Histology Laboratory Exercises

a companion manual for

by Robert W. Ogilvie, Ph.D.

First Edition
2003 Robert W. Ogilvie

Credits
This manual is authored and copyrighted 2003 by Robert W. Ogilvie, Ph.D., Professor, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2204. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the author. WebMic, the program for which this manual is written, is a Virtual Microscope created by a team of educators and programmers. I am very grateful to have had the opportunity of being a part of the team that created WebMic. I wish to express thanks to Dr. Peter Groscurth, Institute of Anatomy, University of Zurich for the opportunity to collaborate to create the content for The Virtual Microscope 2.0, a CDROM playable only on a windows computer, while I was on sabbatical leave in Zurich 1999-2000. Virtual Microscope 2.0 preceded and provided the content for WebMic, a cross-platform JAVA program. Also, I wish to express gratitude to Hanspeter Rohr and his team at the Neocortex Foundation, Basel Switzerland for being the main force behind the production of the creative web interface known as WebMic. Credits for WebMic are:

Concept and Design: Hanspeter Rohr, MD, Professor Neocortex Foundation, Basel Switzerland

Professional Content: Peter Groscurth, MD, Professor, Institute of Anatomy, University of Zurich,
Winterthurerstrasse 190, CH8057, Zurich, Switzerland, Email: gc@anatom.unizh.ch Website: http://www.anatom.unizh.ch. Robert Ogilvie, PhD, Professor, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2204, USA, Email: ogilvieb@musc.edu Website: http://cba.musc.edu Source of Illustrations: Peter Groscurth, MD, Professor, Institute of Anatomy University of Zurich Labels and Legends: Peter Groscurth, MD, Professor, Institute of Anatomy University of Zurich, Zurich Switzerland. Robert Ogilvie, PhD., Professor, Medical University of South Carolina, Charleston, South Carolina, USA Visual and Textual Editor: Birgit Rohr, Neocortex Foundation, Basel, Switzerland Programming and Design: Henning Benecke, Dipl. Ing. and Rolf Konig, Neocortex Foundation, Basel Switzerland Production: Neocortex Foundation for Interactive Media in Education, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland, Email: info@neocortex.ch , http://www.neocortex.ch

Acknowledgments
The author wishes to express thanks to Dr. Luis Filgueira, School of Anatomy and Human Biology, University of Western Australia for many suggestions for improvement of the text. Also, the author is indebted to Dr. Robert Bloodgood, Professor and Course Director for the Histology Course (Cell and Tissue Structure) Department of Cell Biology, University of Virginia School of Medicine, for contributions that have improved format and content. The author would like to express thanks to the following members of the faculty of the Department of Cell Biology at the Medical University of South Carolina (MUSC) for valuable contributions to the text of this manual: Dr. Carol Eisenberg, Associate Professor, Dr. Timothy Fitzharris, Professor, Dr. Steve Kubalak, Associate Professor, and Dr. Karl Karnaky, Jr., Associate Professor. The author is also grateful to Dr. Paul McDermott, Associate Professor, Internal Medicine, MUSC, and to Dr. Gabriel Virella, Professor, Department of Microbiology and Immunology, MUSC, for valuable input on selected chapters. The author would also like to express appreciation to medical students, Stephen Mittelstaedt and Marjorie Turner, who read selected chapters using the program and offered suggestions for improvement. The author is very grateful for editorial assistance by Laura Pomeroy, second year medical student, who read every chapter while using the program and submitted valuable suggestions to improve the integrity of the text with the program, for assisting me in keeping the text in the active voice and for improving the layout of each chapter. Last, but my no means least, I am very grateful to my wife, not only for excellent editorial suggestions, but for the support she has given to me when it has been necessary to be totally consumed by this project. This is only the latest demonstration of her undying devotion to creating an environment at home that has been of great support and encouragement to me over the years.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

Table of Contents
Credits.2 Table of Contents........................................................................................................................3 Preface.........................................................................................................................................4 WebMic Tutorial.........................................................................................................................5 Unit 1: Epithelial Tissue..........................................................................................................15 Unit 2: Connective Tissue.......................................................................................................41 Unit 3: Bone and Cartilage; Bone Development.....................................................................63 Unit 4: Muscle Tissue...................................................... .......................................................87 Unit 5: Nerve Tissue..............................................................................................................105 Unit 6: Peripheral Blood and Bone Marrow..........................................................................121 Unit 7: Cardiovascular Organs...............................................................................................137 Unit 8: Skin and Appendages.................................................................................................161 Unit 9: Lymphoid Organs......................................................................................................189 Unit 10: Gastrointestinal Tract (including gallbladder)...........................................................217 Unit 11: Exocrine Glands (salivary glands, pancreas, liver)....................................................257 Unit 12: Endocrine Glands (pituitary, thyroid, parathyroid, adrenal, Islets)............................281 Unit 13: Respiratory tract and lung...........................................................................................311 Unit 14: Urinary tract and kidney.............................................................................................337 Unit 15: Male Reproductive Tract and Organs.........................................................................363 Unit 16: Female Reproductive Tract and Organs.....................................................................389

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

Preface
This manual is written in the style of a traditional histology manual when students were issued a box of glass slides and used a microscope to study histology. Learning resources for histology have undergone a series of evolutionary steps arriving at the use of technology today. Until the early 1970s, students used only glass slides and a microscope in most courses to learn histology. The students interaction with the glass slide containing the tissue specimen was guided with a written laboratory manual that asked the student to view the specimen at different magnifications. For each magnification the student was asked to find certain structures and received instruction about what they viewed. In the early 1970s, photos taken through the microscope (photomicrographs) were put into 35mm slide format and organized into selfinstructional lessons where the text of the lesson instructed the student about each slide. Today, photomicrographs are incorporated into the powerpoint program and other various interactive applications created by a variety of authoring software and stored on CDs or made available over the internet. WebMic is a program that presents specimens to the student of histology in a manner similar to the glass slide and the microscope. It addresses one of the limitations of the captured photomicrograph, that the student cannot manipulate the image. The specimens in WebMic can be selected, manipulated and studied at several levels of magnification with or without important structures labeled. The user interface allows scanning of specimens that resembles using a microscope to study a glass slide. The image being scanned and the magnified area of the scan are displayed simultaneously, a feature not possible in a light microscope. The specimens in WebMic can be selected with or without important structures labeled. Structural dimensions can be measured. The goals of the companion manual are to: direct the student to important structures, provide a detailed step-by-step guide for examination of each specimen at several magnifications and summarize, using a personal voice to encourage review of what has just been covered. This manual guides the student in interacting with 134 specimens. For each of 3 5 magnified views per specimen the text systematically instructs the student about the histology of important cells, tissues, structures and regions. To engage the reader at the outset, the introduction gets the learner involved in measuring sizes and determining the ratio of areas of the nucleus to the cytoplasm as a way to facilitate learning how to effectively use the program. Each lesson presents a brief overview of the topic followed by the specific objectives for learning. The lesson proceeds to lead the student in using the WebMic virtual microscope in learning the structures and patterns of morphology important to the topic. Each lesson concludes with a summary of what was learned in the lesson as a way of reinforcing the learning exercise. At the end of each lesson, the student is encouraged to utilize two methods of self-assessment to reinforce what has just been learned. Finally, at the end of each lesson, a list of the specimens under the lesson topic is given along with the structures that are labeled at each magnification to facilitate the student quickly finding any structure for review.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

WebMic Tutorial
Your instructor will give you the URL address to access the program. Upon accessing the program, you will first see the title screen. To begin using the program, click anywhere on this window.

The next screen will present you with a menu from which you can select a tissue section to study from either of two categories, namely, General (Tissue) Histology or Organ Histology. In this window, a specimen that illustrates simple columnar epithelium can be seen. A low magnification view of this specimen is given in the lower left rectangle and a higher magnification of a region of this image is displayed in the circular view. Note that when the lowest magnification is selected, usually 5x, the stain and it properties used for the specimen are listed on the screen just below the menu. This specimen was stained with hematoxylin and eosin which stains nuclei blue and the cytoplasm red, blue, or a blend depending on the dominance of acidophilic and basophilic structures.

The best way to begin is to click on the question mark in the upper right of this window which takes you to the help files. The help menu will give you access to a tour of WebMic and tell you the function of actions each button across the top of this window.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

6 The first window seen after clicking on the help button (?) is the shown below. You can select either a guided tour of the sections which are displayed, or you can select the user interface.

The expanded menu under user interface is appears as shown here.

All the buttons that perform functions in WebMic are arranged across the top of the program screen. If the cursor is held over a button, a short text will appear that briefly explains the function of the button.

In the help menu under User Interface, clicking on Buttons will provide a window that explains the functions of all the buttons. This information is given on page 7 of this manual. After reviewing the function of each button, you will be ready to begin the program.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

Explanation of the actions of the buttons in the top menu:

This button activates the WebMic popup menu where the user can choose additional text information, print images and exit WebMic.

This button allows you to move between full screen and microscope screen. The button is disabled when there is no magnified image loaded yet or when the quiz mode has been selected. You should decide before prior to entering the quiz mode whether you want to be tested in the fullscreen or microsope view.

This button restarts the imageloader thereby loading the last image again. A restart is needed sometimes when there is a lot of traffic on the Internet.

This button aborts loading an image. It is enabled while an image is loaded.

This button reveals or hides a grid and a measurement line. Details can be found in the guided tour.

This button activates the Label popup menu where the user can choose the label mode.

This button starts loading a magnified image. There are two buttons for this command, one for a medium size image and one for a large size image. The button for the medium image is always enabled; the other one is enabled only when a large image is available.

This button starts a new browser window to show the WebMic help. This concludes the brief introduction for using WebMic. It would be advisable at this time to go through the entire help menu and familiarize you with the program. The next section is an exercise that will require you to use the program, including the measurement tool.
2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

Determination of the normal nuclear:cytoplasmic ratio

This exercise will ask you to determine the area of the nucleus and cytoplasm for certain cells in various tissues and organs. The purpose of this exercise is twofold: 1) to provide you with a meaningful way to practice navigation within the program and, 2) to guide you into discovering the normal ratio of the nuclear area/volume to the cytoplasm area/volume. The nuclear:cytoplasmic ratio is a constant for each cell type. This ratio is maintained for each cell type because the nucleus contains the instructions and messages for biological functions that will be carried out in the cytoplasm. Cells that are synthesizing protein have more cytoplasm volume. The mass of DNA in the nucleus has to be in proportion to the cytoplasmic organelles it is directing. When the ratio is altered, the cell is considered abnormal as in cancer or neoplasms. An example is given in the paragraph below quoted from a website* of the Baylor College of Medicine Public Awareness, Division of Cytopathology where a presentation of Cervicovaginal Pap Smear is made for the publics information.
The Morphological Basis of Cytology: Cytopathology is based on studying the cells that shed off the surface of epithelial tissues. This tendency is more evident in malignant tumors since they lack cellular cohesiveness and it can be further enhanced artificially by scraping and brushing of the surface. This paper will be limited to discussing squamous epithelium which is the main tissue involved in most cases of cervical dysplasias and malignancies. Normal stratified squamous epithelium exfoliates cells that are uniform. The nuclei are more or less equal in size with a normal nuclear/cytoplasmic ratio and a fine chromatin pattern within the nucleus. The nuclear membrane is smooth and is uniform in thickness. Malignant cells, on the other hand, are generally characterized by irregularity. These cells show increased nuclear/cytoplasmic ratio, are pleomorphic in relation to each other and they vary in their staining characteristics. They are generally dark with a thick nuclear membrane and coarse clumps of chromatin. The irregular distribution of chromatin results in areas of clearing and areas of condensation within the nucleus. The nuclear surface is irregular and shows sharp corners and angles (nuclear bites). In between these two extremes of "black and white," cells showing shades of gray are encountered. These cells are known as dyskaryoti, and although they do not show features of malignancy, their nuclei show slight hyperchromasia associated with wavy, slightly thickened nuclear membrane. The degree of abnormality of these dyskaryotic cells is varies and is classified as mild, moderate and severe with the latter getting very close to the changes associated with malignancy.

* http://www.bcm.tmc.edu/pathology/cytopath/publicaware.htm Note the reference to normal nuclear/cytoplasmic ratio and increased nuclear/cytoplasmic ratio in this text. The contrast of the nuclear/cytoplasmic ratio in normal and malignant cells is most evident in the cervical epithelium obtained by a PAP smear because it presents the diagnostician with the entire cell. The exercise you are going to complete will ask you to estimate the area of the nucleus and cytoplasm in a variety of cells in tissue sections. The entire profile of the cell may not be available to measure because only specimens that have cells sectioned through their middle can be used to accurately determine the real nuclear:cytoplasmic area ratio. This exercise is worthwhile because it will acquaint you with a variety of cells of different sizes. You will be able to observe first hand how the size of the nucleus changes in direct proportion to the size of the cytoplasm. Follow the directions of this exercise beginning on the next page and determine the nuclear cytoplasmic ratio of normal cells by making measurements on several specimens in WebMic. You will be asked to make some observations at the end of the exercise.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

9 Go to General Histology and, under Epithelial Tissue, select the specimen: Stratified Squamous Epithelium (Vagina)- Hematoxylin and Eosin. In this first measurement, you will determine the cytoplasmic and nuclear area of a squamous cell similar to what would appear in a PAP smear of the cervix. Move the cursor until it is over the pale circle on the rectangular frame in the lower left of the window at which point the cursor turns into a hand. Click and hold while dragging and you will be able to scan the lower mag (5x) image and see it magnified 20x in the circular field. This is the way the microscope works. Now click on the 20x bar above the rectangular image and you will be able to scan the 20x rectangular image by clicking and dragging the pale circle. The circular pale area of the rectangular image magnified at 20x is viewed in the circular microscope field at 80x. (The image can be moved also by clicking and dragging in the circular microscope field) Select the 40x image by clicking on the 40x tab. Now move the curser to the label button. Click on the label button to select the menu item show labels. Move the image by clicking and dragging the pale circle in the 40x rectangular image or by clicking and dragging the 160x magnified image in the round window on the right until the label stratum spinosum is in the middle of the microscope field. Find the cell just above this label that contains a black round structure and determine the area of its cytoplasm and nucleus. Click the grid toggle so that the grid and red line with two dots at either end appear in the circular image area. To measure the dimension of any structure click on a circle and drag it to one side of the object you wish to measure. Then click and drag the circle at the other end of the line to the other side of the structure you are measuring. Using the measurement tool, measure the length and width of the cell with the round black structure in it, and the diameter its nucleus. (Note: when making measurements try to place the circles at the end of the measurement line directly over the structure being measured as illustrated below)

Due to the size of the circle, the accuracy of measurements obtained with this tool will decrease as the size of the structure measured decreases. Fill in your measurements and calculations and compare them with those that are in parenthesis. Cell length = ________________ (37 microns) Cell width = _______________(20 microns)

Cytoplasmic area = length x width = ______(37) X ______(20) = ________ (740 m2 ) Nucleus diameter = ____________(8 microns) Nucleus radius = ________ (4 microns)

Nuclear area = r2 = 3.14 (4)2 = 3.14 (16) = 50.24 m2 Nuclear/Cytoplasmic Ratio = 50:740, approximately 1:15

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

10 In this case, the cytoplasmic area is 15 times greater than the nuclear area. Extrapolated to the 3rd dimension this would be the ratio of nuclear to cytoplasmic volume, with one caveot. An error could occur if the plane of section does not pass through the center of the measured cell. Now follow the directions below to measure several other cells and calculate their nuclear/cytoplasmic ratio. Select the next specimen from the list under Epithelial Tissue: Simple Columnar Epithelium (Renal papilla)- Hematoxylin & Eosin stain Observe the specimen at the 5x, 20x and 40x magnification. After you have selected the 40x magnification, turn the labels on and find the cell labeled simple columnar epithelial cell. The label is colored which means if you click on the label, a text will appear to give you information about that cell. The cell itself is delineated by a color. You can temporarily remove the color by clicking anywhere on the cell. Remove the color and measure the cell and nuclear dimensions. Cell Length _____m Cell Width _____m Nucleus radius _____m Cytoplasmic area = ________ m2 Nuclear area = _________m2 Ratio = _______

Select the next specimen from the list available under the nervous system (not nerve tissue) in the organ (not general) histology section: Spinal Ganglion Hematoxylin and Eosin stain This is a specimen taken from a spinal (sensory) ganglion located within the dorsal root of a spinal nerve. Observe the specimen using the 5x, 20x, and 40x magnifications. At the 40x magnification where the specimen in the microscope field is magnified 160x, turn on the labels and find the cell that has its nucleus and nucleolus labeled. Measure the area of the cytoplasm and nucleus. Because the cell and the nucleus are both round, use the radius to calculate the area of a circle in both cases. Cell diameter _____m Cell radius _____m Cell area ___________________m2

Nucleus diameter ______m Nucleus radius ________m Nucleus area _________m2 Nuclear:Cytoplasmic ratio = __________________ Now that you have measured the dimensions of three cells and their nuclei, what observations have you made about cell and nuclear size? Is there any constant between the three cell types as to the ratio of the nuclear area to the cytoplasmic area? Is the nucleus of a large cell larger than the nucleus of a small cell? The nuclear cytoplasmic area ratios you determined should be on the order of:
2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

11 1. Stratum Spinosum Squamous Cell = 50:740 or 1:15 2. Simple Columnar Epithelial Cell = 50:341 or 1:7 3. Spinal Ganglion Cell Body = 346:3846 or 1:11 There is really no firm conclusion that you can reach by inspecting the data from these measuresments and area ration calculations of the three cells. However, you can see that the nuclear:cytoplasmic ratios are different. If you had made the measuresments on a smear of whole cells obtained from a scraping of the cervix as explained in the excerpt from the website at Baylor University, you would have obtained a ratio that would be consistent with normal cells. All the measurements that you made were on tissue slices in which the cells are sectioned at different planes, some through the middle and others eccentric. To make valid comparisons between the nuclear:cytoplasmic ratios of different cells the measurements would have to be made on images in which the cells were sectioned through the middle. One observation that you probably made is that the spinal ganglionic cell is much larger than the other two cells. You probably also observed that its nucleus is larger. As the cytoplasm gets larger, the nucleus gets larger in normal cells. Hopefully, this exercise has 1) helped you in learning to navigate in WebMic and use the meaurement tool, 2) given you some insight into distinguishing between normal and abnormal cells, and 3) provided you with a morphological illustration of the relationship between the nucleus and cytoplasm. There are two functions that will be helpful to know about as you use WebMic. One function is the button that allows you to view the specimen without the microscope. Click on the button to move to full-screen (second button from the left). Now you can view the entire specimen. Hold down the left mouse button and drag the image around to place it where you want it. The print function is also helpful. Return to the microscope view. Now, click on the menu button (first on the left) and select print. Notice that the image without any labels is opened in another browser window. You can save the image to your hard drive, or you can print the image and insert it into this manual in the section where you are working. As you work through this manual, you may wish to print out images to label. If you wish to do that after you have selected the image and the magnification, click on the menu button at the upper left corner of the WebMic window. Choose print and the image presented will open in a new browser window. This will always be an unlabeled image. At this point, you can print it out directly. The images are scaled to print in portrait. You can print the image directly and insert it into your manual. You can also save the image for a photo processing program and arrange to print it portrait or landscape. The next two pages give an example of an image printed in portrait and landscape formats. At this point, if you have not done so, it would be a good idea to go back through all the help menu items to review all functions within this program and testing modes that are available. This will save you time and frustration as you work through the exercises in this manual. The remaining units will guide you through learning the basic microscopic structure of tissues and organs.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

12

The figure below illustrates the new window that appears with the selected image after the print function has been activated. To find this same image, go to general histology and under epithelium select simple columnar epithelium (renal papilla) hematoxylin and eosin at 20x magnification.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

13 This is an example of an image exported out of Adobe Photoshop and placed in a word processor like Microsoft Word in portrait format. To find this same image, go to general histology and under epithelium select simple columnar epithelium (renal papilla) hematoxylin and eosin at 20x magnification. Images like this can be labeled and notes can be written below.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie

14 This is an example of an image turned in adobe photoshop, enlarged, and placed on the page in landscape format. To find this same image, go to general histology and under epithelium select simple columnar epithelium (renal papilla) hematoxylin and eosin at 20x magnification.

2003 Histology Laboratory Exercises for WebMic by Robert W. Ogilvie