Chick Embryo (page 60 of 90) NOTE: You should begin thinking about how the embryo’s body is delimited from underlying extraembryonic tissues and yolk mass, and how the somatopleure and splanchnopleure act to form the various extraembryonic membranes. This is a dynamic process that occurs in three dimensions. You are looking at essentially two-dimensional sections of an “instant” in this process. Part of your challenge is to analyze how the chick embryo’s body and extraembryonic structures are forming by putting together your observations of the various sections (i.e., 18, 24, 33, 48, 72, 96 hr) that you examine.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
Extraembryonic… Embryonic… Both
____ amnion ____ neural ectoderm The following must be ____ amniotic (seroamniotic) ____ epidermis identified as “embryonic” or folds ____ head extraembryonic” when you ____ area opaca vasculosa ____ head mesenchyme identify them on exams. ____ area opaca vitellina ____ coelom (extraembryonic ____ area pellucida coelom = exocoel) ____ blood island Nervous & Sensory System ____ ectoderm (non-neural) ____ chorion (=serosa) ____ alar plate ____ mesoderm ____ proamnion ____ basal plate ____ endoderm ____ primitive streak ____ diencephalon ____ epiblast ____ primitive knot (=Hensen’s ____ diocoel ____ hypoblast node) ____ infundibulum ____ somatic mesoderm ____ subcephalic pocket ____ mesencephalon ____ somatopleure ____ subgerminal cavity ____ mesocoel (aqueduct of ____ splanchnopleure ____ yolk sac Sylvius) ____ splanchnic mesoderm ____ metencephalon ____ metacoel ____ myelencephalon ____ myelocoel ____ neural crest ____ neural ectoderm ____ neural folds ____ neural plate ____ neural tube ____ neurocoel ____ neuromere ____ neuropore (anterior) ____ opticoel
60 Laboratory Identification List
33 hr Chick Embryo (page 61 of 90)
Extraembryonic… Embryonic… Both
Nervous & Sensory System (continued) ____ optic stalk ____ optic vesicle ____ prosencephalon ____ prosocoel ____ sinus rhomboidalis ____ spinal cord
Digestive System ____ anterior intestinal portal ____ midgut ____ oral plate ____ pharynx (=foregut)
Circulatory System Circulatory System ____ endocardium ____ omphalomesenteric veins ____ dorsal aortic roots ____ heart ____ myocardium (epimyocardium) ____ pericardium ____ pericardial cavity ____ ventral aorta (truncus arteriosus) ____ ventral aortic roots
Skeleto-muscular System ____ notochord ____ somite = epimere ____ lateral plate mesoderm (=hypomere)
61 Laboratory Identification List Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
48 hr Chick Embryo (page 62 of 4
Extraembryonic… Embryonic… Both
____ amnion ____ neural ectoderm The following must be ____ seroamniotic (amniotic) ____ epidermis identified as “embryonic” or folds ____ cranial (cephalic) flexure extra-embryonic” when you ____ amniotic cavity ____ head identify them on exams. ____ *area opaca vasculosa ____ head mesenchyme ____ *area opaca vitellina ____ hyomandibular cleft ____ coelom (extraembryonic ____ area pellucida ____ lateral limiting sulcus coelom = exocoel) ____ blood island ____ tailbud ____ ectoderm (non-neural) ____ chorion (=serosa) ____ visceral grooves ____ mesoderm ____ lateral limiting sulcus (= clefts) ____ endoderm ____ proamnion ____ epiblast ____ seroamniotic ____ hypoblast junction/raphe Nervous & Sensory System ____ non-neural ectoderm ____ subcephalic pocket ____ alar plate ____ somatic mesoderm ____ subgerminal cavity ____ basal plate ____ somatopleure ____ yolk sac ____ choroid (optic) fissure ____ splanchnopleure ____ diencephalon ____ splanchnic mesoderm ____ diocoel ____ dorsal isthmus (of brain) ____ infundibulum (of diencephalon) ____ mesencephalon ____ mesocoel (=aqueduct of Sylvius) ____ metacoel ____ metencephalon ____ myelencephalon ____ myelocoel ____ neural crest
62 Laboratory Identification List
48 hr Chick Embryo (page 2 of 4)
Extraembryonic… Embryonic… Both
____ neural ectoderm ____ neural tube ____ neurocoel ____ neuromere ____ neuropore (anterior) ____ opticoel ____ optic stalk ____ optic cup ____ lens vesicle ____ otic (auditory) cup (= forming otic/auditory vesicle) ____ pigmented retina (presumptive) ____ sensory retina (presumptive) ____ prosencephalon ____ prosocoel ____ rhombencephalon ____ rhombocoel ____ spinal cord ____ telencephalon ____ telocoel ____ tuberculum posterius
Digestive System ____ anterior intestinal portal ____ hyomandibular pouch ____ intestinal portal (anterior) ____ *intestinal portal ____ midgut ____ oral plate ____ pharynx (=foregut) ____ pre-oral gut (=Seessel’s pocket) ____ *primordium of liver diverticulum ____ stomodeum ____ visceral pouches
63 Laboratory Identification List
48 hr Chick Embryo (page 3 of 4)
Extraembryonic… Embryonic… Both
Respiratory System ____* laryngotracheal groove
Circulatory System Circulatory System ____ anterior (cranial) cardinal ____ vitelline vein (=omphalomesenteric) arteries ____ aortic arches (1, 2, 3) ____ vitelline ____ aortic roots (dorsal, (=omphalomesenteric) veins ventral) ____ atrium ____ cardinal veins (anterior) ____ cardinal veins (posterior) ____ cardinal veins (common; =ductus cuvieri) ____ bulbus arteriosus (=conus arteriosus, =bulbus cordis, =conotruncus) ____ dorsal aorta ____ endocardium ____ heart ____ *intersegmental artery ____ myocardium ____ pericardium ____ pericardial cavity ____ sinus venosus ____ truncus arteriosus (=ventral aorta) ____ ventricle
Urogenital System ____ mesonephric (=Wolffian) ducts
64 Laboratory Identification List
48 hr Chick Embryo (page 4 of 4)
Extraembryonic… Embryonic… Both
Skeleto-muscular System ____ visceral (=brnachial) arches (1,2,3) ____ dermatome ____ hyoid arch ____ hypomere (=lateral plate) ____ lateral plate mesoderm (=hypomere) ____ mandibular arch ____ maxillary process ____ mesomere (=nephrotome =intermediate mesoderm) ____ *myocoel ____ myotome ____ notochord ____ sclerotome ____ somite = epimere
Endocrine System ____ Rathke’s pouch/pocket ____ thyroid primordium
65 Laboratory Identification List Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
72 hr Chick Embryo (page 66 of 90)
Extraembryonic… Embryonic… Both
____ allantois ____ apical ectodermal ridge The following must be ____ amnion ____ visceral (=branchial identified as “embryonic” or ____ seroamniotic (amniotic) =pharyngeal) cleft/groove extra-embryonic” when you folds (1,2,3,4) identify them on exams. ____ amniotic cavity ____ epidermis ____ coelom (extraembryonic ____ area opaca vasculosa ____ cranial (=cephalic) flexure coelom = exocoel) ____ *area opaca vitellina ____ *cervical flexure ____ ectoderm (non-neural) ____ area pellucida ____ head ____ mesoderm ____ blood island ____ head mesenchyme ____ endoderm ____ chorion (=serosa) ____ hyomandibular cleft ____ non-neural ectoderm ____ lateral limiting sulcus ____ lateral body fold ____ somatic mesoderm ____ proamnion ____ lateral limiting sulcus ____ somatopleure ____ seroamniotic ____ limb buds (wing, leg) ____ splanchnopleure raphe/junction ____ neural ectoderm ____ splanchnic mesoderm ____ subcaudal pocket ____ peritoneal cavity ____ subcephalic pocket ____ tailbud ____ subgerminal cavity ____ *torsion (dextral) ____ yolk sac ____ ventral mesentery
Nervous & Sensory System ____ alar plate ____ acoustico-facialis nerve (CN #7 & #8) ____ basal plate ____ cerebral hemisphere
66 Laboratory Identification List
72 hr Chick Embryo (page 2 of 90)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ choroid (optic) fissure ____ cranial ganglion ____ cranial nerve ____ diencephalon ____ diocoel ____ dorsal isthmus (of brain) ____ endolymphatic duct ____ ganglion (acoustico- facialis, CN #7 & #8) ____ ganglion (Gasserian, CN #5) ____ infundibulum (of diencephalon) ____ lens epithelium ____ lens fibers ____ lens vesicle (lens) ____ mesencephalon ____ mesocoel (=aqueduct of Sylvius) ____ metacoel ____ metencephalon ____ myelencephalon ____ myelocoel ____ neural crest ____ neural ectoderm ____ neural tube ____ neurocoel ____ neuromere ____ oculomotor nerve (CN #3) ____ olfactory(nasal) pit (external nares) ____ optic nerve (CN #2) ____ opticoel ____ optic stalk ____ optic cup ____ otic (=auditory, =acoustic) vesicle
67 Laboratory Identification List
72 hr Chick Embryo (page 3 of 90)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ pigmented retina (presumptive) ____ posterior choroid plexus ____ prosencephalon (telencephalon + diencephalon) ____ prosocoel ____ rhombencephalon (metencephalon + myelencephalon) ____ rhombocoel ____ sensory retina (presumptive) ____ spinal cord ____ spinal ganglia ____ telencephalon ____ telocoel ____ trigeminal nerve (CN #5) ____ tuberculum posterius
Digestive System ____ anterior (=cranial) intestinal portal ____ visceral, (=branchial =pharyngeal) pouch (1,2,3,4) ____ cloaca ____ duodenum ____ esophagus ____ gastrohepatic ligament ____ hindgut ____ hyomandibular pouch ____ intestine ____* laryngotracheal groove ____ liver ____ liver diverticulum ____ mesentery (dorsal, ventral) ____ mesogaster(dorsal, ventral) ____ midgut
68 Laboratory Identification List
72 hr Chick Embryo (page 4 of 90)
Extraembryonic… Embryonic… Both
Digestive System ____ mouth ____ omentum (greater, lesser) ____ peritoneal cavity ____ pharynx (=foregut) ____ posterior(caudal) intestinal portal ____ post-anal gut (tail gut) ____ pre-oral gut (=Seessel’s pocket) ____ stomach ____ stomodeum ____ visceral pouches
Respiratory System ____ *glottis (presumptive) ____ laryngotracheal groove ____ lung buds ____ pleural cavity ____ trachea
Circulatory System Circulatory System ____ aorta (dorsal) ____ allantoic vein ____ aortic arch (1,2,3,4,*6) ____ vitelline ____ aortic roots (dorsal, (=omphalomesenteric) artery ventral) ____ vitelline ____ atrium (=omphalomesenteric) ____ cardinal veins (anterior, vein cranial = jugular veins) ____ cardinal veins (posterior, caudal) ____ cardinal veins (common; =ductus cuvieri) ____ caudal artery
69 Laboratory Identification List 72 hr Chick Embryo (page 5 of 90)
Extraembryonic… Embryonic… Both
Circulatory System ____ bulbus arteriosus (=conus arteriosus, =bulbus cordis, =conotruncus) ____ ductus venosus ____ endocardium ____ heart ____ internal carotid arteries ____ intersegmental artery ____ myocardium ____ pericardium ____ pericardial cavity ____ sinus venosus ____ truncus arteriosus (=ventral aorta, =ventral aortic sac) ____ ventricle
Urogenital System ____ genital ridge ____ mesonephric (Wolffian) duct ____ mesonephric tubule ____ mesonephros ____ nephrotome (mesomere = intermediate mesoderm)
Skeleto-muscular System ____ visceral (=branchial, =pharyngeal) arches (1,2,3,4) ____ dermatome ____ epimere ____ hyoid arch ____ lateral plate mesoderm (hypomere) ____ mandibular arch ____ maxillary process ____ *myocoel ____ myotome
70 Laboratory Identification List
72 hr Chick Embryo (page 6 of 90)
Extraembryonic… Embryonic… Both
Skeleto-muscular System ____ notochord ____ sclerotome ____ septum transversum ____ somite (=epimere) ____ visceral groove
Endocrine System ____ epiphysis (pineal gland) ____ Rathke’s pocket/pouch ____ thyroid primordium
71 Laboratory Identification List Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
96 hr Chick Embryo (page 72 of 90)
Extraembryonic… Embryonic… Both
____ allantois ____ apical ectodermal ridge The following must be ____ allantoic vesicle ____ visceral (=branchial, identified as “embryonic” or ____ amnion =pharyngeal) extra-embryonic” when you ____ seroamniotic (amniotic) cleft/groove (1,2,3,4) identify them on exams. folds ____ epidermis ____ coelom (extraembryonic ____ amniotic cavity ____ cranial (=cephalic) flexure coelom = exocoel) ____ area opaca vasculosa ____ cervical flexure ____ ectoderm (non-neural) ____ *area opaca vitellina ____ head ____ mesoderm ____ area pellucida ____ head mesenchyme ____ endoderm ____ blood island ____ hyomandibular cleft ____ somatic mesoderm ____ chorion (=serosa) ____ lateral body fold ____ somatopleure ____ lateral limiting sulcus ____ lateral limiting sulcus ____ splanchnopleure ____ proamnion ____ limb bud (wing, leg) ____ splanchnic mesoderm ____ seroamniotic ____ neural ectoderm raphe/junction ____ peritoneal cavity ____ subcaudal pocket ____ tailbud ____ subcephalic pocket ____ tail fold ____ subgerminal cavity ____ *torsion (dextral) ____ yolk sac ____ ventral mesentery
Nervous & Sensory System ____ acoustico-facialis nerve (CN #7 & #8) ____ alar plate ____ basal plate ____ brain ventricles (1,2,3,4) ____ cerebellum (presumptive) ____ cerebral hemisphere (cerebrum)
72 Laboratory Identification List
96 hr Chick Embryo (page 2 of 90)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ choroid (optic) fissure ____ choroid plexus (posterior) ____ cranial ganglion ____ cranial nerve ____ diencephalon ____ diocoel ____ dorsal isthmus (of brain) ____ endolymphatic duct ____ facial nerve (cranial nerve VII) ____ ganglion (acoustico- facialis, CN #7 & #8) ____ ganglion (Gasserian, CN #5) ____ ganglion (petrosal, CN #9)) ____ ganglion (nodose, CN #10) ____ glossopharyngeal nerve (CN #9) ____ infundibulum (of diencephalon) ____ lens epithelium ____ lens fibers ____ lens vesicle ____ *medulla oblongata (presumptive) ____ mesencephalon ____ mesocoel (=aqueduct of Sylvius) ____ metacoel ____ metencephalon ____ myelencephalon ____ myelocoel ____ neural crest ____ neural tube ____ neurocoel ____ neuromere
73 Laboratory Identification List 96 hr Chick Embryo (page 3 of 90)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ oculomotor nerve (CN #3) ____ olfactory(nasal) pit ____ opticoel ____ optic nerve (CN #2) ____ optic stalk ____ optic cup ____ otic (=auditory, =acoustic) vesicle ____ pigmented retina (presumptive) ____ posterior choroid plexus ____ prosencephalon ____ prosocoel ____ rhombencephalon ____ rhombocoel ____ sensory retina (presumptive) ____ spinal cord ____ spinal ganglion ____ spinal nerve ____ telencephalon ____ telocoel ____ trigeminal nerve (CN #5) ____ tuberculum posterius
Digestive System ____ anterior (=cranial) intestinal portal ____ visceral (=branchial, =pharyngeal) pouch (1,2,3,4) ____ cloaca ____ cloacal membrane ____ duodenum ____ esophagus
74 Laboratory Identification List
96 hr Chick Embryo (page 4 of 90)
Extraembryonic… Embryonic… Both
Digestive System ____ gastrohepatic ligament ____ hindgut ____ hyomandibular pouch (eustachian tube, presumptive) ____ intestine ____* laryngotracheal groove ____ liver ____ liver diverticulum ____ mesentery (dorsal, ventral) ____ mesoesophagus ____ mesogaster(dorsal, ventral) ____ midgut ____ mouth ____ omentum (greater, lesser) ____ peritoneal cavity ____ pharynx (=foregut) ____ posterior(caudal) intestinal portal ____ post-anal gut ____ pre-oral gut (=Seessel’s pocket) ____ stomach ____ stomodeum ____ tailgut (postanal gut)
Respiratory System ____ *glottis (presumptive) ____ *laryngotracheal groove ____ lung buds (primary bronchi) ____ pleural cavity ____ trachea
75 Laboratory Identification List
96 hr Chick Embryo (page 5 of 90)
Extraembryonic… Embryonic… Both
Circulatory System Circulatory System ____ dorsal aorta ____ vitelline ____ aortic arches (1,2,3,4,6) (=omphalomesenteric) artery ____ aortic roots (dorsal, ____ vitelline ventral) (=omphalomesenteric) ____ atrium vein ____ cardinal veins (anterior, cranial) ____ cardinal veins (posterior, caudal) ____ cardinal veins (common; =ductus cuvieri) ____ caudal artery ____ conotruncus (=bulbous arteriosus,= conus arteriosus,= bulbous cordis) ____ ductus venosus ____ endocardium ____ heart ____ trabeculae carnae ____ *iliac artery ____ internal carotid arteries ____ intersegmental arteries ____ superior mesenteric artery ____ myocardium ____ pericardium ____ pericardial cavity ____ sinus venosus ____ truncus arteriosus (=ventral aorta, =aortic sac) ____ ventricle
76 Laboratory Identification List
96 hr Chick Embryo (page 6 of 90)
Extraembryonic… Embryonic… Both
Urogenital System ____ genital ridge ____ glomerulus ____ mesonephric (Wolffian) duct ____ mesonephric tubule ____ mesonephros ____ nephrotome (mesomere = intermediate mesoderm)
Skeleto-muscular System ____ visceral (=branchial, =pharyngeal) arches (1,2,3,4) ____ dermatome ____ hyoid arch ____ hyomandibular cleft ____ lateral plate mesoderm (hypomere) ____ mandibular arch ____ maxillary process ____ *myocoel ____ myotome ____ notochord ____ sclerotome ____ septum transversum ____ somite (epimere)
Endocrine System ____ epiphysis (pineal gland) ____ *hypophysis (pituitary gland) ____ Rathke’s pocket ____ thyroid primordium
77 Laboratory Identification List Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your instructor to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development. Recall that the first time a term is added to the list, it is italicized, but not subsequently. As you progress to later stages, you will be able to identify the new terms easily by this style.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
6 mm Pig Embryo (page 1 of 5)
Extraembryonic… Embryonic… Both
____ allantois ____ epidermis ____ …coelom (extraembryonic ____ amnion ____ flexure (cephalic, cranial) coelom = exocoel) ____ amniotic cavity ____ flexure (cervical) ____ …ectoderm ____ chorion* (serosa) ____ *flexure (caudal) ____ ...endoderm ____ seroamniotic ____ head ____ …mesoderm junction/raphe ____ head mesenchyme ____ allantoic stalk ____ subcaudal pocket ____ limb bud ____ coelom ____ subcephalic pocket ____ mesoderm ____ ectoderm ____ tail bud ____ endoderm ____ tail fold ____ epidermis ____ visceral (pharyngeal) ____ mesoderm cleft/groove (1, 2, 3, 4) ____ somatopleure ____ visceral (pharyngeal) ____ splanchnopleure pouch (1, 2, 3, 4)
Nervous & Sensory System (Note: CN = Cranial Nerve) ____ abducens nerve (CN 6) ____ accessory nerve (CN 11) ____ accessory ganglion (next to CN 11, but really part of CN 10) ____ acoustic ganglion (CN 8) ____ alar(roof) plate ____ anterior chamber (ocular) ____ auditory nerve (CN 8) ____ auditory (otic) vesicle
78 6 mm Pig Embryo (page 2 of 5) Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ basal (floor) plate ____ choroid fissure ____ diencephalon ____ diocoel ____ facial nerve (CN #7) ____ geniculate ganglion(CN #7) ____ glossopharyngeal nerve (CN #9) ____ hypoglossal nerve (CN #12) ____ jugular ganglion (CN #10) ____ lens ____ mesencephalon ____ mesocoel (aqueduct of Sylvius) ____ metacoel ____ metencephalon ____ myelencephalon ____ myelocoel ____ nodose ganglion (CN #10) ____ oculomotor nerve (CN #3) ____ olfactory bulb ____ olfactory nerve (CN #1) ____ olfactory placode ____ optic cup ____ optic nerve (CN #2) ____ optic stalk ____ optic vesicle ____ otic cup/vesicle ____ petrosal ganglion (CN #9) ____ posterior chamber (ocular) ____ prosocoel ____ retina (pigmented, sensory) ____ rhombencephalon ____ rhombocoel ____ semilunar (Gasserian) ganglion (CN #5) ____ spinal cord ____ spinal (dorsal root) ganglion ____ spinal nerve (dorsal, ventral roots) ____ superior ganglion (CN #9) ____ telencephalon ____ telocoel ____ vagus nerve (CN 10
79 6 mm Pig Embryo (3 of 5)
Extraembryonic… Embryonic… Both
Digestive System ____ cloaca ____ cloacal plate (membrane) ____ colon (large intestine) ____ common bile duct (ductus choledochus) ____ ductus choledochus (common bile duct) ____ duodenum ____ esophagus ____ falciform ligament ____ gall bladder ____ glottis ____ hepatic sinusoid ____ hindgut ____ liver diverticulum ____ mesocolon ____ mesoduodenum ____ omentum (greater, lesser) ____ pancreas (dorsal, ventral) ____ peritoneal cavity ____ pharynx ____ Seessel’s pocket (pre-oral gut) ____ small intestine ____ stomach (cardiac, pyloric) ____ tuberculum impar
Respiratory System ____ bronchus (primary) ____ epiglottis ____ lung bud ____ pleural cavity ____ septum transversum (diaphragm) ____ trachea
80 6 mm Pig Embryo (4 of 5)
Extraembryonic… Embryonic… Both
Circulatory System ____ aorta (dorsal, descending) ____ aorta (ventral, truncus arteriosus, conotruncus) ____ aortic arches (1, 2, 3, 4) ____ cardinal veins (anterior, posterior, common) ____ atrium (auricle) ____ basilar artery* ____ carotid artery (internal) ____ caudal artery ____ celiac artery* ____ conus (bulbus) arteriosus (bulbus cordis) ____ ductus arteriosus (aortic end of 6th aortic arch) ____ ductus venosus ____ epimyocardium ____ heart ____ hepatic portal vein ____ interatrial foramen ____ interatrial septum ____ intersegmental artery ____ interventricular septum ____ mesenteric artery, superior (vitelline artery) ____ myocardium ____ pericardial cavity ____ pulmonary artery (left, right) ____ sinus venosus ____ trabeculae carnae ____ umbilical (allantoic) artery* ____ umbilical (allantoic) vein (right, left) ____ vena cava (inferior) ____ ventricle (cardiac)
81 6 mm Pig Embryo (5 of 5)
Extraembryonic… Embryonic… Both
Circulatory System (continued) ____ vertebral artery ____ vitelline (omphalomesenteric) vein
Urogenital System ____ genital ridge ____ glomerulus ____ mesonephric (Wolffian) duct ____ mesonephric tubule ____ mesonephros ____ metanephros
Skeleto-muscular System ____ dermatome ____ hyoid arch ____ limb bud ____ mandibular arch ____ maxillary process ____ myotome ____ notochord ____ sclerotome ____ somite ____ tail ____ visceral arch (1, 2, 3, 4) ____ visceral groove ____ visceral pouch (1, 2, 3, 4)
Endocrine System ____ adenohypophysis (Rathke’s pocket, anterior pituitary) ____ hypophysis (pituitary gland) ____ neurohypophysis (posterior pituitary, infundibulum) ____ thyroid diverticulum
82 Laboratory Identification List Some terms in the lists have an asterisk (*) in front of them. These terms are usually processes, to which you cannot point, but sometimes they are very small or obscure structures. Each slide set is unique, and not all embryos so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your instructor to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development. Recall that the first time a term is added to the list, it is italicized, but not subsequently. As you progress to later stages, you will be able to identify the new terms easily by this style.
Items appear in ITALICS the first time you are asked to consider (processes) or identify structures, but not thereafter.
Notes on identifying central nervous system structures: In this course we differentiate between cavities and tissues of the central nervous system. The suffix “coel” refers to a cavity, e.g., mesocoel. In such cases, the tip of your pointer must be in the mesencephalon cavity when making an identification. The suffix “encephalon” refers to tissue, e.g., mesencephalon. In such cases, the tip of your pointer must be on mesencephalon tissue when making an identification. Note that the various cavities and tissues of the developing central nervous system are continuous with one and other. Thus, when making an identification, you must point to a definitive example of the CNS tissue or cavity you are asked to identify. “Definitive” means that you must not point to a region where the cavity or tissue is transitioning from one specific tissue region or cavity to another. When you point at a CNS tissue or cavity you are asked to identify on a lab exam, your ID must clearly be the specific tissue or cavity that the identification question asks for.
10 mm Pig Embryo (page 1 of 7)
Extraembryonic… Embryonic… Both
____ allantoic stalk ____ flexure (cephalic, cranial, ____ allantoic stalk ____ allantois cervical)) ____ coelom (extraembryonic ____ amnion* ____ flexure (caudal)* coelom = exocoel) ____ amniotic cavity* ____ head ____ ectoderm (non-neural) ____ chorion* (serosa) ____ head mesenchyme ____ endoderm ____ seroamniotic ____ limb bud ____ epidermis junction/raphe ____ tail bud ____ mesoderm ____ subcaudal pocket* ____ tail fold ____ somatopleure ____ subcephalic pocket* ____ visceral cleft/groove ____ splanchnopleure (1, 2, 3, 4) ____ visceral pouch (1, 2, 3, 4)
Nervous & Sensory System (Note: CN = Cranial Nerve) ____ abducens nerve (CN #6) ____ accessory nerve (CN #11) ____ accessory ganglion (next to CN #11, but really part of CN #10) ____ acoustic ganglion (CN #8) ____ alar(roof) plate ____ anterior chamber (ocular) ____ auditory nerve (CN #8) ____ auditory (otic) vesicle ____ basal (floor) plate ____ brachial plexus © S.R. Haley & S.C. Kempf, 7/25/11
83 10 mm Pig Embryo (page 2 of 7)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ cerebellum (presumptive = metencephalon) ____ cerebral hemispheres (presumptive, =telencephalic vesicles) ____ choroid fissure ____ cornea ____ diencephalon ____ diocoel ____ dorsal isthmus ____ endolymphatic duct ____ependymal layer ____ epiphysis (pineal gland) ____ facial nerve (CN #7) ____ geniculate ganglion(CN #7) ____ glossopharyngeal nerve (CN #9) ____ hypoglossal nerve (CN #12) ____ infundibulum of diencephalon (=neurohypophysis, =pars nervosa) ____ jugular ganglion (CN #10) ____ lens ____ mantle layer ____ marginal layer ____ medulla oblongata (presumptive, = myelencephalon) ____ mesencephalon ____ mesocoel (aqueduct of Sylvius) ____ metacoel ____ metencephalon ____ myelencephalon ____ myelocoel ____ nodose ganglion (CN #10) ____ neural ectoderm ____ oculomotor nerve (CN #3) ____ olfactory bulb ____ olfactory nerve (CN #1) © S.R. Haley & S.C. Kempf, ____ olfactory pit 7/25/11 ____ olfactory placode ____ optic cup 84 10 mm Pig Embryo (page 3 of 7)
Extraembryonic… Embryonic… Both
Nervous & Sensory System ____ optic nerve (CN #2) ____ optic stalk ____ optic vesicle ____ petrosal ganglion (CN #9) ____ posterior chamber (ocular) ____ prosocoel ____ retina (pigmented, sensory) ____ rhombencephalon ____ rhombocoel ____ semilunar (Gasserian) ganglion (CN #5) ____ spinal cord ____ spinal (dorsal root) ganglion ____ spinal nerve (dorsal, ventral roots) ____ *superior ganglion (CN #9) ____ telencephalon ____ telocoel ____ trigeminal nerve (CN #5) (mandibular, maxillary, © S.R. Haley & S.C. Kempf, and ophthalmic 7/25/11 branches) ____ trochlear nerve (CN #4) ____ vagus nerve (CN #10)
85 10 mm Pig Embryo (page 4 of 7)
Extraembryonic… Embryonic… Both
Digestive System ____ appendix ____ caecum ____ cloaca ____ cloacal plate (membrane) ____ colon (large intestine) ____ ductus choledochus (common bile duct) ____ duodenum ____ esophagus ____ falciform ligament ____ gall bladder ____ glottis ____ hepatic sinusoid ____ hindgut ____ liver diverticulum ____ mesocolon ____ mesoduodenum ____ mesogastrium ____ omental bursa ____ omentum (greater, lesser) ____ pancreas (dorsal, ventral) ____ peritoneal cavity ____ pharynx ____ Seessel’s pocket (pre-oral gut) ____ small intestine ____ stomach (cardiac, pyloric) ____ tuberculum impar (root of tongue)
© S.R. Haley & S.C. Kempf, 7/25/11
86 10 mm Pig Embryo (page 5 of 7)
Extraembryonic… Embryonic… Both
Respiratory System ____ lung buds (primary bronchi) ____ pleural cavity ____ septum transversum (diaphragm) ____ trachea
Circulatory System ____ aorta (dorsal, descending) ____ aorta (ventral = truncus arteriosus, conotruncus) ____ aortic arches (3, 4, 6) ____ atrioventricular valves ____ cardinal veins (anterior, posterior, common) ____ atrium (auricle) ____ basilar artery* ____ bulbus arteriosus (=conus arteriosus, =bulbus cordis, =conotruncus) ____ carotid artery (internal) ____ caudal artery ____ celiac (celiac) artery* ____ conus (bulbus) arteriosus (bulbus cordis) ____ ductus arteriosus (aortic end of 6th aortic arch) ____ ductus venosus ____ epimyocardium ____ heart ____ hepatic portal vein ____ iliac artery ____ interatrial foramen ____ interatrial septum ____ segmental artery ____ interventricular septum ____ jugular vein (internal) ____ mesenteric artery, superior (vitelline artery) ____ mitral valve (left) ____ myocardium ____ pericardial cavity ____ pulmonary artery (left, right) © S.R. Haley & S.C. Kempf, 7/25/11
87 10 mm Pig Embryo (page 6 of 7)
Extraembryonic… Embryonic… Both
Circulatory System Circulatory System (continued) ____ umbilical (allantoic) ____ sinus venosus artery* ____ trabeculae carnae ____ umbilical (allantoic) vein ____ tricuspid valve (right) ____ umbilical (allantoic) artery* ____ umbilical (allantoic) vein (right, left) ____ vena cava (inferior; postcaval vein) ____ ventricle (cardiac) ____ vertebral artery ____ vitelline (omphalomesenteric) vein
Urogenital System ____ genital ridge ____ genital tubercle* ____ glomerulus ____ mesonephric (Wolffian) duct ____ mesonephric tubule ____ mesonephros ____ metanephros ____ primordial germ cells* ____ urogenital sinus
Skeleto-muscular System ____ dermatome ____ hyoid arch ____ limb bud ____ mandibular arch ____ maxillary process ____ myocoel ____ myotome ____ notochord ____ sclerotome ____ somite ____ tail ____ visceral arch (1, 2, 3, 4) ____ visceral groove © S.R. Haley & S.C. Kempf, ____ visceral pouch (1, 2, 3, 4) 7/25/11
88 10 mm Pig Embryo (page 7 of 7)
Extraembryonic… Embryonic… Both
Endocrine System ____ adenohypophysis (Rathke’s pocket, anterior pituitary) ____ hypophysis (pituitary gland) ____ neurohypophysis (posterior pituitary, infundibulum) © S.R. Haley & S.C. ____ thyroid diverticulum Kempf, 7/25/11
89 Lab Handout 14 VERTEBRATE DEVELOPMENT BIOL 4410
Tooth Development Each slide set is unique, and not all tissues so labeled are at exactly the same developmental stage. It is possible that a particular structure is too rudimentary to be identified on your slides, but you must still know the pertinent information about that structure. Check with your TA to be certain that you haven’t simply missed seeing it on your slides. Ideally, one would examine several slide sets of a particular stage of development. Adult Tooth Developing Tooth alveolus alveolar bone alveolar bone ameloblasts artifact bell stage blood vessels capillaries cementum dental lamina = dental ledge crown dental papilla dentin dentin enamel space enamal organ gingival enamel space gingival epithelium head mesenchyme junctional epithelium inner enamel epithelium keratinized layer labiogingival lamina lamina propria maxilla marrow cavity mandible odontoblasts nasal cavity periodontal ligament odontoblasts pulp oral cavity root oral epithelium sulcular epithelium outer enamel epithelium sulcus outer sheath Tome’s fibers preameloblasts vetible predentin pulp pulp mesenchyme stellate reticulum tongue vestibular lamina vomeronasal organ
90