1

Microscopic Anatomy Laboratory Manual

1992 Washington University School of Medicine The Microscopic Anatomy Laboratory Manual is a result of the combined efforts of several former and current members of the Department of Anatomy and Neurobiology. The principle contributors include Drs. Allen Enders, Barry King, Milton Goldstein, Nancy Baenziger, Richard Bischoff, David Menton and Adolph Cohen. Credit is also due to the many students down through the years who have contributed useful suggestions for improving the Manual. Indeed, it was at the suggestion of our students that the Manual was written in the first place. Finally, I wish to acknowledge our University, who has steadfastly underwritten the expense of producing the Manual every year, permitting us to distribute it to our students at no further cost to them.

Paul C. Bridgman, Ph. D. Coursemaster of Microscopic Anatomy

Department of Anatomy and Neurobiology Washington University School of Medicine Box 8108 660 South Euclid Avenue St. Louis, Missouri 63110

Contents
Introduction
The demise of the teaching lab The value of the teaching lab Suggestions for success in the histology lab

Slide Collection
Student Loan Collection Box A Box B
Slide Inventory

11

Histological Methods
Fixation
Functions of a fixative

15

Dehydration and Embedding Sectioning Staining Sections and Tissues

The Microscope
Theoretical Considerations
Study questions

19

Cleaning the Microscope

Cleaning the oil immersion lens

Illumination of the Light Microscope The Dual-View Teaching Head

Install the dual body-tube as follows Proper Use of the dual teaching head

The Cell
Basophilic vs. Acidophilic Membranous Structures in the Cell

25

CONTENTS

Filamentous and Tubular Organelles

Epithelial Tissues
Simple Epithelia Pseudostratified Epithelia Stratified Epithelia Transitional

31

Blood
Wrights Stained Smear of Peripheral Blood
Preparation of slide Staining Method Abnormalities of erythrocytes Common artifacts in blood smears

35

Study questions on EMs.

Fibrous Connective Tissue

Loose Connective Tissue Dense Connective Tissue Embryonic Connective Tissue Electron Micrographs

41

Cartilage
Hyaline Cartilage Elastic Cartilage Fibrocartilage Electron Micrographs

45

Bone
Ground Bone Preparations Endochondral Ossification

47

CONTENTS

Intramembranous Ossification

Muscle
Skeletal Muscle Smooth Muscle Cardiac Muscle

51

Peripheral Nerves
Can you identify in slide 92A Integrative questions in neuroanatomy

53

Cardiovascular System
Arteries and Veins Lymphatics Heart

57

Hemopoiesis
Electron Micrographs

59 61

Lymphatic Tissue
Thymus Peripheral Lymphoid Tissue Spleen

Integumentary System
Epidermis and Dermis Hair Sweat Glands

67

Respiratory System
Nasal Cavity Trachea Lung

71

CONTENTS

Electron Micrographs

Oral Cavity
Lips Teeth Tongue Palate Salivary Glands

75

Esophagus and Stomach

Esophagus Stomach

79

Intestine
Duodenum Jejunum Ileum Colon Appendix Rectum Electron Micrographs

81

Pancreas
Exocrine Pancreas Endocrine Pancreas Electron Micrographs

83

Liver and Gallbladder

Liver
Contents of the portal tract

85

Gallbladder

Electron Micrographs of Liver

Urinary System
Kidney Ureter & Bladder
Layers of ureter and bladder

89

Endocrine System
Hypophysis Thyroid Parathyroid Adrenal Gland

93

Male Reproductive System

Testis Accessory Reproductive Structures Penis

97

Female Reproductive System

Ovary Uterus Vagina Oviduct Placenta The Female Breast

103

Glossary

109

Introduction
The demise of the teaching lab
In the last 25 years, most courses at this and other medical schools have either deemphasized or discontinued the laboratory as a learning experience. It is, perhaps ironic that this should occur at the same time that nearly everyone concerned with the medical school curriculum recognizes the pedagogical inadequacy and tedium of countless hours of lectures. While there has been broad agreement that "active learning" should, wherever possible, replace "passive learning," active learning has not always included laboratory study. In many courses, labs have been replaced with discussion groups and problem-solving sessions. Laboratory study has always included these immensely important components, but when discussions and problemsolving are divorced from the lab experience, they too can become more passive than active. There are several reasons for the demise of the lab in medical education. First, labs are expensive. The microscopic anatomy lab at this medical school involves an expenditure of approximately $250,000 in microscopes alone. In addition, several thousand dollars is spent each year in the administration and maintenance of our teaching laboratories, microscopes, slide sets and supplies. The cost of the gross anatomy lab (and its associated body donation program) greatly exceeds the cost of the microscopic anatomy lab. Secondly, labs are demanding and labor-intensive for the faculty. It is relatively easy to give a dozen or so lectures and/or discussions each year on selected subjects closely related to one's field of expertise and research, but far more demanding to serve effectively in a teaching laboratory where wide-ranging questions emerge spontaneously from both the lab and the lecture. Finally, many students find the lab an "inefficient" way to learn, given the great demands on their time and energy. It is much easier and quicker to either read or hear what you "need to know" than to discover it for yourself in a lab.

The value of the teaching lab

Perhaps then, a defense of the teaching laboratory is in order. Your microscopic anatomy faculty believes that the expense, labor, and putative inefficiency of a lab are easily compensated by a sense of discovery and depth of insight not easily obtained by any other means. Even lectures become tolerable if they are followed by an opportunity to examine for oneself the very subject of the lecture. Lectures and discussions remain only mental exercises, until one has an opportunity to both use and reinforce that information with personal experience. More importantly, the information that can be gleaned from the study of actual biological tissues and organs is essentially without limits. In contrast, the prepared information to be found in a lecture, book, photograph, etc., is finite and necessarily reflects the bias of its author. For biological knowledge to increase, there must be some point where the closed loop of codified

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INTRODUCTION

information is open to the discovery of new information from nature itself. There are many examples in the biomedical research literature of observations that could have been made by an astute observer of our class set of microscope slides and electron micrographs. We are convinced that if you have made a good career choice in medicine and/or biomedical research, you will appreciate the opportunity to discover for yourself the marvelously complex world of microscopic anatomy. Your faculty is enthusiastic about assisting you in this discovery, and, indeed, we will be learning with you.

Suggestions for success in the histology lab

First, we urge you to regularly attend lab. Only by regular practice and experience will you learn to grasp the three-dimensional world of microscopic anatomy from the nearly (but not quite) two-dimensional specimens on your microscope slides. Before you come to lab, you may find it helpful to read the relevant section of your lab guide and atlas. Also, the Menton collection in the library has proved useful to many as a lab orientation or preview. This collection is available in three forms: color 35-mm transparencies, Kodak Photo CDs and a computer tutorial on the network. All of these comprise the same specimens you will be studying in the lab. When you come to lab, we suggest that you bring your textbook or atlas as you will often find a photo or drawing that assists you in understanding the specimens on your slides. Perhaps the most important suggestion we can give you is that you not leave the lab until you have seen several examples of each of the structures printed in boldface in this manual. This is best accomplished by keeping your eyes open for structures you have already seen as you search for new ones. Typically, the same structure will appear in several places on your slides, giving you a chance to see them in several planes of section. The double-viewing attachment we provide gives you an opportunity to view your slides with another observer -- both seeing the same specimen right-side-up and with a common pointer. This encourages analysis and discussion of histological structures with your lab partner or the faculty, and teaches you to use the terminology you will encounter in your medical career. Finally, we urge you to consider drawing many of your specimens in an unlined notebook. Even a bad drawing teaches you to be a careful observer.

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Slide Collection
Student Loan Collection
Two students must share a set of slides comprising Box A and Box B. Slides in Box B are distinguished by the presence of a line below the slide number; those in Box A have no underline. Each slide also bears the number of it's slide set - please do not mix them with other sets. You will be held responsible for the slides in the set you are issued.

Abbreviations used
AB AF BS CCH CIV FG H&E H & OGE IH MC MT OG OT PAS PTAH RF SB VH Alcian blue Aldehyde-fuchsin Bodian silver Copper-chrome hematoxylin Carmine injection - vascular system perfused with gelatin and carmine Fast green Hematoxylin and eosin Hematoxylin and orange G-erythrosin Iron hematoxylin Muci-carmine Mallory trichrome Orange green Osmium tetroxide Periodic acid - Schiff Phosphotungstic acid hematoxylin Resorcin-fuchsin Sudan black Verhoeffs hematoxylin

All specimens are human tissue unless otherwise indicated.

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SLIDE COLLECTION

Box A
1A 2A 3A 4A 5A 6A 7A 8A 9A l0A llA l2A l3A l4A l5A l6A l7A l8A l9A 20A 2lA 22A 23A 24A 25A 26A 27A 28A 29A 30A 31A 32A 33A 34A 35A 36A 37A 38A 39A 40A 4lA 42A 43A 44A 45A 46A 47A 48A 49A 50A Skin, digital, monkey; H & E; AF; OG; FG; CIV Skin, digital monkey; Bodian silver (carmine) Scalp, human L.S. Scalp, bald; H & E Skin, scalp; H & E Skin, adult thigh; H & OGE; VH Skin, palmar, monkey; H & E; plastic section Skin, axillae; H & E Tendon; H & E Musculotendinous junction; H & E Musculotendinous junction; PTAH Skeletal muscle, monkey; H & E; plastic section Skeletal muscle, dog; H & OGE Skeletal muscle, dog; IH Heart, mammal; H & E Heart, monkey; H & E; plastic section Heart, monkey; H & E Heart, monkey, base of ventricles; AF, OG, FG Heart, ventricle, dog; PTAH Heart, beef, endocardium, Purkinje fibers; H & E Heart, human, left ventricle; H & E Vena cava; H & E Aorta; H & OGE; VH Arteries, monkey; H & E Artery, vein, nerve; VH Mesentery; whole mount, CIV Artery and vein, monkey; H & E; plastic section Tibia, cross section, rabbit; H & E Bone marrow, hyperplastic; H & E Tonsil, dog; H & OGE Blood smear, normal; Wright's stain Bone marrow, human; Wrights stain Aorta, monkey; H & E; plastic section Lymph node, monkey; H & E; plastic section Lymph node, dog; silver impregnation; H & OGE Thymus, human; H & E; plastic section Thymus, infant; H & E Areolar tissue spread; RF Thymus, newborn; H & E Thymus, involuting; H & E Fatty thymus, adult, small mammal; H & E Spleen; silver impregnation; H & OGE Spleen, engorged; H & E Spleen, washed through artery, dog; H & OGE Spleen, phagocytosed carbon, rat; H & E Spleen, monkey; H & E; plastic section Spinal ganglion, cat; H & E Tendon, monkey; H & E; plastic section Elastic cartilage, ear, dog; H & OGE; VH 5lA 52A 53A 54A 55A 56A 57A 58A 59A 60A 6lA 62A 63A 64A 65A 66A 67A 68A 69A 70A 7lA 72A 73A 74A 75A 76A 77A 78A 79A 80A 8lA 82A 83A 84A 85A 86A 87A 88A 89A 90A 9lA 92A 93A 94A 95A 96A 97A 98A 99A l00A Vertebral column, cat, longitudinal section; H & E Fibro-cartilage, intervertebral disc; H & E Vertebral column, cat; H & E Jaw bone, infant; H & E Thorax, human fetus (40 mm); tetrachrome Ribs, stillborn; M (with fast green rather than aniline blue) Foot, 5-month fetus; H & OGE Finger, stillborn; and 5-month fetus; H & OGE Compact bone, ground section Knee joint, cat; H & E Developing membrane bone; H & E Fracture, l3 days, rabbit; H & E Lip; H & E Tooth and mandible, monkey; H & E Tongue, monkey; H & E Tongue with taste buds, monkey; H & E Parotid gland, dog; H & OGE Parotid gland, monkey; H & E; PAS Submandibular gland, monkey; H & E; aldehyde fuchsin Submandibular gland, monkey; PAS Submandibular gland; H & E Submandibular gland, monkey; H & E; plastic section Larynx, frontal section; H & E Sublingual gland, monkey; H & E Esophagus; H & E Esophagus and trachea; H & E Gastro-esophageal junction, monkey; CCH; Muci Stomach, fundus; H & OGE Stomach, fundus, monkey; H & E; plastic section Stomach, fundus, dog; IH; PAS Mesothelium, rabbit; silver stain Pyloro-duodenal junction, monkey; H & E Pyloro-duodenal junction, monkey; CCH; Muci Jejunum, 40 years; H & E Jejunum, monkey; H & E; PAS (carmine) Jejunum, monkey; H & E; plastic section Duodenum, human; H & E; plastic section Ileum, human; H & E; plastic section Ileum; H & E Ileum, cat, Peyers patch; H & E Nerve, dorsal root ganglion in culture; OT and SB Enteric nerve plexus (Auerbach), cat; Richardson silver Colon, monkey; H & E Rectum, human; H & E Appendix, human; H & E; plastic section Appendix, carmine-injected vessels Appendix, infant; H & E Recto-anal junction; H & OGE Adipose tissue, monkey; H & E; plastic section

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SLIDE COLLECTION

Box B
lB 2B 3B 4B 5B 6B 7B 8B 9B l0B llB l2B l3B l4B l5B l6B l7B l8B l9B 20B 2lB 22B 23B 24B 25B 26B 27B 28B 29B 30B 3lB 32B 33B 34B 35B 36B 37B 38B 39B 40B 4lB 42B 43B 44B 45B 46B 47B 48B 49B 50B Liver, bile canaliculi; silver impregnation Liver, infant; H & E Liver, dog; intravenous carbon injection Liver, rat; intravenous trypan blue; H & E Liver, cat; carmine injected vessels Liver, rat; regenerating; H & E Liver, monkey; H & E; plastic section Gallbladder, monkey; H & E; plastic section Gallbladder; H & E Pancreas, human; H & E Pancreas, cat; H & E; guinea pig; aldehyde fuchsin Pancreas, monkey; H & E; plastic section Thyroid gland, monkey; H & E; plastic section Thyroid and parathyroid, dog; H & OGE Neck organs, monkey; H & E Thyroid, rabbit, hyperplastic; H & E Adrenal and kidney, fetal, human; tetrachrome Adrenal, dog; H & E Adrenal, monkey; H & E Adrenal, monkey; H & E; plastic section Adrenal, infant; H & E Parathyroid, human; H & E Hypophysis; H & E Hypophysis, human; Masson Hypophysis, rabbit; PAS; orange G; alcian blue Pituitary and hypothalamus, cat; H & E Nasal cavity, cat; H & E Soft palate, cat; H & E Lung, monkey; H & E; plastic section Lung, monkey; VH and picro-ponceau Epiglottis, elastic cartilage; VH Palate, cat; H & E Trachea; H & E Trachea, monkey; H & E; plastic section Lung, rabbit; H & E; aldehyde fuchsin Lung, cat collapsed, pneumothorax; H & E Lung, thick section Lung, dog; carmine injected vessels Kidney, 40 years; H & E Kidney, rabbit; carmine injected; orange G, fast green Kidney, monkey; H & E; plastic section Kidney, rabbit; carmine injected; PAS; H & E Kidney, guinea pig; silver impregnation Ureter, monkey; H & E; plastic section Kidney, mouse; alkaline phosphatase 5lB 52B 53B 54B 55B 56B 57B 58B 59B 60B 6lB 62B 63B 64B 65B 66B 67B 68B 69B 70B 7lB 72B 73B 74B 75B 76B 77B 78B 79B 80B 8lB 82B 83B 84B 85B 86B 87B 88B 89B 90B 9lB 92B 93B 94B 95B 96B 97B 98B 99B l00B Bladder, relaxed and stretched; H & E Testis, monkey, mature; H & E or tetrachrome Testis and epididymis, monkey; H & E Testis, monkey; H & E; plastic section Testis and epididymis, infant; H & E Hela cells, mitosis; H Spermatic cord with ductus deferens, human; H & E Seminal vesicle, monkey; H & E; plastic section Seminal vesicle; H & E Bulbo urethral, adult rabbit; H & E Prostate, monkey; H & E Prostate; H & E Prostate, monkey; H & E; plastic section Penis, stillborn; H & OGE Bladder, monkey; H & E; plastic section Ovary, monkey; H & E; plastic section Ovary, corpus luteum, 25th day menstrual cycle; H & E Ovary, corpus luteum, first trimester; M Ovary, infant; H & E Ovary, senile; H & E Ovary and uterine tube, small mammal; H & E Uterine tube, human, l7 years old; H & E Uterine tube; H & E Uterine tube; senile; H & E Uterus, early and late luteal phases; H & E Uterus, menstruating and follicular phases; H & E Uterus, 2 months pregnant, human; H & E Uterus, monkey; H & E; plastic section Cervix and uterus, term human baby; H & E Cervix, adult; H & E Vagina, monkey; H & E; plastic section Vagina, 20 years; H & E Placenta, human, 2-month; H & E Placenta, human, full term; H & E Oviduct monkey; H & E; plastic section Placenta, human at parturition; tetrachrome Placenta, human, lst trimester; H & E Placenta, human, early 2nd trimester; H & E Mammary gland, nonpregnant; H & E Placenta, human, 3rd trimester; H & E; plastic section Mammary gland, senile; H & E Mammary gland, lactating; H & E Umbilical cord, human, tetrachrome Eyelid, rabbit; H & E Mammary gland, monkey; resting; H & E Mammary gland, monkey; lactating; H & E

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Indicate damaged or missing slides in the appropriate spaces below. A blank space after a number indicates that slide is present in the box and serviceable.

Box A
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.

Box B
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.

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Histological Methods
To intelligently use the slides in your slide collection, you should have at least some knowledge of the histological technique that has gone into their preparation.

Fixation
The first step in preparing slides of a specimen for light microscopy is fixation. Ideally this accomplishes the following important requirements for subsequent sectioning and staining: Functions of a fixative Kills the tissue quickly. Preserves much of the chemical composition of the cell. Minimizes tissue swelling or shrinkage. Inactivates tissue proteases. Imparts rigidity to preserve the shape and location of the tissue components during sectioning. Mordant for some dyes used in staining. Most fixatives act on the proteins of the tissue to render them insoluble. The following fixatives are commonly used for light microscopy:

Formalin
Formalin is an aqueous solution of formaldehyde. It is easily the most common fixative for routine light microscopy, and functions by binding to certain side groups of amino acids to form methylene bridges between protein molecules. Aldehydes allow lipid extraction, but they penetrate tissues quickly and preserve structure quite well.

Alcohol
Alcohol coagulates protein and penetrates rapidly but dehydrates tissue and causes shrinkage. Alcohol preserves glycogen and other water soluble components of tissues better than aqueous fixatives.

Bouin's Fluid
Bouin's fluid is a mixture of the following three fixatives: Formalin - same function as described above. Picric acid - offsets the tendency of formalin to harden tissue excessively and makes cytoplasm more basophilic (affinity for basic dyes). The exact chemical effect of this fixative is not known although it probably forms additive compounds with amino groups. Acetic acid - offsets tissue shrinkage associated with picric acid by breaking salt linkages between protein chains and exposing hydrophilic groups to water.

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HISTOLOGICAL METHODS

Special Fixative Ingredients

Chromium salts
Certain chromium salts such as potassium dichromate, produces oxidation and chromium linkages between proteins and also binds phospholipids.

Mercuric chloride
Mercuric chloride acts on sulfhydryl; carboxyl and amino groups of protein, producing mercury linkages between the molecules.

Immersion and Perfusion Fixation

Fixatives are occasionally perfused into tissues in vitro by way of the blood stream but in most cases, small blocks of tissue are simply immersed in the fixative. This latter method often results in unequal fixation in the block. When you study slides, you will often notice that tissue preservation, shrinkage and even staining will vary from the periphery of the section to the center as a result of the rate of penetration and varying exposure to the fixative. Slide 23B in your collection shows an example of this; note how the shrinkage and staining artifact at the periphery of this pituitary gland differs from the better-preserved and stained interior. The periphery was over fixed.

Dehydration and Embedding

All of the sections in our slide collection were cut from tissues embedded in either a paraffin or plastic matrix. Some sort of solid embedding matrix is essential for slicing the tissue into sections of about l0 m or less in thickness. One can simply freeze a block of fresh tissue in water (or other aqueous medium) and cut frozen sections in a cryostat microtome. This method has certain advantages: it avoids extracting lipids, allows histochemical procedures for localizing enzymes in tissues, and is a very quick method attractive to surgical pathologists. Ice as an embedment has several disadvantages however, including rather severe ice crystal artifact in the tissue (though very quick freezing will minimize this). Fixed tissues are typically dehydrated in a graded series of alcohol and are cleared in xylene or other nonpolar solvent. Xylene makes the tissue more translucent to light, but even more importantly, xylene is miscible with the embedding medium and thus allows its penetration into the tissue block. Paraffin is the most common embedding medium, although many slides in your set have been embedded in celloidin (nitrocellulose). You can distinguish slides prepared by these two embedding methods by looking carefully at the section with the unaided eye. In a celloidin section you will often see a rectangular field of celloidin that extends slightly beyond the perimeter of the specimen. Unlike paraffin, celloidin is not dissolved from the section before mounting (compare slide 6A [celloidin] with 8A [paraffin]).

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HISTOLOGICAL METHODS

Sectioning
Paraffin and Celloidin Sections
Most of the slides in your class set are sections cut from a block of embedded tissue. Paraffin and celloidin sections are typically cut about 5-l0 m thick with a razor sharp steel knife mounted on a microtome. Sectioning is necessary if one is to examine the interior of a block of tissue, rather than just its outer surface. More importantly, a thin section allows us to transilluminate the specimen with light in the microscope. In a sense, the light microscope optically sections your section even thinner due to the limited depth of focus of the high power objectives. At 400x magnification, for example, the depth of field is only a little over l m. It is for this reason that one generally keeps focusing up and down through the specimen as you study, say, a 7 m thick section. After sectioning, one or more sections are carefully placed on a microscope slide where they are made to adhere as wrinkle-free as possible. Wrinkles are often confused with some structural features of tissues by the beginning student. You can easily detect a wrinkle because they have very straight edges and are both thicker (often three thicknesses) and darker than the rest of the section. Naturally, you will also find occasional tears or separations in your sections. After the paraffin is dissolved from the section, it is stained with one or more of a wide variety of stains.

Thin Plastic Sections

Some slides in your class set are plastic sections. In this case the tissue was embedded in a rather hard epoxy resin and sectioned about l.5 m thick with knives made from broken pieces of thick plate glass. These very thin sections offer greater clarity as well as greater detail of cell components. Compare a l.5 m thick plastic section of monkey testis (55B) with a 7 m thick paraffin section of monkey testis (54B).

Non-Sectioned Tissue
There are a few slides in your class set that really are not sections at all. Blood smears and marrow smears (32A), for example, are not sections. The intact white blood cells of a smear look substantially larger than they would in sections of these same cells why is this so? Cells cultured on a coverslip may also be observed without sectioning (57B). Slide 39A is a teasedout spread of areolar connective tissue which, of course, has not been sectioned.

Staining Sections and Tissues

While the examination of unstained viable cells and tissues is frequently informative (particularly with polarizing, phase, dark-field and fluorescence microscopy), most sections of tissue must be stained with dyes to reveal detailed structure under the light microscope. Often, more than one stain is employed on the same slide to differentiate two or more tissue elements. It must be remembered that no one stain or combination of stains can satisfactorily differentiate all tissue elements. Whereas the hematoxylin and eosin method is perhaps the most commonly used stain to show general tissue morphology, specific or selective stains are frequently required to show certain elements to best advantage. It is, for example, impossible to see mitochondria with most

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HISTOLOGICAL METHODS

procedures, but they can be stained by Janus green B with supravital techniques. Frequently even the fixation must be carefully selected to either adequately preserve the desired tissue elements, or to promote their staining with a particular dye. The slides loaned to you for this course have been stained with a wide variety of stains. It will be helpful to consider the possible reasons the indicated stain was used for each tissue and what you might expect to see in it. Histological specimens are most easily interpreted when you understand the specific staining characteristics of the stain used in preparation. The glossary at the end of the lab manual includes all of the stains you will encounter in this course and details their dye affinities for cell and tissue components. The two most common stains are hematoxylin and eosin (H & E) and hematoxylin and orange G-erythrosin (H & OGE), described below. Hematoxylin and eosin (H & E) - The most common histologic stain used for routine study of general morphology. Stains nuclei blue and practically all cytoplasmic structures red. Those constituents staining blue with the basic dye hematoxylin are commonly called basophilic and those staining red with the acidic dye eosin are calledacidophilic. Pronounced basophilia in the cytoplasm of cells usually indicates a high level of RNA and protein synthesis such as is observed in developing organs in the embryo (or in cells of the adult organism that are very actively engaged in protein synthesis, e.g., the pancreatic acinar cells). As a general rule, the basic components of a tissue stain with acidic dyes and so are called acidophilic, whereas the acidic components stain with basic dyes and are called basophilic. Hematoxylin and orange G-erythrosin (H & OGE) - A general purpose stain for morphology. The hematoxylin primarily stains nuclei and other basophilic constituents of the cell, if any. Orange G is a rather strongly acid dye which stains acidophilic components such as the cytoplasm, an orange-red color. The erythrosin is also an acid dye, but stains some structures such as smooth muscle, a light pink.

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The Microscope
Theoretical Considerations
Resolution
Resolution is the closest distance that two points may be separated and still observed to be two separate points. The resolution of the unaided eye is normally about .2 mm. The resolution of the light microscope depends on the wave length of light used and the numerical aperture of its lens (the latter is stamped on the side of the objective). The ultimate resolution of the light microscope using visible light is about 0.2 m. The maximum theoretical resolution of a light microscope with a given objective lens is determined by the following formula:

r=
Where r= = NA = resolution wave length of illumination numerical aperture of lens

0. 6l NA

The wave length of visible light is typically not under the control of the microscopist. The numerical aperture (NA) is equal to the sine of the angle of aperture () times the refractive index (n) of the medium through which the light passes and varies with the objective used. The angle of aperture is the angle between a point in focus and the margins of the first lens of the objective.

NA = n sine
As can be seen by an examination of this formula, objectives designed to resolve smaller objects must either have a very broad diameter (impractical) or a closer working distance to increase the size of . In any case, the sine of the angle of aperture can only approach a value as great as 1. To achieve a NA greater than 1, an immersion medium with a high refractive index, typically oil, must be placed between the specimen and the front lens of the objective. What is not as obvious is that the effective NA of the microscope is the average of the NA of the objective and that of the condenser. We should then write the resolution formula as follows:

NA = n sine

r =NA

l + Cond NA OBJ

Put simply, unless the condenser is able to fill the objective lens with light, the NA of the lens will be reduced because the angle of aperture will be reduced. Consequently, over-closing the iris diaphragm of the condenser will reduce the effective NA.

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THE MICROSCOPE

Study questions: 1. Assuming a mean wave length for light of 0.5 m, calculate the optimum resolution of your microscope for each of the objectives (4x, 10x, 40x, and 100x), using the NA value marked on each objective. 2. What effect would the magnification of the eyepieces have on resolution? 3. Could you resolve any of the following with your microscope: cell membrane cilium mitochondrion ribosome

Cleaning the Microscope

A dirty objective lens is a common cause of lack of resolution in microscopes. If you suspect a dirty objective, unscrew it from the lens turret and examine the surface of the lens with an eyepiece from your scope. If you look through the eyepiece the wrong way it can be used as a fine quality loupe. This will reveal any deposit on the lens. The most common way of dirtying an objective lens (especially the 40x one) is to accidently drag it through oil. This can be avoided by never rotating the lens turret after you have the oil in place and by using only the oil lens (l00x objective) on oiled slides. This lens, by the way, has a black ring around the lens barrel to make it easily distinguishable from the high dry lens (40x). If you must look at an oiled slide with the high dry lens, then lower (focus) the slide away from the lens, blot up the bulk of the oil from the slide with lens tissue and then carefully rotate the lens turret to bring in the 40x lens. The l0x and 4x objectives should clear any puddle of oil. You must clean the oil off the oil immersion objective (or any accidently soiled objective) after each days use. If you leave it on, it will collect dust in the oil making a grinding compound when you finally wipe it off. The l00x oil immersion objective is fortunately easy to clean as follows: Cleaning the oil immersion lens 1. Turn the lens turret so you can get at it. 2. Wipe off the oil as well as possible with dry lens tissue (lens tissue only please). 3. Slightly dampen a clean piece of lens tissue with lens cleaning fluid and gently wipe lens with this. You might finish with another dry wipe. It is exceedingly important that you do not use an excess of lens cleaning fluid

21

THE MICROSCOPE

as this can dissolve the cement between the lens elements and thus ruin the objective.

If you get oil or residue of any kind on your dry lenses (the 40x is particularly susceptible to this), lens tissue will usually not suffice. All of the dry objectives on your scope are of a special flat field design (image is flat edge to edge) in which the first lens element isconcave . Lens tissue just glides over the central portion of this type of lens. You can see this with your eyepiece loupe. We have found that the dry lenses (especially l0x and 40x) are best cleaned with styrofoam. We will provide each lab with white styrofoam peanuts, the kind used by shippers to pack fragile equipment. You simply break the styrofoam in two and use the freshly exposed fracture face to gently clean your objectives. These surfaces will absorb oil and clean the lens quite nicely if you use a few clean areas of styrofoam. It should not be necessary to use lens cleaner with this method. Do not use the styrofoam method on eyepieces. Finally, you should never have to clean the lenses or prisms in the interior of your microscope. If you think you have a problem here, call it to the attention of your lab instructor. It shouldnt even be necessary to clean the inner surface of the lens on your eyepiece, though you may have an occasion to clean the outer surface of your eyepiece lens. If so, first remove the sliding eye cup from the ocular. The cup (more accurately, tube) will not come completely off until you line up a pin with a keyway - if you twist as you pull, the eye shade will come off. Naturally, you will use lens tissue on this lens as well and if absolutely necessary your tissue may be dampened with a small amount of lens cleaner. NEVER use the air jets on the lab benches to blow dirt out of a lens - these air lines are full of oil and will inevitably make matters worse.

Illumination of the Light Microscope

Improper illumination of a microscope generally causes the greatest amount of difficulty encountered in the use of this instrument (dirty lenses run a close second). In order to properly illuminate a microscope, it is necessary to understand that the function of a condenser is to focus the light on the specimen in such a manner that the specimen acts as the source of illumination. The iris diaphragm of the condenser serves to eliminate stray light, not to reduce intensity! The Olympus CH is relatively noncritical in its illumination, but the following procedure will insure that you are getting the best possible image: 1. Focus on a specimen with the 4x l0x or 40x objective. 2. Focus the condenser on the light source. Focus the condenser up and down until you see the sharpest possible image of the ground

22

THE MICROSCOPE

glass diffusing plate (in the light source) superimposed on your focused specimen. When you have accomplished this you should see a grainy field over your specimen. This will be easy to see if you close the condenser diaphragm as far as it goes. While this is the ideal condenser focus you will want to eliminate the grainy field by defocusing your condenser above its focal point until the grain disappears. 3. Adust the condenser diaphragm. Now open the condenser diaphragm wide open. From its open position, slowly close the condenser diaphragm until it just begins to darken the field. You might want to check your adjustment by removing one of the eyepieces, looking down the tube and insuring that the diaphragm is just beginning to encroach upon the disk of light at the back of the objective. To insure maximum resolution, this procedure should be repeated for each objective as the required adjustments are slightly different. This is especially true for the condenser diaphragm setting. For special purposes you may wish to close the condenser diaphragm more than usual to increase contrast. This will result in some loss of resolution, but can actually help to visualize low contrast features of the specimen such as fine fibers. To appreciate this effect, examine your slide of heart muscle (l6A) with the condenser diaphragm wide open. This will produce light flare that degrades specimen contrast, making it difficult to see the striations and intercalated disks of this tissue. Now slowly close the diaphragm. You will notice an increase in contrast that makes the muscle striations and intercalated disks quite easy to see.

The Use of the Oil Immersion Objective

The 100x objective, and only this objective, must be used with immersion oil. This objective is clearly marked with a black ring around it. Failure to use immersion oil with this objective will result in substantially less resolution than with the dry 40x objective. Never begin studying a slide with the 100x objective. The small field of view of this lens will make the location of areas of interest in your slide very difficult. Always start your study with a lower power objective and work your way up in power as you focus your attention on details of interest. If you find that the magnification and resolution of the high dry lens (40x objective) is inadequate for resolving the required detail in a particular field, then, and only then, go to oil. Assuming you have a field of interest in focus under the 40x objective, check to see that your pre-focusing lever is set so as to prevent further upward travel of the stage with the coarse focus. This lever is just medial to the coarse focusing knob on the side opposite the light switch. To set this lever, simply loosen it and retighten it. When correctly set, this will allow you to quickly find focus under oil without risk of running the 100x objective into the coverglass. The condenser should now be focused at its uppermost position. Lower the stage as far as it will go with the coarse focus and turn the lens turret so that the objective lenses are out of the way for the application of the oil to the slide. Then, carefully apply one small drop of oil to the coverslip over the area of interest illuminated by the condenser. Try to avoid making bubbles in the oil as this can greatly reduce resolution. Turn the turret to put the 100x objective in viewing position and slowly focus the stage up with the coarse focus until it comes to a stop at the position previously set by your pre-focus lever. Look through the eyepieces and cautiously make any necessary fine focusing adjustments with the fine focusing knob to get the sharpest image. Finally, remove an eyepiece and look down the body tube of the microscope. Adjust the

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THE MICROSCOPE

condenser diaphragm so that it is just outside the field of the objective aperture. If you have any bubbles in the oil, they will be easily seen (with the eyepiece removed) as refractile spheres. Noticeable bubbles should be removed from the oil as they can seriously degrade resolution. Bubbles can be removed by lowering the stage and gently sweeping the surface of the oil droplet on both the slide and the objective with a wisp of lens tissue. It is important to clean the oil off the objective before you put the scope away (explained in next section). Also, wipe the oil off the slide unless it is a blood smear with no coverslip.

The Dual-View Teaching Head

Two students sharing a set of slides will typically share a single dual-view teaching head. This equipment consists of a dual body-tube (looks like rectangular boxes on either end of a 10" long tube), a special binocular head with dual focusing eyepieces, an Olympus electrical transformer (model TDO) and a pointer light/socket assembly with electrical cord (may already be attached to the dual body-tube). Remove any plastic covers from the dual body-tube assembly and save them with the bag or box for repacking when you turn it in. Install the dual body-tube as follows: 1. 2. Remove the binocular head from one of your microscopes by loosening the large knurled knob. Carefully place the end of the dual-body tube with the protruding black "joy-sticks" on the opening where you removed the binocular head (note that the silver label next to the longer black joy-stick must be right side up for this assembly to fit properly). Orient the tube so that it extends out over the arm of the microscope (the part you grab when you pick up the microscope) and tighten the knurled metal knob to attach the dual-body tube to the microscope (you may need to tilt the assembly to get it seated properly before you tighten the knob). Place the binocular head you removed (it should have only one focusing eyepiece) on top of the dual-body tube and tighten the knob on the dual-body tube to secure the head in place. Slide the small beige plastic "kick-stand" on the dual-body tube so that it wedges between the tube and the microscope arm (this is important because it relieves strain on the body tube). We discourage orienting the dual-body tube in a direction that does not permit this support. Attach the special binocular head with two focussing eyepieces to the other end of the dual-body tube. Insert the bulb/socket assembly into the hole on the side of the dual-body tube (at the end with the black joy-stick) and plug the other end of the wire into the back of the transformer.

3.

4.

5. 6.

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THE MICROSCOPE

Proper Use of the dual teaching head 1. Place the scope with teaching head either on one of the center tables or on the pull-out "bread board" between adjacent carrels. The binocular heads should ideally be facing away from one another so that both observers are face to face (this will ensure that each observer sees the same field in the same orientation). The host (person operating the controls on the side of the tube to which the microscope is directly attached) should focus on a slide with the nonfocusing eyepiece then adjust the focusing eyepiece on the same field. The partner should then focus both eyepieces on the other end of the dual-body tube so that this same field is in focus (no further adjustment should be necessary by the partner as the host focuses on slides). The pointer transformer should be plugged in and adjusted to low, medium or high light intensity as desired. The pointer should be visible to both host and partner in the same orientation. The color of the pointer can be changed between red and green using the black plastic slide near the long joy-stick. If the light does not light, first check that the transformer is plugged in. If the pointer still doesn't light up when you move the joy-stick about, then pull the pointer light out of its socket (follow the wire to the microscope) after loosening the set screw on the light socket. New lamps are available in the supply room (see your instructor). Both host and partner can move the pointer. The host pointer is the longer of the two black joy-sticks. The partner may use the short joy-stick that protrudes at an angle from the host end of the dual-body tube. Both should find all controls, including the main focus and mechanical stage, to be easily accessible. To avoid wear and damage, leave the dual viewing apparatus on the microscope throughout the semester (unless you do not wish to use it all). You will find that the microscope with its teaching head will fit in your carrel locker if you merely turn the two binocular heads 180 degrees so that their eyepieces are close to each other.

2.

3.

4.

5.

6.

7.

25

The Cell
Histology is the study of tissues, but no intelligent study of tissues can be made without at least a basic knowledge of the cells of which tissues are comprised. Although there are many types of cells unique to each organ (and these will be considered as the various organ systems are studied), you will benefit at this stage by a brief consideration of the structure of cells in general. You will be studying cells as imaged in the light microscope, as well as in the transmission and scanning electron microscopes. In most cases you will be looking at sections through cells and tissues; however, there are a few exceptions. Slide 57B , for example, contains whole HeLa cells in culture. Scanning electron micrographs also generally show whole cells rather than sections. It is not always easy for a beginning student in histology to discern even such basic structures as the nucleus, nucleolus and cytoplasm. This may be a good place for you to start. Pick any slide from your slide boxes and first familiarize yourself with what iscellular and what is extracellular, (i.e., connective tissue and ground substance), and then finally what is nucleus, nucleolus (not always visible), and cytoplasm. Look through the electron micrographs available to you (including, of course, your textbook) and study these same basic features. Dont dismiss this as too obvious until you have tried a variety of samples.

Basophilic vs. Acidophilic

Throughout this course cells will be frequently described as beingbasophilic or acidophilic with regard to the cytoplasm as stained by hematoxylin and eosin respectively. If you have not already done so, you might read through the sections of this lab guide on Histologic Stains, particularly under hematoxylin and eosin (H & E). Generally, acidic groups bind basic stains (like hematoxylin) and basic groups bind acidic stains (like eosin). Cellular basophilia usually indicates the presence of the RNA-containingfree ribosomes or ribosomes in association with endoplasmic reticulum (rough endoplasmic reticulum). A high ribosome content generally typifies a cell that is actively engaged in protein synthesis. Look at the section of pancreas on slide l2B. Pancreatic acinar cells often appear to be arranged in circular groups, much like the pieces of a pie. These cells exhibit basophilia, i.e., the acidic RNA stains with the basic hematoxylin dye (bluish) in the region where ribosomes are concentrated around and below the nucleus. The area of the cell oriented toward the center of the circle is filled with secretion granules; these granules are eosinophilic. Other cells associated with the duct system of this gland will be distinctly nonbasophilic and are almost colorless. If rough endoplasmic reticulum predominates, this usually indicates a cell which is synthesizing proteins for export out of the cell; conversely, if free ribosomes (polyribosomes in the cytoplasm but not associated with membranes of endoplasmic reticulum) predominate, the proteins are generally kept within the cell. Look through the electron micrograph (EM) collection and determine for yourself if this is a good rule of thumb. See if you can find an EM of a plasma cell in your textbook. This is a basophilic cell; are the ribosomes free or bound to the endoplasmic reticulum? Does this fit our rule of thumb? Why? Ribosomes, by the way, are often confused with glycogen in electron micrographs; in particular polyribosomes are often confused with alpha glycogen. Your EM collection will provide examples of cells with glycogen and polyribosomes; learn to distinguish these by differences in size, staining, and aggregation of the particles. It should be pointed out that the nucleolus and the chromatin in the nucleus generally are basophilic. Why? Some extracellular material is also basophilic (look at the cartilage models of bones in the foot of a human embryo on slide 57A). These cartilaginous bones stain so blue with the hematoxylin that you can see them clearly on the slide with the unaided eye. This

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THE CELL

is due to the glycosaminoglycans in cartilage which contain strongly acidic sulfate groups.

Membranous Structures in the Cell

When you look at EMs, you will soon realize that nearly everything you see consists of membranes, filaments or granules. Most of the organelles of the cell are largely membranous structures. Membranes as such are not visible in the light microscope, being generally in the range of 7-l0 nm thick whereas the resolution of the light microscope is only 200 nm. You can see where the border of the nucleus is in the light microscope, but you cannot see the membranes of the nuclear envelope. Study the nuclear envelope (a double layer of membranes) in EMs. Can you find nuclear pores? Notice that the chromatin, which generally tends to adhere to the nucleoplasmic side of the nuclear envelope, is usually lacking wherever there is a nuclear pore. The nuclear membrane appears to fold back on itself around each nuclear pore. Ribosomes are usually attached to the cytoplasmic side of the nuclear envelope, but not to the nucleoplasmic side. Occasionally you may see a continuity of the endoplasmic reticulum with the nuclear envelope. Nuclear pores probably function as channels for the passage of materials such as messenger RNA, ribosomal subunits, etc., between nucleus and cytoplasm during interphase (i.e., when the cell is not dividing). You will notice that in EMs of any membranous structure, the membrane may vary in sharpness and apparent thickness from place to place. This is due to the plane of section; bear in mind that while the membrane is only about l0 nm thick, the thickness of the section you are looking at is generally between 60 and l00 nm thick. If a membrane is oblique to the plane of the section, it will appear thicker and very blurred. Look through the EMs; you will find abundant examples of this in the plasma membrane, nuclear envelope and endoplasmic reticulum membrane. Occasionally, you will find a portion of membrane that is cut virtually parallel to the plane of section and appears as a broad sheet. The cell membrane itself (plasma membrane) has a number of interesting specializations that you will encounter in your studies. If you look at EMs of closely packed cells, you will notice that their plasma membranes rarely actually touch one another. There is generally an intervening space no less than l5-20 nm. This space results at least in part from the presence of glycoproteins and polysaccharides called the glycocalyx. Such coats may have antigenic properties, as well as other roles in relation to the micro-environment around each cell. In some cells (i.e., on the apical ends of absorptive cells of the intestine), this glycocalyx is very highly developed. The glycocalyx is part of the plasma membrane, in contrast to a layer of basal lamina (to be described later) which is not considered to be an integral structure of the plasmalemma.

Membrane Junctions
The cells of a tissue, particularly those of an epithelium, tend to adhere to one another, both selectively and tenaciously. While the glycocalyx is not easily seen, there are visible attachment structures including desmosomes, gapjunctions, tight junctions, and apical junctional complexes (combination of different junctions). Only at tight junctions do the cell membranes actually touch, often becoming fused in those areas. Your EM collection of cell organelles will help you get started in discerning these junctional structures. These specialized cell junctions are either at the limit of or below the resolution of the light microscope, though evidence of their existence may be seen in some epithelial tissues. In the epidermis of the skin, for example, there is a layer of cells called the spinous layer (so called because of the spiny shape of the epidermal cells).

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THE CELL

The spiny shape results from shrinkage artifact in the histological technique. When the individual cells shrink, they continue to hang on to one another at many points on each cell by means of the desmosomes, thereby producing the spiny appearance. Look at the epidermis on slide 7A with either your 40x or 100x (oil) objective.

Microvilli
Many cells have, to a greater or lesser extent, small finger-like membrane bound cytoplasmic processes extending from the surface of the cell which apparently serve to amplify its surface area. These structures, called microvilli, are particularly abundant on absorptive cells such as those in the small intestine and proximal convoluted tubules of the kidney. Very long specialized microvilli can be seen lining the lumen of the epididymis of the testis (54B). Look at the slide with the unaided eye; you will see a large blue circular field that is the testis proper, and on one side of this you will see a smaller red circular field. It is this field that will have the cross sections of the ducts, which are lined with long microvilli. These microvilli of the epididymis are called stereocilia (non-motile cilia), but are actually not cilia.

Cilia
True cilia are motile structures that can be seen lining the irregular lumen of the oviduct (88B). Cross sections of cilia in the EMs will be particularly useful in studying the interesting fine structure of the cilium. Unlike microvilli, cilia have a core of microtubules in a 9+2 arrangement. Flagella are similar in structure to cilia, but they are longer and usually there is no more than one per cell. The best example of flagella can be found on the testis slide (54B). The lumina of the epididymis contain flagellated spermatozoa. Flagella also have the 9+2 microtubular arrangement. Curiously, many epithelial cells have been found to bear a single flagellum.

Endoplasmic Reticulum
There are two types of endoplasmic reticulum (ER): (l) rough ER and (2) smooth ER. The ER can be considered a series of potentially interconnected labyrinthine channels running throughout the cytoplasm. The rough ER is studded with polyribosomes, which you should be able to see in both cross sections and glancing surface views in appropriate EMs. These surface views often show numerous small clusters of polyribosomes. The ribosomes of each cluster are in a coiled linear arrangement that is related to one molecule of mRNA, and allows the simultaneous production of several polypeptides from one messenger RNA. The flocculentappearing content of the rough ER represents synthesized protein that has gained access to the cisternae of the ER, and from here may be carried to the Golgi complex for further processing and/or packaging. Cells in a very active state of protein synthesis will often have greatly distended rough ER cisternae. Rough ER often has a tendency to occur in packed parallel arrays, while smooth ER has a much more irregular arrangement. Look for this in EMs. Nissl substance in the neuron is a particularly striking example of this. Though most cells have a mixture of rough and smooth ER, those cells producing largely proteins will have predominantly rough ER, whereas those cells producing steroids and with high lipid and cholesterol metabolism will have largely smooth ER. Smooth ER lacks ribosomes, rarely occurs in the long flattened cisternae typical of rough ER, and is more likely to be found forming continuous and discontinuous contacts with the Golgi complex. You will find several examples of smooth (agranular) ER in your EM collection.

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THE CELL

Golgi
Identification of the membranous organelle known as the Golgi complex frequently gives the beginning student trouble. The Golgi complex is big enough to be seen easily in the light microscope, but unfortunately it resists being stained with nearly every dye and can be demonstrated convincingly only with a special silver stain. One can, with experience, take advantage of its chromophobic nature and discern the Golgi zone as a clear or negative image in many stained sections of cells. The Golgi will appear as a clear zone next to the nucleus. Even in the EM it takes some experience to quickly find the Golgi complex. The EM collections and your textbook should be helpful in this regard. The Golgi resembles smooth ER, but unlike the smooth ER, its agranular membranous cisternae are stacked in closely associated layers. These stacks are often curved and close to the nucleus, and occasionally, even in a pocket indented into the side of the nucleus. Many cells, particularly those of a simple epithelium, have a polarity that is often indicated by the position of the Golgi in the cell. The Golgi apparatus will usually be on the side of the nucleus toward that end of the cell from which the secretory products leave.

Mitochondria
One of the most striking of the membranous organelles of the cell is the mitochondrion. Though mitochondria are within the resolution of the light microscope, they are not usually stained in common histological preparations. The iron hematoxylin-stained section of stomach (80A) does show mitochondria as small granular or rod-shaped structures in the parietal cells. Of the two sections on slide 80A, look at the black one. Look for cells arranged in rows. You will see two types of cells in these rows: white foamy-looking cells and grey-looking cells. The latter are the parietal cells containing the black-staining mitochondria. If the cytoplasm contains a high proportion of mitochondria and few ribosomes, the cytoplasm often stains with an acid dye primarily because of the mitochondria. In the EMs, mitochondria can be seen to have a highly characteristic structural organization and frequently have a characteristic size, morphology and location for a particular tissue. This fact will help you later to identify tissue in EMs, as well as understand the cellular physiology of the tissues. For now, it will be helpful to look at a wide variety of EMs and learn to identify mitochondria, paying particular attention to their variations in morphology. Whereas most mitochondria have lamellar-shaped cristae, those of steroid-secreting cells often have tubular cristae; your EM collection has an example of such a cell. Cardiac muscle mitochondria often have cristae that are angular. In some cells (e.g., liver), the matrix of the mitochondrion is very dark. In many cells most mitochondria will occupy a particular position in the cell. For example, in some secretory and absorptive cells, the mitochondria are sandwiched between the folds of a greatly infolded basal cell membrane. You will encounter examples of this when you study the proximal tubules of the kidney and the striated ducts of salivary glands. You might want to glance at these in your textbook now.

Vesicles and vacuoles

In EMs most cells contain a variety of essentially round vacuoles or vesicles. These may form by an inpocketing of the plasma membrane or by budding off from other membranous organelles within the cell. Large invaginations of the surface of the cell often result in the uptake of particulate matter from the exterior (phagocytosis). In many cases, cytoplasmic processes (called pseudopodia) extend out from the cell surface and surround particulate material in the

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THE CELL

extracellular environment. The vacuoles formed as a result of phagocytosis are called phagosomes. Invaginations at the surface of cells that imbibe fluid, participate in a type of endocytosis called pinocytosis or micropinocytosis, thereby producing pinocytotic vesicles. The reverse of this process of pinocytosis, where vesicles fuse with the cell membrane and lose their contents to the exterior of the cell, is called exocytosis. Such a process takes place in all secretory cells in which the secretory product resides in vesicles which are derived from the Golgi complex. A special type of micropinocytosis involves coated vesicles. The area of the invaginating cell membrane is unusually thick, due to additional material (clathrin) on the cytoplasmic side of the cell membrane. Proteins in the exterior environment of the cell appear to selectively bind to the cell membrane at these coated portions of the cell membrane, and are subsequently taken into the cell by micropinocytosis. Examples of coated vesicles can be found in the EM collection.

Lysosomes
The lysosome is a membrane-bounded vesicle in the cell which contains a number of hydrolytic enzymes active at an acid pH. This organelle has received considerable attention in recent years as a result of the interest in storage diseases. Lysosomes are electron dense, membranebounded structures about the size of the narrowest dimension of a mitochondrion. They originate as separate structures in the region of the Golgi complex. Lysosomes can fuse with the phagosomes to form a phagolysosome or, as it is often called, a digestive vacuole. Lysosomes can also fuse with vacuoles containing organelles of the same cell, an autolysosome or autophagic vacuole. Both phagolysosomes and autolysosomes show evidence of membranous debris within them. In this way, the cell (by means of lysosomes) can digest both materials brought into the cell from outside as well as endogenous materials and effete organelles within the cell. Many examples of lysosomes and digestive vacuoles will be found in your study of EMs. Occasionally, cells will accumulate indigestible material within a vacuole, which is then called a residual body. This indigestible material often includes the so called myelin figures, which look like rolled up membranes. Granules in neutrophils and eosinophils of the blood have been shown to be lysosomes. Those of the acidophilic leukocyte in particular are of sufficient size, number and staining quality to be easily seen with the light microscope.

Filamentous and Tubular Organelles

All cells contain various classes of fibrillar organelles, but you may not always see them in a particular plane of section. The three main types in order of increasing diameter aremicrofilaments, intermediate filaments, and microtubules. These organelles are of indefinite length and, in general, appear to participate in various types of mechanical activity within cells (such as locomotion, particle transport, cytokinesis, chromosome movement and structural rigidity). Microfilaments are about 5 nm in diameter, and often occur in skeins or bundles near the plasmalemma. They are similar, if not identical, to the actin filaments found in muscle cells. The drug cytochalasin B interferes with the operation of several mechanochemical processes that appear to be dependent upon microfilaments. Cells treated with cytochalasin B fail to undergo cytokinesis, and cell locomotion is reversibly inhibited. Intermediate filaments are about l0 nm in diameter and usually occur singly or in loose tangles. They are prominent in neurons (neurofilaments) but can also be found in most other cells. Their composition in cells of different types is being investigated. Although they may appear similar

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THE CELL

morphologically in different cell types, their chemical composition varies. Their function includes a mechanical-structural role as is indicated by the great abundance in the epidermis. Microtubules are tubular structures of indefinite length, are remarkably straight, and measure about 24 nm in diameter. Microtubules are generally surrounded by a zone of low electron density, free of ribosomes and other organelles. Microtubules are composed of the protein, tubulin, a dimer with a MW of ll0,000. The tubulin monomers (MW, 55,000) are about 5 nm in diameter. Thirteen subunits are arranged in a ring to form the cross section of the microtubule. Drugs such as colchicine and vinblastine block many microtubule-dependent processes by binding to the tubulin dimers and preventing their assembly into microtubules. In addition to their role in the movement of cilia and flagella, microtubules have been implicated in a variety of other mechanical processes such as chromosome movement, transport of cytoplasmic organelles and axoplasmic flow. Microtubules also serve a mechanical function as a sort of cytoskeleton. The maintenance of the biconcave shape of some red blood cells, for example, is due in part to a ring of microtubules just under the cell membrane. Examples of microfilaments and microtubules will be found in your EM collection.

Centrioles
The centriole is an organelle of the cell that consists primarily of microtubules. This curious organelle is discussed at length in your textbook. A fortuitous plane of section is required to find one of these in an EM, as usually, there are not more than two per cell. Your EM collection includes a section of a centriole. Centrioles that are related to the bases of cilia are called basal bodies. Some rather oblique sections of basal bodies may be found in your EM collection. Return to slide 88B,where the basal bodies may be seen as pink staining material just beneath the apical cell surface.

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Epithelial Tissues
In this laboratory, as in subsequent ones, be sure to examine the slide first by eye to gain familiarity with the orientation of the tissue. Then scan the tissue under the lower magnification lenses. Only when you have found an area of interest should you proceed to the oil immersion lens, and then only if really necessary. If you are not familiar with the use of a light microscope, study the section on Illumination of the Light Microscope included in this laboratory manual. An epithelium is an avascular sheet of contiguous cells (with very little intercellular substance), resting upon an extracellular basement membrane. An epithelium may consist of a single layer of cells (simple epithelium), or may have two or more layers of cells (stratified epithelium). An oblique section through an epithelium may give the impression that there are more layers than actually exist. Also, the presence of lymphocytes among columnar cells often gives an erroneous impression of a pseudostratified rather than simple epithelium.

Simple Epithelia
Simple epithelia can be divided into four basic types for which there are many representatives in your slide collection. It should be noted that there is continuous gradation from type to type.

Simple Squamous Epithelium

This type of epithelium comprises a single layer of flat cells in which the nuclei form a prominent bulge in the mid-portion of the free surface of each cell. Mesothelium is the name given to the type of simple squamous epithelium lining the body cavities. Peritoneum consists of mesothelium and a thin layer of connective tissue. Slide 8lA gives a surface view of an unsectioned whole mounted sheet of mesentery with mesothelium on both surfaces of a connective tissue layer. In favorable areas the cells are outlined by a silver deposit. One of the commonest forms of simple squamous epithelium is the type known as endothelium which lines the heart, blood vessels and lymphatics (l00A). You should be able to find blood vessels on just about any slide in the collection - - you might try your hand at identifying the endothelium on a few random slides.

Simple Cuboidal Epithelia

Cuboidal - height of each cell is approximately equivalent to its width. In slide l3B,the best example is found surrounding the pink stained colloid in the thyroid follicles. Cells may vary between cuboidal and columnar, depending upon the activity of the gland. In slides 44B and 46B, cross sectioned kidney tubules provide an opportunity to compare squamous, cuboidal and tall cuboidal to columnar epithelium. Slide 46B is stained with PAS, which shows the basement membrane (in red) surrounding each tubule. Study the parietal (or outer) layer of Bowmans capsule as an example of squamous epithelium.

Simple Columnar epithelia

In this type of epithelium, cells are taller than wide and often prismatic in shape. Frequently, there may be a specialization of the apical cell surface where one may find cilia or microvilli. The simple columnar absorptive epithelium (which lines the greatly folded inner surface of the intestine), can be seen on slide 86A. Due to the many different planes of section, some areas may appear multi-layered. In slide 88B,we see an example of the ciliated simple columnar epitheliumlining the uterine tube (oviduct). Stop down the condenser diaphragm to increase contrast in order

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EPITHELIAL TISSUES

to see cilia. Basal bodies may be seen in some areas of the epithelium.

Pseudostratified Epithelia
Comprises two or more layers of nuclei in simple columnar epithelium, and gives the appearance of stratified epithelium. For example, in slide 36B the bases of all cells lining the trachea touch the basement membrane, but not all the cells reach the surface. These cells are also ciliated.

Stratified Epithelia
This type of epithelium is characterized by two or more layers of cells, and subdivided into the squamous, cuboidal or columnar types (depending upon the shape of the surface cells as seen in cross section).

Stratified Squamous
In most cases, the lower layers of stratified squamous epithelium are more cuboidal, or even columnar as in skin (7A). In the case of skin, the uppermost squamous cells are cornified and dead. Note the gradual transition from columnar cells in the lower most basal layer to squamous in the superficial cornified layer. The lining of the esophagus (75A) is similar, even though the superficial squamous cells are living and contain nuclei. Compare lining epithelium of esophagus and trachea. How is each particularly suited for its function? Slide 83B of vaginal epithelium is an example of stratified squamous epithelium that may prove confusing to you. Here the superficial cells are greatly swollen, and only lightly stained due to the dissolution of great quantities of glycogen that had occupied these cells.

Stratified Cuboidal
The ducts of sweat glands in the skin consist of a two-layered cuboidal epithelium. Slide 7A has many highly coiled sweat glands deep in the dermis, near a layer of skeletal muscle. The duct is that portion of the gland extending from the epidermis to the highly coiled secretory portion deep in the dermis. The relatively straight sweat gland ducts will be occasionally observed in cross section when their stratified (i.e., two-layer) nature will be more clearly seen.

Stratified Columnar
Examples of both stratified cuboidal and stratified columnar may be found in the ducts of salivary glands (7lA). Stratified cuboidal and stratified columnar epithelium typically occur as a transition between a simple epithelium and a stratified squamous epithelium. Does this rule of thumb fit the examples you have observed in slides 7A and 71A? Where else in the body might you expect to find such a transition between a simple epithelium and stratified squamous epithelium?

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EPITHELIAL TISSUES

Transitional
This epithelium is limited to the urinary tract and is especially suited for the vast surface expansion that develops in bladder, ureters and upper urethra. When the bladder is relaxed (67B), the epithelium appears multilayered and resembles stratified squamous, but the diagnostic superficial cells (calledfacetcells) are more biscuit-shaped. The facet cells are large polyploid cells that are highly modified to increase their membrane surface, while retaining their ability to serve as an ionic barrier. Several investigators have reported that all of the cells of transitional epithelium (including the facet cells) are in contact with basement membrane and thus, should be categorized as a type of simple epithelium. While you are on slide 67B, note the mesothelium (simple squamous) that covers the border of the bladder opposite the transitional epithelium. What is this layer called? In the distended bladder the superficial cells are flatter (5lB). There may be more than one nucleus per epithelial cell.

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Blood
Wrights Stained Smear of Peripheral Blood
Preparation of slide: 1. Clean slide with 95% alcohol. Slide must be completely dry. 2. Clean finger with 70% alcohol and cotton and allow to dry. 3. Puncture finger with a clean stylet. Wipe away the first drops. 4. Take the clean slide and gently touch it to a small drop of blood so that the blood is l-2 mm from right end of slide. Do not touch the slide with this finger. Spread drop right away. 5. Place slide on flat surface and hold edges of slide with fingers of your left hand. Place one end of the spreader slide to the left of the drop of blood and pull it to the right until it just comes into contact with the blood. Allow the blood to spread along the end of the spreader slide. Then, with a firm, steady movement and holding the spreader slide at 30 angle, push the spreader slide to the left. All of this must be done rapidly, before the blood clots. It is important to realize that you are pulling or dragging the blood across the slide -- not pushing it. It is important that the edge of the slide not pass over the top of the blood, or it will damage the cells. 6. The thickness of the smear will depend upon (l) the angle of spreader slide and (2) the rapidity of the spreading movement. The smaller the angle of inclination, the thinner the smear will be. The film is thicker if the spreading is done rapidly. Thinner smears are preferable. 7. Air-dry before staining. Staining Method: 1. Place slide on a rack with the blood side up. 2. Add just enough Wrights stain to cover the surface and allow to stand from l to 3 minutes, depending on the stain. 3. Add an equal amount of water and blow on surface to mix until a greenish metallic sheen appears on the top of the stain. Allow to stand 4 or 5 minutes. Do not allow this to dry out at the edges; add more buffer if necessary. 4. Rinse with distilled water. Be sure to flood the stain from the preparation,

Proper position and angle of spreader slide relative to blood specimen.

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BLOOD

because pouring it off will cause a precipitate to be found on the smear. Once there, it cannot be washed off. 5. Drain with the thin end of the smear pointed upward. 6. Air dry. Remember, water and immersion oil to not mix.

Examination
Using the low and high dry lenses, find a relatively thin area of the smear (i.e., where the red cells are plentiful but not touching or aggregated). Then switch to the oil immersion objective. The details necessary for adequate assessment of the formed elements in the peripheral blood can only be seen at this magnification. The oil drop should be placed directly on the smear. No coverslip is needed. Begin by examining the red cells. Normal erythrocytes appear as uniformly sized, smooth contoured discs (7.2 to 7.9 m in diameter), with a rim of pink stain (due to hemoglobin) and a clear central area. The clear area represents the thin central portion of the biconcave disc, which contains less stained hemoglobin; it normally occupies just a little less than half the cell diameter. Abnormalities of erythrocytes . Size (larger than normal, smaller than normal, or nonuniformity) Shape Staining properties reflecting one or more of the following: Shape changes Altered hemoglobin concentration and distribution within the cell Unusual elements, such as RNA in reticulocytes Inclusions

These abnormalities can contribute to the diagnosis of many disease states. Examples of many such abnormalities are shown in the Upjohn monograph. It should be noted that true abnormalities of these red cell parameters typically do not affect 90-l00% of the cells, and those cells that are abnormal are found roughly evenly distributed on a slide. If you find red cells uniformly deviant in a field, check other fields to rule out artifact. Artifacts may appear in some part of nearly every blood film. Common artifacts in blood smears Precipitated stain

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BLOOD

Red cell vacuolation, usually central Loss of central pallor due to overstaining Irregularly occurring abnormally shaped red cells.

Platelets
Next, look for platelets (thrombocytes). These tend to occur in clumps, since contact with glass triggers aggregation. They appear as small blue bodies ( diameter of RBC), with striking red or purple granules. The granules are usually central. There should be roughly one platelet per l0-20 RBC. The platelet content is judged to be adequate or not by this criterion in the routine examination of a blood smear; an actual platelet count is not routinely done, unlike RBC and WBC counts. If you look carefully for the presence of the granules and the surrounding blue cytoplasm, you are not likely to mistake precipitated stain for platelets.

Leukocytes
Leukocytes are often found towards the edge of the blood film. Begin by identifying examples of each type of leukocyte. You may not find a basophil. Neutrophils are the easiest to identify. Their granules are fine and inconspicuously stained against a background of pink cytoplasm. The nuclear morphology is not obscured by overlying cytoplasmic granules. Mature neutrophils have highly segmented nuclei (2-3 lobes) while the bands have horseshoe-shaped nuclei. The drumstick in neutrophils of females represents the shut off X chromosome. Not finding it after viewing l00-200 neutrophils is no denial or confirmation of your phenotypic sex. Sometimes, when the entire blood film is overstained, the neutrophil granules may look dark brown or black like basophil granules. The small size of the granules, three lobed nuclei, evidence of overstaing (in RBCs, platelets and neutrophil cytoplasm) and excessively high "basophil" counts, should tip you against erroneous identification.

Eosinophils and Basophils

Eosinophils and basophils contain coarse granules, stained bright red-orange and brown-black respectively. The granules tend to fill up the cytoplasm and often overlie the nucleus, obscuring their bilobed morphology. When you encounter these cells, there is generally no doubt in your minds as to their identity.

Lymphocytes and Monocytes

The most difficult distinction you will have to make is between lymphocytes and monocytes. Start by finding the classical small round lymphocyte, with its nucleus filling up most of the volume and only a thin rim of blue cytoplasm. The chromatin is distributed in coarse masses, which confers the typical clumpy dark purple staining of the nucleus. Other lymphocytes may have more cytoplasm, but the nuclear and cytoplasmic staining will be generally similar to that of this clearly identifiable small lymphocyte. The large lymphocyte (infrequent in blood) has a relatively less clumped chromatin pattern, but the cytoplasmic features can often differentiate it from a monocyte. The monocyte has more abundant cytoplasm of a grayish-blue cast, and the nucleus has a relatively finely-dispersed chromatin pattern (which is stained red rather than

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BLOOD

deep purple). The presence of cytoplasmic vacuoles usually suggests a monocyte, if the nuclear and cytoplasmic appearance fit. A truly folded nucleus identifies a monocyte, but both cell types can show nuclear indentation. Both cell types can have cytoplasmic granules. Monocyte granules are numerous, purple or lilac, and fine (hence hard to see), while lymphocyte granules are red (azurophilic), few, and coarser.

White Blood Cell Differential

Now that you can recognize each cell type, classify l00-200 successive WBCs and record their numbers in the table provided at the end of this chapter. This means that every WBC in each randomly selected field (in a good area) must be included in the count. Do not include broken cells, where either the cytoplasmic or the nuclear membrane has ruptured. Such a count is called the differential count. An increase in the relative percentage of particular WBC types is characteristic of many disease categories. The specific examination of blood cells is routinely done with special stains (Wrights and Giemsa). You have performed such an examination. You also need to be able to identify these
White Blood Cell Differential Distribution
Cell Type Size: Microns
10-13 9-12 10-14 8-10 6-10 12-15

PerCent

Neutrophilic bands Neutrophils Eosinophils Basophils Lymphocytes Monocytes (saddle nucleus) Erythrocytes

2-10 60-70 1-3 0.5-1 20-40 2-10

7.5

---

cells when they are located within tissues you will be studying under routine H & E staining. For all practical purposes, platelets cant be seen, and RBCs simply appear as eosinophilic spots of appropriate size. They can be seen edge on, in stacks or singly, and their shapes can be deformed when squeezed by other structures. Of the WBCs, only lymphocytes and neutrophils are seen with frequency. Monocytes are difficult to distinguish from lymphocytes, unless they show an unequivocal folded nuclear morphology or cytoplasmic vacuolization. Lymphocytes and neutrophils are easy to spot by their nuclear morphology, regardless of stain. Eosinophils retain the bright red stain of their granules under H & E; they differ from mast cells by their nuclear morphology. The similar granules of mast cells are displaced to the side of the nucleus, unlike the eosinophil granules. Look at slide 27A. Within the arterial lumen, you should be able to see several examples of lymphocytes and neutrophils among the RBCs.

39

BLOOD

Study questions on EMs.

l) How can an eosinophilic leukocyte be distinguished from the other granulocytes? 2) At the LM level, small lymphocytes do not show nucleoli. Does the EM suggest otherwise? 3) In what ways would you expect the EM of a large lymphocyte to differ from that of a small one? 4) The Golgi apparatus is situated in a characteristic site in all the WBCs. One of the EM pictures shows this location. Do you know the location?

Score 100 leucocytes by making counter marks in the table provided below. Assuming your blood sample is normal, your count should fall in the range indicated in the "expected" column. Your Differential Count Cell Type Score Expected

Neutrophilic bands Neutrophils Eosinophils Basophils Lymphocyte Monocytes

2-10 60-70 1-3 .5-1 20-40 2-10

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Fibrous Connective Tissue

Fibrous connective tissue is arbitrarily classified as either loose or dense. Loose connective tissue is found virtually throughout the body (serving as the excelsior of life), while dense connective tissue is more confined to the capsules, tendons, ligaments and dermis of the body. Both tissues are characterized by abundant collagen fibers and, usually, scattered elastic and reticular fibers. The difference is primarily one of packing density of the constituent fibers. Loose connective tissue is also much more cellular than dense connective tissue, and it is through the ground substance in the interstices of its fibers that water, salts, gases and various metabolites freely diffuse. Dense connective tissue serves more of a mechanical function than a diffusional one. Examine slide 8A of skin with your lowest power objective to appreciate the difference between dense and loose connective tissue. Just deep to the stratified squamous epithelium (epidermis) is a layer of eosinophilic loose connective tissue (papillary dermis) and deep to this is a thicker, denser and less cellular layer of irregular dense connective tissue (reticular dermis). Within the dermis you will see cross sections of numerous glands and hair follicles; all of these are invested in a layer of loose connective tissue. Can you think of any reasons for this mixture of dense and loose connective tissue? Deep to the dermis is a fatty layer (hypodermis) that is known as an adipose connective tissue, but is really a special case of loose connective tissue.

Loose Connective Tissue

Slide 39A is a whole mount of loose connective tissue (perhaps fascia) that has been teased out on a slide, and stained with H & E and Weigerts resorcin-fuchsin for elastin. The loosely tangled collagen fibers are eosinophilic. In contrast, the dark purple elastic fibers are straight and branching and appear to be under tension in this teased specimen. These elastic fibers do not stain well in the typical H & E section and, thus, are usually not obvious in sections. The tissue is very cellular, but cytological detail is not very good in a whole mount of this type. Slide 83B from the vagina provides an excellent opportunity to see the loose fibrous architecture of sectioned loose connective tissue. Look just deep to the stratified squamous epithelium. The rich cellularity of this connective tissue contrasts sharply with dense connective tissue. The most abundant cell is the fibroblast with its elongate nucleus and very elongate (though indistinct) cytoplasm. Numerouslymphocytes are also in evidence with small dark round nuclei and no visible cytoplasm. Plasma cells are much less common, but should be easy to identify. They are among the largest cells in the connective tissue, with a round to oval nucleus having a distinctive clock face chromatin pattern. The nucleus is typically eccentrically placed in an abundant basophilic cytoplasm. Mast cells are not numerous in this preparation, but stand out quite well with their distinctive small red cytoplasmic granules. The mast cell nucleus is similar to the plasma cell nucleus, but is usually more centrally-placed in the cell. Macrophages can be very large irregularly shaped cells, but are not always easy to identify, unless the cytoplasm contains ingested material which appears as irregular eosinophilic granules. Now look at 88A of small intestine. Loose connective tissue forms much of the wall of the gut; however, do not confuse the collagen with the brighter red staining smooth muscle cells in the gut wall. You should find all of the cell types previously mentioned. Mast cells are particularly abundant in the gut and often appear close to blood vessels. In many preparations (particularly paraffin sections), mast cell granules are often lost or dissolved in the common histological fixatives making them difficult to identify. In these 1.5 m thick plastic sections the granules have lost heparin, but have retained histamine and are thus eosinophilic.

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FIBROUS CONNECTIVE TISSUE

Reticular Connective Tissue

This is another special case of loose connective tissue. Reticular tissue is characteristic of lymphoid and hematopoietic organs and can be seen in slide 43A of the spleen (where the reticular fibers have been stained black with silver). Reticular fibers are a type of collagen and can only be identified in sections, with certainty, after special silver techniques.

Adipose Tissue
A specialized type of loose connective tissue that is widespread in the body. Plastic sections of unilocular (common/yellow) adipose tissue (100A) show a dense-packing of bubble-like fat cells (adipocytes) in a scant loose connective tissue stroma. The adipocytes consist of a large leached-out lipid droplet which has pushed aside the nucleus, and compressed the cytoplasm to a very thin periphery. Can you distinguish the adipocyte nuclei from nuclei of endothelial and fibroblast cells? The large framework into which the fat cells are packed consists of dense irregular connective tissue septa carrying muscular arteries, other blood vessels and myelinated nerves. A set of finer partitions extend out from these septa in the form of a rich network of capillaries and reticular fibers (not stained in this preparation).

Dense Connective Tissue

Dense connective tissue is of two types: irregular dense connective tissue and regular dense connective tissue. The former is found in the dermis and organ capsules; the latter in ligaments and tendons. In regular dense connective tissue, the collagen fibers are tightly packed and all are oriented in approximately the same direction; in irregular dense connective tissue, there often appears to be no special organization of the fibers (In fact, the fibers are often highly organized in irregular dense connective tissue as well, but are woven in a complex way that influences the important anisotropic biomechanical properties of the tissue).

Dense Regular Connective Tissue

In dense regular connective tissue (such as ligaments and tendons), the only cellular elements (fibroblasts), are highly flattened, lying in interrupted rows between the fibers. The cytoplasm is scanty and attenuated so that only the nuclei are visible. In longitudinal section, the nuclei are plate-like, whereas when they have been transversely sectioned they are oval. Examine plastic section 49A in which small amounts of cytoplasm may be seen adjacent to the nuclei of the tendon fibroblasts. Slide 10A shows a tendon in a musculotendinous junctions. Most of the tissue is muscle, with thin bands of tendon attached in several areas, especially at the tapering end of the tissue. The muscle is slightly pinker than the collagen, and the nuclei of muscle and dense regular connective tissue differ in shape. Try to visualize the cross striations of muscle by closing the iris diaphragm of the condenser. Compare this section with slide 12A of skeletal muscle. Ligaments can be seen connecting the bones of the knee joint in slide 60A (This slide is diagrammed in the section on Cartilage). You may find separating the different types of connective tissue difficult in this preparation until you gain some familiarity with cartilage,

43

FIBROUS CONNECTIVE TISSUE

fibrocartilage and bone.

Irregular Dense Connective Tissue

Examples of irregular dense connective tissue may be found in any slide of skin (1A) by examining the dermis, and in the capsule of the testis on slide 54B. This type of connective tissue is more cellular than regular dense connective tissue but much less cellular than loose connective tissue. There are two sections on most specimens of slide 1A. One section is stained with H & E and the other is stained with aldehyde-fuchsin, fast green and orange G. In the latter preparation, elastin fibers are stained purple by the aldehyde-fuchsin, collagen fibers are stained by the fast green (as usual) and also orange G (thus, collagen may be green or yellow). Comparing these two sections will show you how unnoticeable elastic fibers can be if they are not specially stained. The blood vessels have been injected with red carmine. Again, portions of the dermis are loose connective tissue but the bulk of the dermis is dense.

Embryonic Connective Tissue

This type of tissue (found in embryos), is also calledmesenchyme, and develops from mesoderm and neural crest ectoderm. Many examples of this type of tissue can be found in slide 55A of the fetal thorax. These stellate-shaped cells are enmeshed in very fine and scattered collagen fibers together with extensive ground substance. The mesenchymal cells are particularly evident in the developing kidney and just deep to the epidermis in this specimen. Mucous connective tissue is similar to mesenchyme, but the ground substance is more abundant and the fibrous component even less obvious. One of the few examples in the body is the Whartons jelly of the umbilical cord (slide 96B), which helps to keep the umbilical vessels from being pinched off.

Electron Micrographs
The fine structure of fibroblasts and collagen is illustrated in the notebook of electron micrographs, as is that of tissue macrophages. What are two striking characteristics of such a macrophage? Which peripheral blood granulocyte does the mast cell resemble? Osteocytes, osteoclasts, and osteoblasts are shown in a light photomicrograph included in the notebook.

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45

Cartilage
Hyaline Cartilage
Cartilage is generally classified by the amount and type of its fiber content. The most ubiquitous type is hyaline, in which the collagen and elastic fiber content of the matrix is insufficient to give it a fibrous appearance in ordinary preparations. Start your examination of this type of cartilage with slide 56A of the ribs of a stillborn. Ignore (for now) the bony ribs, and study only the costal cartilages. The two costal cartilages on the slide are large greenish-blue elliptical areas as seen by eye. In this slide (as for others), examine by eye first, and then study at low magnification before using high dry lens. In young cartilage, the chondrocytes are small and close together, but do not cluster in groups. In growing cartilage, there is a gradation from small closely-packed cells in the area of appositional growth beneath the perichondrium(dense fibrous connective tissue), to the older and larger cells in the center of the cartilage. In older cartilage, the chondrocytes and theirlacunae are larger and typically tend to cluster inisogenous nests. The cells of these nests are daughter cells, and reflect interstitial growth. The fetal foot (57A), and knee joint (60A) (see diagram of 60A at the end of this section), show excellent examples of hyaline cartilage with all stages of chondrocyte maturation. Articular cartilages are also hyaline (as can be seen in these slides), but are unusual because they lack a perichondrium. Slide l8A of the bronchi shows the more intense staining of the chondroitin sulfate immediately surrounding the individual lacunae; this is referred to as the capsule. You can spot the location of the cartilage by eye by finding the areas of darkest staining on the slide. The presence of the sulfated mucopolysaccharide in cartilage ground substance is responsible for the staining with hematoxylin or basic dyes; the ground substance is therefore said to be basophilic. Aldehyde fuchsin is not specific for chondroitin sulfate or elastin, but for the sulfate or disulfide moieties in these substances.

Elastic Cartilage
Elastic cartilage is not widely distributed, but is important for its flexibility in the external ear and epiglottis. Slide 33B of epiglottis shows the elastic fibers of this elastic cartilage particularly well. You will notice a broad lamina of lipid containing cells in the center of this cartilage, crossed by strands of collagen (red) and elastic fibers (black). A lipid filled center is common in elastic cartilage plates (such as the ear and epiglottis), and may contribute to the mechanical properties of these structures. Without a special stain for elastin, elastic cartilage may be difficult to distinguish from hyalin cartilage.

Fibrocartilage
Fibrocartilage may be difficult to discern in histological sections, since it resembles dense fibrous connective tissue. There is no perichondrium. The major clue to its identification is the presence of lacunae and capsules around the chondrocytes. The presence of these lacunae indicates that the matrix is cartilaginous. In slides 5lA and 52A of the intervertebral disc, fibrocartilage joins the ends of adjacent vertebrae. This band of fibrocartilage (annulus fibrosus) encloses a cavity filled with gelatinous material (nucleus pulposus). In slide 60A the ligament or tendon inserts into the bone via fibrocartilage. A unique feature of cartilage is that it is not vascularized, and yet in slide 57A you will find

46

CARTILAGE

blood vessels which have invaded this cartilage. These vessels occupy areas that will soon become centers of ossification as the cartilage is replaced by highly vascularized bone. You should also have noticed that bone stains a very different color than cartilage.

Electron Micrographs
Electron micrographs of cartilage cells may be found in the connective tissue notebook. They show that cartilage cells contain large amounts of glycogen and have a rather distinctive appearance. Note that the collagen fibrils in cartilage matrix are extremely fine. How does the collagen of cartilage differ chemically from that of, say, the dermis or bone?

47

Bone
Ground Bone Preparations
Slide 59A is a specimen of ground dried bone, and is particularly good for demonstrating the inorganic matrix. This is a rather thick piece of bone and, thus, should be examined mainly with the 4x and l0x objective. If you use the 40x objective be very cautious -- the working distance will be close. It will help you in the examination of this slide if you increase the contrast by closing the condenser diaphragm a little more than usual. You can identify osteons, lacunae, and canaliculi within these osteons, as well as the Haversian canals, and an occasional Volkmanns canal. Note the circumferential arrangement of the lamellae at the periosteal surface of the bone. Using the vernier scale on your mechanical stage (graduated in millimeters) and the pointer in your eyepiece, calculate the maximum distance you can find between an outermost osteocyte of a circular (cross sectioned) osteon and the center of its Haversian canal. This is probably close to the upper limit for metabolite diffusion in the canalicular system of compact bone. The other preparations which you will examine are sections of decalcified bone. When bone is demineralized, one is left with mostly collagen; this bone stains about the same color as the surrounding fibrous connective tissue.

Endochondral Ossification
Most bones of the body develop first as hyaline cartilage models. These cartilage models are eventually invaded by blood vessels, and calcification of the cartilage quickly follows. Calcified cartilage then serves rather like a scaffolding on which true bone matrix and, finally, bone is deposited. All calcified cartilage is eventually eroded away by osteoclasts. The development of the bones of the fetal foot (57A) provide a good example of these stages. With your 4x objective, you will note that the calcaneus and cuneiform bones are still largely or entirely hyaline cartilage, whereas the metatarsals and phalanges are part bone (red) and part cartilage (blue). One of the first stages in bone formation may be seen in the calcaneus and cuneiform bones, where blood vessels may be seen to invade these cartilage models. The lighter color of the matrix surrounding these vessels is calcified cartilage. There is significant bone development in the diaphyseal portions of the metatarsals, and most of the phalanges. The epiphyseal ends are cartilaginous, and the articular surfaces in the joints will remain so in the adult. Some of the phalanges may show an early stage of bone formation as a bone collar develops around the mid diaphysis of the cartilage model; note that the chondrocytes are very hypertrophic and vacuolated in this region. Notice how holey and irregular the bone appears compared to the cartilage. The general mechanism for bone development you have seen in slide 57A is called endochondral ossification. By holding slide 60A up to the light and using your eyepiece as a loupe, you can readily see the marrow cavity, diaphysis, epiphyseal plate, epiphysis and articular cartilage. The drawing of 60A in the previous section will aid you in orienting yourself on this slide. Using your microscope, examine the epiphyseal plate and identify the zone of proliferating cartilage cells, maturation zone of hypertrophic cartilage cells and zone of provisional calcification. Identify the periosteum, osteocytes, osteoblasts, osteoclasts, and, if possible, lamellae and Haversian systems. The sequence of maturation during growth in length of a long bone is nicely exhibited in the epiphyseal and metaphyseal parts. There are rows of chondroblasts, showing progressive proliferation and maturation in the epiphyseal plate. These chondrocytes become hypertrophic, and the plates of cartilage between them calcify. Notice the irregular trabeculae of calcified cartilage (grey) on which bone (red) is being deposited by osteoblasts. Osteoclasts

48

BONE

in Howships lacunae can usually be seen in relation to the trabeculae proximal to the epiphyseal plate. The calcified cartilage matrix may stain intensely with hematoxylin, depending upon the preparation; if the proteoglycan has been modified during decalcification, there will be little basophilic staining. The thin band of uncalcified matrix recently produced by osteoblasts (osteoid), stains with eosin, but to a lesser extent than does the mature bone matrix.

Intramembranous Ossification
In membrane bone formation, first osteoblasts and then bony spicules begin to appear in a region of mesenchymal tissue without any cartilaginous precursor. Slide 6lA provides an example of membrane bone. Here, a lamina (membrane) of mesenchymal cells has differentiated into very basophilic osteoblasts, which have begun to produce bone by a process calledintramembranous ossification. No cartilage model is formed in this type of bone formation. The different colors (blue and red) probably reflect a difference in the maturation of the bone matrix. What bone do you think this specimen came from? Membrane bone formation is also shown in slide 54A of the infant jaw (see diagram on next page for help in orientation). A modified form of membrane bone formation is called periosteal ossification, because, the osteoblasts are restricted to a region just under the fibroblastic connective tissue of the periosteum. In this process, osteoblasts appear surrounding a volume of cartilage, and begin to form an enlarging jacket of bone without invading the volume of cartilage (although the latter generally breaks down). The connective tissue around the bone-jacketed cartilage changes from perichondrium to periosteum, since it now contains bone forming cells. In long bones, growth in the length is by the endochondral method through growth and maturation of the epiphyseal plate. Circumferential enlargement of the diaphysis, however, is technically a form of membrane bone formation. Periosteal ossification can be seen at the periphery of the developing ribs of a fetus in slide 55A. In this slide, a red basic dye stains cell nuclei and a green acid dye stains the bone (collagen). Do not be misled just because the warm color represents the basic dye and the cool color an acid dye (in contradistinction to hematoxylin and eosin). You can observe normal hyaline cartilage in some developing ribs, but in others the cartilage is being enveloped by forming bone (within the periosteum), and the cartilage cells have begun to swell and die. A later stage in the development of the ribs may be seen on slide 56A.

49

BONE

50

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Muscle
Skeletal Muscle
Slide l0A illustrates the junction of skeletal muscle fibers with tendon. Note the position of the nuclei. What is the actual nature of the junction between tendon and muscle fiber? In slide l3A (a cross-section of dog skeletal muscle), the shape of the individual skeletal muscle fibers is readily seen. The differential effects of fixation can also be appreciated. Note for example, the difference in fibers at the periphery, as opposed to those at the middle of the bundle. Since the vessels and nerves of skeletal muscle tend to parallel fiber direction, cross sections of these structures are seen in the perimysium. Many of your slides will also have muscle spindles in this location. If your slide does not have one, look at that of a neighbor. In cross section, you should be able to recognize the connective tissue capsule and intrafusal fibers of the spindle. The muscle spindle acts as a sensor in the feedback system that serves to maintain muscle tone. While studying slide l3A, compare the skeletal muscle fibers with the smooth muscle cells found in blood vessels. The plastic section (l2A, H & E) of skeletal muscle in longitudinal section shows the in-register patterning of myofibrils and also fibers (cells) of the same muscle. In most sections, part of the perimysium septa surrounding groups of fibers can be seen; frequently, the septa, contain blood vessels. This specimen allows you to see the various bands of striated muscle. You should be able to see the H and Z bands, as well as A and I bands. The H stands for Hensens band (or zone), the light area in the center of the A band. The Z is taken from the German word Zwischensheibe, and is used to denote the band or line (disc) defining the terminal boundaries of the sarcomere (the disc between 2 sarcomeres). The designations A and I refer to the terms anisotropic and isotropic, and are derived from observations made with the polarizing microscope. The A bands appear dark in the light microscope, and correspond to areas in which both actin and myosin filaments run parallel to one another; the I bands are light in appearance, and are regions of the sarcomere adjacent to the Z line containing only actin fibers. In your electron micrographs, please take note of these more overt cytological features of striated skeletal muscle (as well as those structures such as transverse tubules and the sarcoplasmic reticulum that are not visible at the light microscope level, but are essential to muscle function).

Smooth Muscle
Slides 86A (intestine), and 94A (colon) are probably the best preparations in which to observe the shape of smooth muscle fibers. Both smooth and skeletal muscle can be seen on slide 76A; smooth muscle is associated with the trachea cartilage (completing the circle) and, depending upon the level at which the esophagus was sampled, skeletal muscle may be present in the outer wall of the esophagus. Another good preparation for comparing smooth and striated muscle is slide 99A of the recto-anal junction. In some regions of this slide, you will see some contracted or partially-contracted smooth muscle fibers in which the nuclei have assumed a characteristic spiral or corkscrew configuration. This nuclear configuration is one of the criteria that can be used to distinguish smooth muscle cells at the light microscope level. One of the fine structural characteristics of smooth muscle is the presence of numerous surface invaginations or caveolae. What other features would you use to distinguish smooth muscle cells from fibroblasts at the electron microscope level? How would you distinguish between areas of smooth muscle and dense connective tissue on the same slide?

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MUSCLE

Cardiac Muscle
Slide l7A (monkey heart) contains portions of both the atrium and ventricle, and may include part of an atrioventricular valve. The atrial myocardium is thinner and contains more connective tissue than the ventricle. Look for a place where there is a good cross-section of the cardiac muscle fibers. Note the centrally-situated nuclei, the irregular curved outlines of the fibers, and variation in fiber size. The cardiac muscle plastic sections (slide l6A) are longitudinal sections with occasional cross-sections, and frequent oblique sections at the edges. That the irregular columns of cells branch and anastomose can be seen by carefully following a length of fiber. The cells are joined end-to-end at the intercalated disk. The richness of the capillary supply is apparent at l0x. At 40x, the one or two central nuclei in their spindle-shaped packet of sarcoplasm (rich in eosinophilic mitochondria) are well seen. Note also that the individual myofibrils are separated by rows of mitochondria. Many of the longitudinally sectioned nuclei have indented margins. Why? Intercalated disks and the out-of-register sarcomeric banding of myofibrils are obvious at 40x. At one edge of slide 20A (embedded in the loose connective tissue of the endocardium), are the Purkinje fibers (cells designed more for conduction than for contraction). They consist of a group of large cells several times the diameter of ordinary myocardial cells. The myofibrils are sparse and confined to the periphery of the cell. The central region contains the nucleus and cytoplasm, rich in glycogen (not seen in this preparation). The large, basophilic structures in the myocardium of this slide are sections of parasites.

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Peripheral Nerves
Peripheral nervous tissue, like that of the central nervous system, is composed of cells actively involved in impulse conduction and neurotransmitter production, and release (neurons) and helper cells (termed satellite cells if adjacent to neuron cell bodies, and Schwann cells if related to axons). Schwann cells are also called neurilemmal cells. Peripheral nerve tissue differs from central nerve tissue because (with the exception of the enteric plexuses), it is interspersed with connective tissue components, giving it its substantial tensile strength. Peripheral neurons are generally gathered in aggregates called ganglia. Within ganglia, individual neurons are recognized by their large cell bodies, or perikarya, usually containing a large vesicular nucleus containing a prominent nucleolus. In the case of autonomic ganglia, these cell bodies will support both dendrites and an axon; in the case of sensory ganglia a single process is dispatched from the soma (which then divides into a peripherally and a centrally directed axon). You will encounter neuronal perikarya in many of your histology slides in the autonomic ganglia associated with visceral organs. Slide l8B, of most sets, contains a large ganglion external to the adrenal gland (this is one of the coeliac ganglia that receives preganglionic input from the splanchnic nerve; it provides adrenergic innervation for the gut and does not innervate the adrenal gland). Note that each perikaryon is surrounded by encapsulating cells. The innermost of these are the satellite cells. They, in turn, are surrounded by flattened connective tissue cells. To gain a better idea of the actual shape of autonomic neurons, examine slide 93A (which is a magnificent flattened, silverstained preparation of Auerbachs plexus). In spite of poor preservation, you can observe the general organization of a spinal ganglion on slide 48A; the nerve cell bodies and their investing satellite cells have been separated artifactually. The ganglia contributing to the Myenteric plexus of Auerbach are located between the inner circular and outer longitudinal muscle layers of the alimentary tract (slide 94A). The plastic section of the intestine (86A) shows these ganglion cells particularly well, and also shows the ganglion cells of Meissners plexus (located in the connective tissue just inside the muscle layers). Do you see typical satellite cells here? Most of the nerve trunks that you will encounter in your slides contain large numbers of myelinated nerve fibers. See slide 25A (carotid sheath). In a typical H & E tissue preparation (e.g., in the perimysium of muscle, slide l3A) you should be able to see axons, myelin sheaths, Schwann cell nuclei, perineurium and epineurium. After standard fixation procedures, the axon of myelinated peripheral nerves is surrounded only by remnants of myelin sheath, which has had its lipids extracted. This remnant material is termed neurokeratin. Thus, in order to see fine details of nerve trunk structure, you must study your class electron micrographs. Only here can it be seen that unmyelinated axons are also surrounded by the cytoplasm of a Schwann cell. Note that in contrast to the one-to-one relationship seen in the myelinated nerve fiber-Schwann cell unit, a single Schwann cell may invest several unmyelinated fibers. Both myelinating and nonmyelinating Schwann cells are surrounded by a basal lamina. The endoneurium consists of the scattered connective tissue cells and fibers (collagen and reticular) which immediately surround the nerve fiber-Schwann cell unit of peripheral nerve. Nerve fibers are grouped or bundled together, and are surrounded by a connective tissue layer termed the perineurium (derived from fibroblasts). Larger and thicker nerves consist of several perineurium-bound smaller nerves, held together by a coat of connective tissue (termed the epineurium). In nerves containing only one fascicle, the perineurium cannot be distinguished from the epineurium. When peripheral nerve fibers penetrate epithelia, they typically lose their Schwann cell sheath and basement lamina. Slide 92A is a Sudan black fat stain of a sensory ganglion in tissue culture containing some myelinated and many unmyelinated nerve fibers in the outgrowth zone. DO NOT USE OIL ON

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THIS SLIDE (40x objective is adequate for viewing). The outgrowth also contains many other cell types (including many phagocytes) scattered over the top of the culture, and underlying layers of fibroblasts. The culture was prepared from a single dorsal root ganglion taken from a mouse fetus. No myelin was present at the time of explantation into culture. Several hundred neurons of the ganglion survive in the central mass of tissue, often stained too intensely to see. Sometimes, individual neurons of distinctively different sizes can be maintained in culture. Axons grow outward to form an outgrowth, along with accompanying Schwann cells, proliferating fibroblasts and many phagocytes. The myelin present has formed in culture.

Can you identify in slide 92A: 1. Individual myelin segments (called myelin internodes). 2. The myelin-related Schwann cell nucleus related to each segment. Note that this is a fat stain and the nucleus is not stained. It appears as a vesicular void in the Schwann cell cytoplasm adjacent to the myelin. 3. Nodes of Ranvier between myelin segments. 4. Myelin segments still being formed (found in the more peripheral regions), or (rarely) extremely short myelin segments (called intercalated segments) interposed between myelin segments of normal length. 5. Branching in a myelinated fiber. What happens to the myelin sheath here? 6. Non-myelinating Schwann cell nuclei related to fascicles of unmyelinated nerve fibers. These are best found in nerve fiber fascicles in the peripheral regions. 7. Regions in the most peripheral parts of the outgrowth where the axons have not yet acquired Schwann cell ensheathment and are growing over the surfaces of very flattened fibroblasts. 8. Fat cells (sometimes present); phagocytes; stress fibers in the fibroblasts at the culture periphery; satellite cells of neurons (if nerve cells are individually visible). Before leaving todays laboratory also review the fine structure of the myoneural junction (see the muscle set of EMs). In what ways does this structure resemble a synapse terminating on another neuron? Correlating your knowledge of the peripheral nervous system from Gross Anatomy, the Neuroscience Course and Histology, can you answer the following:

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Integrative questions in neuroanatomy 1. Some Schwann cells form myelin, and others only loosely ensheathe non-myelinated axons. Does the axon or the Schwann cell specify this relationship? 2. What is the relationship between nerve fiber diameter and myelin internode length in vivo? Why does cultured nerve tissue have only very short myelin segments? 3. Which ganglionic neurons (sensory, sympathetic, or parasympathetic) are sources of myelinated (as opposed to unmyelinated) nerve fibers? 4. What embryonic structures are the sources of Schwann cells? What cells form the perineurium? 5. There are several types of axons which are myelinated by both Schwann cells and oligodendrocytes. Where are these located? 6. What neurotransmitters would you expect to find in the neurons of the dorsal root ganglion culture you have examined? How would you demonstrate the presence of a neurotransmitter system in these neurons? 7. Which ganglion would you culture in order to obtain noradrenergic neurons in a culture preparation? 8. Which neurons of the peripheral nervous system are not related to typical Schwann cells? 9. How would you expect the histology of the vagus nerve and the phrenic nerve to differ? 10 How would you expect the histology of the dorsal and ventral root to differ from each other and from the standard peripheral nerve trunk?

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Cardiovascular System
Arteries and Veins
Begin your study of vessels with the largest elastic artery, the aorta. There should be two sections on your slide of the human aorta (23A). Study the H & OGE section first. All blood vessels (with the exception of capillaries) are constructed of three main layers or tunics: the tunica intima, the tunica media and the tunica adventitia. These layers stand out distinctly on 23A; starting from the lumen, there is a thick (pathologically thick) t. intima containing the endothelial lining, and a significant amount of connective tissue. Surrounding this, there is the t. media which is comprised mainly of circularly-arranged smooth muscle, interspersed with fenestrated laminae of elastin. Surrounding all is the t. adventitia, which is composed of a rather loose connective tissue with scattered elastic fibers. Large vessels such as these are also supplied by blood vessels called the vasa vasorum; these small vessels may be seen in both the t. adventitia and t. media. Why do these vessels extend no deeper into the wall of the aorta than the outer t. media? Now, if you look at the other section on slide 23A stained for elastin, you will see the elastic components of the t. media and t. adventitia quite easily. Slide 33A of the monkey aorta is a plastic section and shows more cytologic detail. Note the tenuous endothelium and very thin connective tissue layer of the t. intima. The outer most limit of the t. intima is clearly indicated by a wavy pink-stained lamina of elastic tissue (called the internal elastic membrane). The t. media comprises most of the wall of the artery. You can see the nuclei of the smooth muscle cells that make up the wall; some run circumferential and some run longitudinal. The elastic laminae of the media are wavy (why?), and stand out quite well even though this is an H & E. The adventitia shows some evidence of the vasa vasorum. Slide 24A has several muscular arteries and an elastic artery; can you tell the difference? You will probably find a vein or two, as well. How might you identify the vein? Slide 27A will serve well for demonstrating the differences between an artery and a vein. Fortunately, arteries and veins usually run in parallel throughout the body, so that when you see one in section you usually see the other (as you do here). In general, veins are distinguished by their deformability, their comparatively thin and indistinctly layered wall, and the presence (in large veins) of valves. In 27A, the vessel with the thicker wall and yet smaller lumen is the artery. Compare the relative thickness and composition of the tunics between these vessels. Slide 25A is nicely stained for elastin. Note the striking difference in the development of the internal elastic membrane between the artery and vein. There are significant differences in the elastic laminae of the t. media as well. Many of the slides of specific organs provide excellent examples of smaller blood vessels. Slide 82A (pyloro-duodenal junction), for example, contains a wide range of vessel sizes. You should be able to find longitudinal sections of arterioles and capillaries in this preparation (An arteriole has only one or two layers of muscle cells outside the intima). What type of section of the individual smooth muscle fibers would you expect to obtain in a cross section of a medium sized vein? Slides l5B (neck organs) and l00A (adipose tissue) contain blood vessels of different sizes.

Lymphatics
Lymphatic vessels can be recognized in most tissues by their relatively large size, irregular shape, and thin wall (which may consist of little more than an endothelium). The small lymphatic vessels can be distinguished in different tissues by their irregular contour and thin walls with attenuated endothelial cells. Nice examples of lymphatic vessels are present in the plastic sections of monkey ovary (68B) and the human ileum (88A); they are readily distinguishable

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from the blood cell-filled blood vessels in these preparations. They can be found in the dermis in slide 50A of the dog ear. Lacteals are fat-absorbing lymphatic capillaries located in the villi of the small intestine. Lacteals in the collapsed state can be found with difficulty by careful examination of slides from the series on the intestine (e.g., slide 9lA of the cat ileum). Lymphatic vessels can also be found in the area of the lymph node in slide l5B of neck organs.

Heart
Slide l5A (from the heart of a small mammal) shows a portion of the left ventricle, the left and right auricles, and segments of the aorta and pulmonary artery between the right and left auricles. The ventricle is covered by a layer of mesothelium and connective tissue, the epicardium. The endocardium lines the inner surface of the heart. Note its dense layer of connective tissue immediately below the endothelial cells. A portion of papillary muscle extends into the ventricle. Between these two layers, one can see the arrangement of the myocardial cells. Note the difference in the thickness of the walls of the right and left auricle. Aortic valves project from the inner walls of the aorta. The surface of the valves is covered by endothelial cells. Dense connective tissue and a few cells which resemble chondrocytes are seen in these valves. The connective tissue and chondroid (the substance secreted by the chondrocyte-like cells), provide rigidity for the valves. Slide 16A. Note the distribution of myofibrils, and location of nuclei in the cardiac muscle cells. The intercalated discs which join one muscle cell to another are very well preserved in16A. Note the wavy contour of the discs and the many small blood vessels among the myocardial cells. From what large vessel are they derived? Slides 20A and 21A demonstrate Purkinje cells. Note their location below the endocardium in the intraventricular septum. The cells are very easy to distinguish in the section of beef heart. They are shorter and broader than typical cardiac muscle cells, and have fewer myofibrils than the typical cardiac muscle cell. Can you find any abnormalities in the structure of the cardiac muscle cells or in the blood vessels in the section of the human heart?

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Hemopoiesis

Slides 28A and 32A

This laboratory is intended to familiarize you with the myeloid elements, and to introduce you to the differentiating cells in bone marrow which give rise to cellular elements of the peripheral blood. Identification of the various cell stages depends upon cell size, the size and shape of the nucleus, the presence of cytoplasmic basophilia and the number and staining characteristics of the cytoplasmic granules. The goal of this laboratory is to be able to identify various stages in the development of erythrocytes and granulocytes on the basis of understanding the changes in differentiation. Study slide 28A to gain an impression of the organization of marrow in conventionally stained and sectioned material. Cells are clustered between sinusoids. You will note numerous holes in the marrow; what causes this? You might be able to identify some cells, but do not spend much time in this endeavor; the marrow stained with Wrights stain is best for cell identification. Megakaryocytes are very large and may be easily seen in any preparation of marrow. Slide 29A is a section through an intact plug of dog marrow, which has been separated from its investing bone. With your 4x objective, note the large central longitudinal vein into which the sinusoids drain. Next to this vessel is a large central longitudinal artery and other smaller arteries. Note that the central vascular compartment of the marrow is more fatty, and the peripheral compartment is more hemopoietic. At higher magnification, you can find numerous megakaryocytes in this hyperplastic marrow. With high dry, you ought to be able to discern sinusoids, fat cells and the reticular cells that (together with reticular fibers), make up the stroma of the marrow. You will be able to discern various stages in the development of granulocytes (neutrophils and eosinophils), but a detailed study of hemopoiesis is much better done on marrow aspirates or touch preparations. Most of your laboratory time should be spent looking for examples of different developmental stages in the red and white blood cell series in the slide of human bone marrow (32A). This preparation was made by aspiration, which destroys much of the normal relationships you observed in sections but gives much better cytological detail. The reticular stroma and fat cells are either missing or destroyed. Use oil immersion. Do not try to identify every cell you encounter; instead look for good examples of each cell stage. Choose thin areas where cells are nicely spread out for your perusal. It is sometimes helpful to start identifying the more mature stages first and then carefully work your way back through the more immature stages. Dont forget that, in the case of the more advanced developing granulocytes, the term basophilic or eosinophilic refers to the cytoplasmic granules, not the cytoplasmic staining per se. The overall cytoplasm is more basophilic for any type of granulocyte in the early developmental stages. Refer to the lecture handout for aid in identifying the various stages in erythrocyte and granulocyte differentiation. By examining 32A under oil, you should be able to find basophilic erythroblasts, polychromatophilic erythroblasts, normoblasts and both immature and mature erythrocytes. Look for promyelocytes with only a small cluster of granules and otherwise basophilic cytoplasm. What is the name of these granules? Their nuclei are not indented. Myelocytes show a wide variation in numbers of granules, are often quite large, and display a less basophilic cytoplasm. Metamyelocytes with horseshoe shaped nuclei are readily seen, and are a terminal stage which forms the mature polymorph by differentiation without further division. The basophilic metamyelocytes have large purple granules and are relatively rare, but can be found

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in occasional preparations. Look for band forms. Other cell types that you will find are megakaryocytes, lymphocytes, fat cells, plasma cells and reticulum cells. The cells with the most prominent dark blue cytoplasm might be either plasma cells or basophilic erythroblasts. At what stage can you first distinguish between developing neutrophils, eosinophils and basophils? There are several characteristics which distinguish a plasma cell. The eccentricallyplaced nucleus has a cartwheel appearance due to the arrangement of the chromatin and a prominent nucleolus. The cytoplasm is highly basophilic (why?) except for a light area in the center of the cell next to the nucleus. What is in this lighter region? The plasma cell often contains a prominent vacuole in the cytoplasm. After studying plasma cells, how would you distinguish them from basophilic erythroblasts? How can you distinguish immature, but anucleate, erythrocytes from mature ones? Do not confuse the term reticulum cell with reticulocyte. The latter refers to an immature erythrocyte, which has been made highly visible for quantitative purposes by staining with brilliant cresyl blue or other dyes which stain ribonucleoprotein (RNP). The name reticulocyte derives from the fact that these special stains precipitate the RNP in immature erythrocytes into a darkly staining network (i.e., reticular arrangement), which otherwise would have stained homogeneously for hemoglobin (e.g., pink) in more mature cells. The reticulum cell, on the other hand, is one of the larger cells you will see in a marrow smear (the even larger megakaryocytes and fat cells are often destroyed in preparation). The reticulum cell has abundant and rather irregularly granular cytoplasm and a large, lumpy nucleus, often with a distinctive pale blue nucleolus. The reddish-blue chromatin of the nucleus generally has a ropy appearance. Reticulum cells are believed to represent the reticular cells of intact marrow.

Electron Micrographs
Try to work out the characteristics of the differentiative processes in these cell lines from both your observations in the light microscope and your perusal of the electron micrographs. For instance, how does the chromatin change as the cells mature? The compaction of the nuclear elements is correlated with decreasing synthetic activity of the cell. In picture #l, you should compare the nuclei of the myelocyte and the polymorphonuclear leukocyte. What organelles reside in the cytoplasm that occupies an area indenting the nucleus of a monocyte? Correlate the light microscopic appearance and the fine structure of the plasma cell. Do the same for the developing erythrocytes.

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Lymphatic Tissue
Thymus
Begin by looking under low power (4x) at the plastic section of a young human thymus (37A). Note that the connective tissue septa separating the lobules are very thin. The intensely blue cortex is thick, relative to the medulla. In some areas, the medullary regions of several lobules can be seen to coalesce (actually all the medullary regions connect with each other). Under l00x, you can see that the homogeneously-stained pink spots and streaks represent vessel lumens containing plasma and RBCs. There are also numerous cells with bright red granules, which are mast cells. They are found mostly in connective tissue around vessels near the corticalmedullary junction, where the larger vessels distribute (drain) small branches to (or from) the cortex and medulla. The medulla appears lighter in color because of the relatively larger percentage of the big epithelial-reticular cells. First, find one of the larger Hassalls corpuscles. These consist of concentrically arranged, flattened epithelial-reticular cells in various degrees of degeneration. Many contain dark staining granules. In some of the larger ones, the outermost few epithelial reticular cells appear quite intact. These will show you the very euchromatic, large, roughly oval nucleus and bright red nucleolus (or 2 nucleoli) characteristic of these cells. Now you can identify many other such nuclei throughout the medulla as probably belonging to epithelial- reticular cells. Most of the other cells in the medulla are lymphocytes (small and medium), endothelial cells, and macrophages. The macrophages also have rather euchromatic, large nuclei, and are almost impossible to distinguish from epithelialreticular cells with certainty unless you see intracytoplasmic debris or vacuolization. At the EM level they can be distinguished. Do you know the criteria? Within the cortex, the lymphoblasts, macrophages and epithelial-reticular cells cannot be distinguished from each other with certainty, since they all have large, euchromatic nuclei. The predominant cell type is the small lymphocyte, which has very clumped chromatin and scant cytoplasm. Intermediate sized lymphocytes (between the small and the blastic lymphocyte) exhibit intermediate degrees of chromatin clumping and amount of cytoplasm. Do you see Hassalls corpuscles in the cortex? Do you see mitotic figures? Do you expect to see mitotic figures within the medulla? Could the metaphase mitotic figure you think you see be a neutrophil or a pyknotic nucleus? Can you see any plasma cells? Should there be any? In slide 38A from a human infant, it is rather difficult to distinguish the cortex from the medulla. Slide 42A shows an involuted thymus from a small mammal. Under low power, observe the considerable widening of the interlobular connective tissue spaces and their replacement by fatty tissue. The cortex is thinner (relative to the medullary region) and total thymic tissue is decreased. Under high power, you can see the same distribution of cell types. The cortex is still packed with small lymphocytes. Occasional clear-cut mitotic figures can be found, consistent with the current view that the thymus continues to function well into adulthood and, perhaps, old age.

Peripheral Lymphoid Tissue

Diffuse Lymphocytic Accumulations
The simplest form of lymphoid tissue consists of mere unorganized accumulations of lymphocytes and some associated cells (such as macrophages, plasma cells). These may be diffuse or nodular. They are found in many organs, e.g. the submandibular salivary gland (7lA), the

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gastrointestinal tract (99A) and the bronchial tree. You are not yet familiar with the histology of these organs, but the lymphoid accumulations can be easily spotted under low power as relatively dense concentrations of very dark, heterochromatic nuclei. Under high dry, you can appreciate that the small and medium lymphocytes predominate, thereby conferring that characteristic appearance. From these slides, you will note that lymphoid accumulations often develop beneath mucous membranes and in connective tissue surrounding ducts of glands.

Lymphoid Nodules
Next in order of complexity are the lymphoid nodules (also called follicles) that are found in specific locations associated with the gut. Germinal centers are frequently present in these nodules. The tonsils are such nodules of lymphoid tissue underneath the mucosal epithelium. They are massively developed in the oral pharynx (slide 30A). The pharyngeal tonsils contain extensive epithelial crypts (tonsillar crypts) which are infoldings of the epithelium. The epithelium can be very thin over the large lymphoid nodules. Peyers patches constitute the other main group of such accumulations in the ileum (9lA). Similar collections can be seen in the colon (94A) and the appendix (96A).

Lymph Nodes
Lymph nodes represent highly organized and encapsulated collections of lymphoid and related cells. The silver impregnated section (the black and blue one) on slide 36A shows the general architecture of lymph nodes best. The reticular fibers appear as black threads and the cell nuclei have been counterstained blue. The subcapsular, intermediate and medullary sinuses stand out as relatively more open spaces, containing fewer reticular fibers and fewer cells. In contrast, the cortex and medullary cords are densely packed with cells. All the vessels are clearly outlined by a ring of reticular fibers, as are the connective tissue trabeculae. In the cortex, there are many germinal centers. One can see the typical appearance of a cap (or ring) of densely packed small lymphocytes over the more lightly stained oval area. Within the light area of the germinal centers, many large lymphocytes and blasts are found. The medullary cords constitute a highly branched network of solid tissue going from the cortex to the hilum of the node. In some sections, small segments of the afferent lymphatics can be seen to empty into the subcapsular sinus, and the lymphatic valves, sometimes can be seen outlined by reticular fibers. In some areas of the sinuses, you can clearly see rosettes of grayish-tan color attached to the reticular fibers criss-crossing the sinus lumen. These are sinus macrophages engorged with phagocytosed red cells. Such macrophages are seen in the subcapsular, intermediate and medullary sinuses. RBCs do not normally enter the lymph, so this node represents a pathological situation. Lymph node macrophages will phagocytose RBCs, although many other types of macrophages will not gobble up normal RBCs (absolutely essential in the liver since the macrophages are in blood sinuses). Now turn to the other section on the same slide. You will again see the RBC (bright orange) engorged sinus macrophages. Within the sinuses you can identify some of the reticular cells as having large, oval, euchromatic nuclei and cytoplasmic processes which stretch some distance across the lumen. They can be most easily seen in the subcapsular sinuses. Consistent with the abnormal blood extravasation into the lymph sinuses, you can also find many neutrophils in the sinus lumen. They are also not normally found there. Within the cortex, you can see a full range of euchromatic and heterochromatic nuclei. The larger and more euchromatic nuclei belong to lymphoblasts, large lymphocytes, macrophages and reticular cells. The ones

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with smaller and more clumped chromatin belong to small and medium lymphocytes. The large lymphocytes and blasts are mostly found within germinal centers. Mitotic figures are also found primarily there. True plasma cells are rare around germinal centers, but some cells with abundant basophilic cytoplasm and heterochromatic nuclei (representing transitional cells on their way to becoming plasma cells) can be found within the cortex near the corticomedullary border. The medullary cords can be clearly seen to be covered by a layer of reticular cells. The cords contain lymphocytes and many plasma cells. The finding of neutrophils outside of vessels in the cords (as seen here) is abnormal. Reticular cells forming the scaffolding meshwork for holding the lymphoid cells within the cortex, and cords are not distinguishable in most instances from lymphoblasts, large lymphocytes and macrophages. Distinctive structures found in lymph nodes are the high endothelial postcapillary venules, which are the sites where blood lymphocytes enter the lymph node parenchyma. These are found within the cortex, largely in the deeper portion of the cortex. The height of the endothelial cells vary somewhat with tissue preparation. Therefore, on any given slide, you should first locate some typical capillaries and note their lumen size, the flatness of their endothelial cell nuclei and the almost indiscernible layer of cytoplasm. Usually you can see only one (at most two), very flat nuclei in a capillary cross section. Then, within the deep cortex, find vessel lumens of similar size which are lined by thicker cells. Since these endothelial cells are roughly cuboidal and cover less territory than the usual squamous endothelial cell, there are usually 2-3 or more of these nuclei per cross section of these vessels. In longitudinal section, the nuclei can be seen to be spaced more closely together than the usual endothelial cell nuclei, and have a more rounded shape (rather than the usual long oval shape of endothelial nuclei). Moreover, you can make out some cytoplasm around the nuclei, unlike the usual endothelium. Sometimes lymphocytes can be found in passage between the endothelial cells, which may make the initial recognition of the endothelium harder, but help the identification in the end. Slide 34A is a plastic section of a lymph node. If your section is cut near the hilum, you can see a concentration of muscular arteries and veins and, perhaps, a collapsed thin walled lymphatic vessel, all within rather extensive connective tissue spaces. As is characteristic of plastic sections, the open spaces seen on paraffin sections seem to have disappeared. However, one can still appreciate relatively less cellular appearance of the various sinuses. Apply the description of slide 36A to this section except for the following: i.e. there are only rare RBC-engorged macrophages and a few neutrophils in the lymphatic sinuses. The few RBCengorged macrophages must be carefully distinguished from the well preserved and conspicuous mast cells. Are the latter present in sinuses, in both the cortex and medullary cords? Unlike the paraffin section, the many macrophages can be clearly identified in the cortex and cords by their cytoplasmic debris and vacuolization (especially in germinal centers). The identification of the high-endothelial postcapillary venule is both easier and harder. Although the cuboidal nature of the endothelial cells is better preserved, the lumen in most instances are filled with lymphocytes (and some other blood cells). Many are caught in longitudinal-oblique section and show lymphocytes in passage through the endothelium.

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Spleen
Histologically, the spleen is the most complex lymphoid organ. The general structural plan can best be seen on the H & E section on 43A. Under very low power, you can easily identify each of the following regions and then examine them under high dry:

White Pulp
The white pulp areas (WP) are the dense collections of small lymphocytes around a relatively small muscular artery, i.e., the periarterial lymphatic sheath (PALS), its central artery and lymphatic nodules (with or without germinal centers). The PALS is the collection of mainly small lymphocytes, which are roughly symmetrically arranged around the central artery. The nodules are roughly spherical structures, arranged to the side of the central artery. So in a cross section through a nodule, the central artery appears eccentric within the total area of lymphocyte accumulation (nodule + PALS). How do T and B lymphocytes distribute within the nodules versus the PALS? The nodules contain densely packed small lymphocytes. When germinal centers develop within them, a lighter center is seen in the nodules, as is typical of all germinal centers. Why are germinal centers more lightly stained? There are very few on this side. Under high power, you can see the smooth muscle cells of the central artery wall and its endothelium. With careful focusing you can also discern some of the pink streaks of reticular cell cytoplasm between the numerous lymphocytes. The lymphocytes are mostly small and medium types, except in germinal centers. The large, euchromatic nuclei belong to macrophages and reticular cells. Where and when are you likely to find plasma cells in the spleen?

Red Pulp
The Red Pulp (RP) consists of the bulk of the space between the WP areas. They include the splenic sinuses (venous sinusoids) and the splenic cords (of Billroth) in between the sinuses. The former are blood sinuses with large and free lumen (no fixed cells or fibers crossing it). The blood has been largely removed from this spleen before fixation, so the sinus and vessel lumens appear as empty spaces, only occasionally containing a few blood cells. The sinuses can be distinguished from the pulp veins which drain them by their peculiar endothelial cells. The round endothelial nuclei are irregularly spaced around the sinus, and tend to protrude into the lumen. The pink cytoplasm of these endothelial cells can be seen in the spaces between the nuclei. The endothelial cells of the splenic sinusoid are long spindle-shaped cells that are arranged in the wall of the sinus like the staves of a barrel. In some oblique sections, you can see the candy stripe appearance of these cells. Many blood cells can be seen in passage between the endothelial cells. This is called diapedesis. Immotile cells (RBC and platelets) pass through also, but the term is not applied to them. The pulp veins are easily identified by their usual hardto-see squamous endothelium, the scarcity of connective tissue surround, and their large lumen (especially relative to wall thickness). Between the sinuses are the cords. These are filled with all the formed elements of blood, as well as resident macrophages and free plasma cells. The stroma is formed by the reticular cells and their meshwork of fibers. Many of these various cells can be identified by criteria already familiar to you.

Vascular Organization of the Spleen

Slide 45A of the dog spleen is particularly good for showing the vascular organization of the

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spleen. Scattered throughout the spleen are sections through the numerous connective tissue trabeculae. These radiate from the splenic capsule and branch throughout the substance of the spleen. Major vessels enter and leave in them. In the connective tissue trabeculae of this dog spleen, one can find many smooth muscle cells running parallel to and mixed in the connective tissue collagen, as well as smooth muscle cells forming the walls of the trabecular arteries and trabecular veins. In human spleens, the smooth muscle component is much less outside the vessel walls. The trabecular arteries give rise to branches which leave the trabeculae as central arteries, while the pulp veins drain into the trabecular veins. Found within the cords are the arterioles and capillaries of the red pulp. The central artery terminates as it leaves the white pulp, branching to form the penicillar vessels of the red pulp. Different portions are given specific class names, as the penicillar vessels branch and rebranch to supply the red pulp. The initial portion (right after they leave the WP) is the pulp artery. These short segments still possess a thin muscular coat, but much attenuated relative to that of the central artery. As their branches become capillary sized, they often acquire a sheath of concentrically-arranged phagocytic cells. These are called the sheathed arterial capillaries. They are plentiful in the dog spleen, as can be seen on this slide. They stand out by virtue of the intensely eosinophilic staining of the cytoplasm of the sheathing cells, and the remarkable thickness of this coat of cells around a capillary-sized clear lumen. In human spleens, there are fewer of these sheathed capillaries and the coat is not as well developed. The terminal portion of the penicillar vessels are simply ordinary capillaries, although they are specifically designated asarterial capillaries. The sheathed arterial capillaries terminate as arterial capillaries after they emerge from the sheath. Usually such terminations are bell-shaped and open-ended, so that the lumen may appear somewhat wider than the usual capillary seen in other organs. Alternatively, the arterial capillaries may derive directly from the pulp artery without an intervening segment of sheathed arterial capillary. (Note: Unfortunately pulp arteries is also sometimes used loosely to designate all arteries and capillaries of the red pulp, i.e., synonymous with penicillar vessels).

Marginal Zone
The marginal zone (MZ) is the area immediately surrounding the White Pulp areas. It represents the transition between WP and RP. It is mostly easily discerned under low power (4x and l0x), as the relatively cellular zone surrounding the densely cellular WP area but which also contains some small sinusoids. Under high power, the MZ can be seen to contain cords between small sinuses. The cells in the cords include blood cells (conspicuously, neutrophils and RBCs) lymphocytes, macrophages, reticular cells and plasma cells. Now turn to the silver stained section on slide 43A for a quick look at the dense mesh-like stroma formed by the reticular fibers. Relate the outlined structures here to the other section which you have now examined in great detail. All the trabeculae and larger vessel walls are darkly stained. Why is that? The white pulp areas have a looser mesh of fibers (especially the lymphoid nodules). The sinusoid lumens, like the vessel lumens, are free of fibers. One can also appreciate that the MZ has a denser mesh of fibers than the WP, and the sinusoids outlined by the reticular fibers are smaller than in the RP. Of particular interest is the circular investment of the sinuses by the reticular fibers, which assume a relationship to the endothelial cells like that of the strapping to barrel staves. The basement membrane of the sinusoid contains reticular fibers, so this strapping arrangement holds for the basement membrane as well. This discontinuous arrangement of the basement membrane may aid diapedesis.

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Sinusoids
Next, look at slide 47A (the plastic embedded section of a primate spleen). Identify the WP, RP and MZ under low power. In some of your slides, there is one trabecula with a large vessel in it. Note that the WP areas appear lighter because the RP sinusoids and vessel lumens are filled with deeply eosinophilic RBC and plasma. The MZ is less distinct than in the previous slide, but can be seen as the area with smaller sinusoids roughly concentrically placed around the WP areas. Under high power, the cellular components of all the regions can be seen in greater detail in this section. Starting with the WP, you can see that the central arteries are all partly collapsed. The lumens are constricted and appear only as small areas of deep pink in the middle of a thick wall of smooth muscle cells. The endothelial cells appear thick and crowded rather than squamous due to the collapse. Find the germinal centers present in some lymphoid nodules. Within the germinal centers, you should be able to identify some macrophages, mitotic figures and many small lymphocytes. An occasional reticular cell can be identified with certainty by their large euchromatic nucleus, and long extended cytoplasmic processes. Within the RP, you should be able to identify the complete outline of many sinusoids with their thick endothelium and wide (but filled) lumen. Identify some of the many examples of leukocytes undergoing diapedesis. The cords can be found immediately adjacent to each sinusoid you delineate. They can be seen to contain many more cells, and the spots of pink (RBC) are less continuous than in the lumen of the sinusoids (where plasma between the RBC also stain). Some of the cord cells can be clearly identified as lymphocytes, neutrophils, eosinophils, plasma cells and macrophages. Slide 44A is an unwashed spleen which is still full of blood. If you compare the amount of blood in the red and white pulp areas, you will readily understand the derivation of these names.

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Integumentary System
Epidermis and Dermis
The most obvious feature of cross sections of this organ is that it consists of two distinct strata, a superficial epidermis and the underlying dermis. The epidermis is a stratified squamous epithelium in which the most superficial cells are dead squamous cells, devoid of most cell organelles and filled with filamentous and amorphous proteins called keratins. The general process by which keratins are produced in the cytoplasm of cells is called keratinization, and may also be seen in the epithelium of the vagina, esophagus, oral cavity and thymic corpuscles. In these cases, however, keratinization normally does not result in a discrete dead layer of cells devoid of organelles (called the stratum corneum), but rather the cells retain their nuclei and may be living. This type of incomplete keratinization is calledparakeratinization and may occur in human skin in certain pathological conditions. Since a form of keratinization occurs in many epithelial tissues, it is helpful to use the wordcornification for that process that results in a dead and relatively impermeable stratum corneum. Epidermal cells which synthesize keratin are called keratinocytes. Slide lA is digital skin from a monkey. The H & E section shows the greatly thickened epidermis in this area. Note the well developedstratum corneum which is virtually free of nuclei or basophilia. Just deep to the stratum corneum is the stratum granulosum. The cells in this layer are filled with basophilic (blue) keratohyalin granules, which participate in the formation of keratin that will fill the horny cells of the stratum corneum. Note how sudden the transition is from living cells in the granular layer to the dead flattened cells in the stratum corneum. The epidermis, deep to the stratum granulosum, is called the stratum spinosum (stratum Malpighii). The deepest layer of the epidermis consists of low columnar cells, with nuclei squeezed very close together. This layer is called the basal layer, or occasionally the stratum germinativum since most (if not all) of the mitotic activity occurs here. The irregular form of the deep rete ridges is due to the plane of section. Melanin granules are abundant in the basal cells of the rete ridges. Stem cells are located in these areas, and are protected from actinic radiation by the supernuclear location of their melanin. You may occasionally see the dendritic processes of a melanocyte in the basal layer. The dermis is stained very lightly in this section, but in the other section on this slide (stained with aldehyde fuchsin, orange G and fast green), the yellow stained collagen and purple elastic fibers are evident. Most of the nuclei are in fibroblasts. Note the many cross sections of blood vessels in the dermis. The ridges formed on the under surface of the epidermis are called rete ridges. Note that the ducts of the sweat glands always contact the epidermal surface at the crest of these ridges. Coiled sweat glands and blood vessels are abundant. Can you find the sweat ducts in the epidermis? The duct goes through the stratum corneum in a spiral path (Some sections of the aldehyde fuchsin specimen on lA will show a parasitic round worm embedded in the epidermis). In some sections, there are a few onion-like nerve endings (called Pacinian corpuscles) located in the dermis. These pressure receptors consist of concentric layers of squamous epithelial cells around a central nerve fiber. Finger-like dermal extensions extending up between the rete ridges are calleddermal papillae. Within these papillae are blood capillaries and nerve endings. In the Bodian silver preparation of monkey digital skin (slide 2A), specialized nerve endings (Meissners corpuscles) will be seen at the apex of some dermal papillae. Also notice that the deep dermis has many nerve trunks consisting of many fibers in bundles, and that fibers often terminate in association with the eccrine sweat glands (probably representing the sympathetic innervation of these structures). If you compare the two sections on this slide,

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you will find that it would be impossible to identify elastic fibers in this section without a suitable stain such as aldehyde fuchsin. The plastic section (7A) is best for observing keratohyalin and melanin granules, the spiny cells of the stratum spinosum, and both the secretory and duct portions of the eccrine sweat glands. The occasional clear cells in the basal layer containing scattered melanin granules are melanocytes. These cells are responsible for the synthesis of melanoprotein. You will note that most of the melanin in the basal layer is actually in pink stained keratinocytes, rather than in clear melanocytes or the more superficial keratinocytes. The keratinocytes of the basal layer phagocytize the tips of the dendritic process of melanocytes and, in this way, take on melanin granules. Once inside the keratinocyte, there is a directed movement of melanin granules, such that they come to occupy a supranuclear position; this provides an effective solar screen for the DNA in these cells. You should see evidence of this preferential location in your slides of pigmented skin. As the keratinocytes ascend to a higher level in the epidermis, most of their melanin is broken down and is no longer visible. Some pigment may persist in the stratum corneum of darker skinned humans and animals. The spiny-looking cells of the stratum spinosum are easily discerned in the middle layers of the epidermis. This spiny appearance is actually a shrinkage artifact which reveals the scores of desmosomal attachments on the surface of each keratinocyte (keratin-producing cell in epidermis). The stratum granulosum is easily visible. Between this granular layer and the stratum corneum, you will find occasional transitional cells with pyknotic nuclei. These cells are dying and will become corneocytes. Notice that the corneocytes have little peg and socket elaborations between cells that appear to give mechanical integrity to the stratum corneum; this is typical of epidermis from the palms or soles which are high wear areas.

Hair
The bodies of nearly all mammals are covered with hair. Slide 3A shows growing (anagen) hair follicles in the human scalp. The hairs (like the sweat glands) are of epidermal origin though a little tuft of dermis, called the hair papilla, in the deepest part of the hair follicle, is necessary for growth and differentiation of the hair. Longitudinal sections of hair follicles often show the dermal papilla surrounded by the bulb of the hair follicle like a claw. Hair follicles grow at a relatively uniform angle to the epidermis. Because of the plane of section, it is not always possible to see the whole length of some hair follicles. The hair itself is yellow with black pigment granules in the cortex, the living portion of the follicle stains blue, and the inner root sheath stains red. The melanocytes that produce the melanin are adjacent to the hair papilla, as can be observed in a longitudinal section through the hair bulb(bulb-shaped end of follicle). The mitotic activity that supports hair growth occurs only in the hair matrix in the interior of the bulb. As these matrix cells move up the follicle, they differentiate into the keratinized layers of the inner root sheath as well as the hair shaft. As in the stratum corneum, part of this differentiation involves cell death (Consult your histology book for details of hair follicle structure; Ham is particularly good in this respect). About one-third of the way down the follicle, on the side that forms an obtuse angle with the epidermis there is generally a bulge of vacuolated cells comprising the sebaceous gland. When the cells in the interior of this gland break down, their detritus (called sebum) slips out between the inner root sheath of the follicle and the hair ( pilosebaceous canal) shaft to the surface of the skin. Some of the cytological details of this breakdown process can be seen in electron micrographs in the integument collection. You may observe some resting (telogen) hair follicles. These follicles are shorter than anagen follicles and the deep end of the

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follicle is enlarged and has a rather spiny appearance from which it gets its name -- club hair. Slide 3A also shows the cutaneous blood supply to good advantage. Note the capillary loops in the dermal papillae and the close association of blood vessels with both hair follicles and the eccrine sweat glands in the subcutaneous fat. Slide 63A (lip) has well stained cross and longitudinal sections of hair follicles. The largest hair follicles in the lip are sinus hairs which derive their name from the fact that the bulbous portion of the follicle is suspended in a large blood sinus. These follicles produce the vibrissae or whiskers of rodents, cats, etc., where they have a highly developed sensory function. Slide 4A is from the bald human scalp and reveals that hair follicles and growing hairs are as numerous as in slide 3A. The hairs and hair follicles of the bald scalp are much smaller, however, and are called vellus hairs. These are the kind of hairs we find on our nose and forehead. Note the well-developed sebaceous glands that add insult to injury in baldness by making a shiny pate. These tiny hairs are common on the human body and give the mistaken impression that man is largely hairless. Slide 6A has two sections of adult thigh skin, the red staining H & OGE for collagen and the black Verhoeffs hematoxylin for elastic fibers. The epidermis is stained poorly on both sections. On the H & OGE preparation, the superficial papillary layer of the dermis is rather sharply demarcated from coarser fibers in the reticular layer of the dermis. The reticular layer merges with the subcutaneous fat.

Sweat Glands
Eccrine Sweat Glands
Slide 7A is particularly well suited to study the histological detail of both the secretory and duct portions of eccrine sweat glands. The secretory portion of the sweat glands is readily distinguished by the presence of myoepithelial cells, which give a scalloped appearance to the tubules in cross section. This is the only slide in which the mucus-producing dark cells and the serous fluid-forming light cells can be readily discerned. The dark cells appear to be near the lumen and have distinct red granules, whereas the light cells comprise the bulk of the secretory segment. Actually both the dark and light cells rest on the basement membrane. The duct, on the other hand, is clearly a stratified cuboidal epithelium. Note the various cell types (including mast cells) that populate the dermis. In slide 6A, the coiled secretory portion of the sweat glands and a good portion of the sweat ducts lie in the reticular layer of the dermis. Note that the epithelium of the duct of the sweat glands is two cell layers thick and is darkly stained, while the epithelium of the secretory portion is one cell layer thick (although it may appear thicker) and lightly stained. Also notice the myoepithelial cells that are associated with the secretory portion. What function do they have?

Apocrine Sweat Glands

Slide 8A of axillary skin has been included in your collection to show the two types of sweat glands that exist in this area. You have seeneccrine sweat glands in most of the previous slides. Apocrine sweat glands are much more limited in distribution. There are two obvious differences in the histology of these glands. The secretory part of the apocrine gland is larger in diameter and consists of a simple columnar epithelium. You will occasionally see a yellow lipofuscin

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material in these cells. The myoepithelial cells are well developed and, therefore, can be easily seen on the apocrine and eccrine sweat glands. Apocrine glands are also found on the slide of the recto-anal junction (99A); this slide is also very good for studying sebaceous glands, hair follicles, and Pacinian corpuscles. The Meibomian glands (slide 98B) of the eyelid are of interest because they develop in association with hair follicles and are greatly enlarged and modified sebaceous glands. What function might these glands serve?

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Respiratory System
Nasal Cavity
In examining the respiratory system from nares to alveoli, try to think of the ways in which the structural adaptations of each part serves its function(s). The nose contains two nasal cavities separated from one another by the nasal septum. Each nasal cavity is divided into two parts: (l) the vestibule, a slight dilation inside the opening of each nostril, and (2) the respiratory portion, the remainder of the cavity.

Slide 28B is a frontal section through half of a cat nose. Orient yourself by examining the slide without the microscope (see diagram). Note that the borders are the nasal septum (medial) and the wall of the maxillary sinus (lateral), while the hard palate forms the floor of the nasal cavity (as well as the roof of the oral cavity). The conchae (turbinates) are curled plates of bone extending from the lateral wall into the cavity and greatly increase the surface area over which air passes; they are more complex in the cat than in the human. Evidence for continuing bone deposition can be found on this slide. The epithelium covering most of the nasal mucosa is pseudostratified ciliated columnar epithelium with numerous goblet cells. Venous plexuses are prominent beneath the epithelium of the conchae.

Olfactory Epithelium
Olfactory epithelium covers a portion of the surface of the upper conchae. This epithelium is tall pseudostratified columnar, and consists of supporting (sustentacular) cells whose nuclei are generally nearest the free surface and bipolar nerve cells (calledolfactory cells) whose nuclei occupy a wide zone deep to those of the supporting cells. Venous plexuses, the olfactory glands of Bowman (serous), and bundles of nerve fibers are found beneath the epithelium.

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RESPIRATORY SYSTEM

Palate
The palate extends posteriorly so as to form a partial division between the nasal and oral portions of the pharynx, and consists of an anterior 2/3 (hard palate) and a posterior l/3 (soft palate). Slide 34B is a section through a portion of the palate. The hard palate can be identified by the presence of bone, covered superiorly by the mucous membrane of the nasal cavity and inferiorly by the oral mucosa. Proceeding from above downward in the hard palate, one finds: l) nasal epithelium; 2) a lamina propria containing a few small, primarily mucous, glands; 3) bone; and 4) stratified squamous epithelium of the oral surface. Posteriorly in the soft palate, bone is absent and dense regular CT, muscle and large mucous glands are found. Palatine tonsils indicate that this section is lateral and caudal. Note that the passageways which carry only air have a pseudostratified columnar epithelium with goblet cells, while those which convey food or both food and air are lined by a stratified squamous epithelium.

Trachea
Slide 35B represents a portion of the wall of the trachea cut in cross section. Although the epithelium is poorly preserved, the mixed nature of the tracheal glands is well shown. Study the plastic section (36B) in which the trachea is much better preserved, including the cilia.

Lung
Slide l8A shows the lobar bronchi, which supply air to one lobe of the lung. These bronchi are in the lung, but the histology of the lung parenchyma is poorly preserved. Slide 37B is particularly useful for studying the intrapulmonary branching of the bronchial tree. Bronchi can be easily discerned under low power by the presence of isolated pieces of cartilage deep to the submucosa. More distally in the tree, the cartilage is lost giving rise to the bronchioles. The accompanying diagram will be of assistance in locating the various portions of the peripheral airway. Respiratory ducts and alveolar ducts are easily visible in this preparation. The aldehyde fuchsin section is especially useful since it emphasizes the cartilage, elastin and mucus of the goblet cells. All of the blood vessels in the lung are rather thin-walled, making arteries difficult to distinguish from veins. In general the pulmonary and bronchial arteries are close to and branch with the bronchial tree, whereas the veins (carrying oxygenated blood) run in the septa between adjacent bronchi. The pulmonary arteries have better developed elastica interna and externa than do the bronchial arteries. Slide 32B is a thinner section of lung and, therefore, best for studying many of the finer details; in this preparation elastin and cell nuclei are black, cell cytoplasm is yellow, and collagen and cartilage are red. Look for the various air passages; cilia are particularly well preserved. Study the alveolar wall; how would you characterize the pattern of the fine elastic fibers which are very nicely shown? Leukocytes also can be identified easily in this preparation. Of what is the visceral

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pleura composed? Slide 3lB (a plastic section) will reward your careful study with an appreciation of many structural details of the alveolus and alveolar ducts. The aggregates of smooth muscle cells that comprise the helical wall of the alveolar ductare very well shown as a knob-like structure between alveoli (when the muscle cells are cut in cross section). Greater alveolar cells (septal cells, type II pneumocytes) are also clearly visible among the type I pneumocytes in the alveolar wall. The greater alveolar cell bulges into the lumen of the alveolus, and is highly vacuolated due to extraction of its lipid rich multilamellar bodies (cytosomes). Note how closely the capillaries may approach the alveolar lumen. Elastin is unstained in this slide, but may still be discerned near the smooth muscle in the wall of the alveolar ducts. Ciliated cells may be present in some of the slides. Leukocytes are easily seen. Slide 37B is a relatively thick section from a lung collapsed by pneumothorax. However, more of the blood has been retained in the capillaries, and the pulmonary epithelium is in a better state of preservation. Knobs of smooth muscle marking the walls of alveolar ducts can be recognized. Study the thick section (39B) in order to obtain a three-dimensional impression of the terminal airways. Try to distinguishalveolar ducts from alveoli. Under high-dry, the cellular composition of some of the alveoli can be seen in a whole mount view. If you study the surface of the alveolar wall, you will be able to see the alveolar pores which are holes l0-l5 m in diameter between adjacent alveolus; these serve as accessory communications between alveoli. Alveolar macrophages (dust cells) may also be seen on the luminal surface of the alveolar wall.

Electron Micrographs
In EMs of the lung, identify the squamous pulmonary epithelial cells, greater alveolar cells with their laminated bodies, and endothelial cells of the capillaries. Examine the air-blood barrier and identify all the components of that barrier in thick and thin areas. EMs of the trachea show the typical appearance of ciliated columnar epithelial cells. Note the appearance of cilia and microvilli in longitudinal and cross section. Also present are portions of goblet cells with many mucigen droplets in the apical part of the cell.

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Oral Cavity
The digestive system consists of a long epithelial-lined muscular tube, beginning at the lips and ending at the anus. At both ends of the tube, the moist mucosal epithelium of the digestive tract abruptly joins the cornified integument at a mucocutaneous junction. That portion of the tube extending from the pharyngeoesophageal junction to the recto-anal junction is known as the alimentary canal, and has a common structural plan throughout.

Lips
Slide 54A shows the mucous and cutaneous surface of the lip of a 4-5 month fetus. The epithelium on the cutaneous surface is cornified (though most of the stratum corneum is lost), whereas that on the mucous surface is simply keratinized. The epidermal cells are much larger on the mucous surface than the cutaneous surface and, naturally, lack hair follicles and sweat glands. The mucous surface of the lip andgingiva is quite similar. Mucus-secretinglabial glands are just developing under the mucosa of the lip. A diagram of this slide appears in the section on Bone. Slide 63A shows the mucocutaneous junction of the lip in the adult human. Note the sudden change in the thickness and appearance of the epidermis at the vermilion border of the lip (the unhaired portion of the cutaneous surface of the lip). Some of the hairs of the lip are sinus hairs, having a large loose capsule filled with blood. Note the mature labial salivary glands . Try to find their ducts.

Teeth
Slide 54A also shows the early development of the teeth. The tooth is embedded in a connective tissue space of the alveolar bone. You should be able to find the outer epithelium (simple cuboidal) of the enamel organ which surrounds the stellate reticulum. The enamel organ is initially shaped like a ball with its bottom pushed in. The resulting concave surface is the inner enamel epithelium. The stellate reticulum is derived from the internal cells of the enamel organ through an accumulation of gelatinous intercellular substance. In your slide, the inner enamel epithelium cells have elongated into tall columnar cells called ameloblasts (enamel producing) which are separated from the stellate cells by a layer of cuboidal cells, the stratum intermedium. The whole cap-shaped enamel organ sits on the dental papilla. The mesenchymal cells of this papilla immediately adjacent to the ameloblasts also form tall columnar cells called odontoblasts (will form dentin). Dentin production precedes enamel production, and a thin gray area of dentin may be visible just under the ameloblasts. As dentin is laid down, these odontoblasts recede centripetally, narrowing the pulp cavity. The enamel will grow centrifugally. In neither case are cells incorporated into the calcified tissues, although the odontoblasts leave a cell process in the dentin. A mature tooth is visible in slide 64A. Note the cornified gingiva. The enamel has been lost in this decalcified preparation (enamel is about 95% inorganic). The pulp cavity, dentin, dentinal tubules, cementum and the periodontal ligament are visible.

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ORAL CAVITY

Tongue
The dorsal epithelium of the anterior part of the tongue has two types of papillae. Both the coneshaped and cornified filiform papillae and the mushroom-shaped fungiform papillae can be observed on slide 65A. The junction of the epithelium andlamina propria is greatly folded into papillae, much as in the skin.

Taste Buds
Taste buds will be visible in the epithelium at the apex, rather than on sides, in most fungiform papillae. The sustentacular and taste cells are difficult to distinguish. In fact, the taste buds are difficult to distinguish from secondary papillae in this preparation. In some taste buds you will be able to see the taste pore. The lamina propria of the tongue is clearly delineated from the muscular layer. Both serous and mucous lingual salivary glands are prominent in the posterior portion of the tongue. You may observe lymphoid tissue surrounding epithelial crypts at the posterior of the tongue; these are the lingual tonsils. The muscle layer of the tongue is greatly interwoven, facilitating complex movements. Slide 66A is a particularly fine section of the tongue showing a circumvallate papilla. Note the taste buds on the lateral wall of this papilla. You should be able to see a taste pore in a favorable section. The papilla is surrounded by a marginal cleft into which the ducts of the serous glands of von Ebner secrete their watery product.

Palate
The hard palate (slide 34B) consists of a bony roof over the mouth with its connective tissue and epithelium. The stratified squamous and cornified epithelium has conspicuous posteriorlydirected folds called rugae. The lamina propria is continuous with the periosteum of the bone. The soft palate continues posteriorly from the hard palate. This transition is visible on slide 34B. Note the difference in the epithelium on the oral side of the soft palate, and on the nasal side. For further explanation of the palate refer to the lab exercise on the respiratory system.

Salivary Glands
The three major salivary glands are parotid gland (slides 67A ), the submandibular gland(70A72A) and the sublingual gland (74A).

Parotid Gland
The parotid gland is described as a pure serous gland, but it does secrete some glycoproteins. Two classes of ducts are readily visible: the interlobular ducts which occur in the connective tissue septa, and the intralobular ducts surrounded by acini. Note the relationship of the vasculature to these ducts. Myoepithelial cells (basket cells) are particularly evident on the ducts and acini which have been superficially nicked in the plane of section. The nuclei of these contractile cells are spindle-shaped. Distinguish the secretory striated ducts and the intercalated ducts. Both of these ducts are intralobular in location. The basal striations of the

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ORAL CAVITY

striated duct are best seen in slide 67A. What causes the striation and what is its functional significance? Accumulation of fat cells in the connective tissue septa is characteristic of the parotid gland.

Submandibular Gland
The submandibular gland is a mixed gland having both serous and mucous components, though it is largely serous. The alveoli are either entirely serous, or they are mucous with a crescent-shaped patch of a few serous cells (called serous demilunes and best seen in slide 72A). In slide 70A, the mucous cells show a strongly positive response to PAS. Slides7lA and 72A are good for studying the ducts. You will find all of the types of ducts that you found in the parotid glands, except that in the submandibular gland, the striated ducts are longer, and thus more frequently observed. The intercalated ducts are quite short. Note the abundance of lymphoid tissue around some of the ducts in slide 7lA.

Sublingual Gland
The sublingual glands (74A) are mixed glands that are largely mucous, with some areas being entirely mucous. Serous demilunes, separated from the lumen by mucous cells, are frequently found. The gland lacks a capsule. The secretory portions of the ducts are very short and, therefore, rarely seen; the striated ducts are not found at all.

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79

Esophagus and Stomach

The alimentary tract consists of a basic structural plan throughout its length. From the lumen outward this includes mucosa, submucosa, muscularis and adventitia. The mucosa differs in each segment and is, hence, useful for identification. It is subdivided into a superficial epithelium, lamina propria, and a thin muscularis mucosae. The epithelium is often elaborated into glands extending into the lamina propria, which often contains accumulations of lymphocytes. The submucosa is a connective tissue layer containing blood vessels. The muscularis may be differentiated into as many as three layers, characterized by the orientation of the muscle fibers. The thin adventitia is often lost during preparation of the sections.

Esophagus
Slides 75A and 76A. Beginning from the lumen there is: l) a thick stratified squamous epithelium with a papillated lower border; 2) a thin lamina propria with an occasional accumulation of lymphocytes; 3) a broad muscularis mucosa consisting of longitudinal muscle fibers; 4) thick submucosa with an abundance of elastic fibers; 5) a muscularis externa having an inner circular layer and an outer longitudinal layer. At what level of the esophagus were these sections taken? Slide 75A may include ducts or secretory cells of the submucosal mucous glands.

Stomach
Slide 77A is a longitudinal section of the junction of the esophagus and stomach. The transition from stratified squamous to simple columnar epithelium of the gastro-intestinal tract is abrupt. The stratified squamous epithelium of the esophagus may appear uncommonly tall because of the oblique plane of section. For the same reason, the gastric pits may appear as small round pink-lined holes rather than tubes. These pits lead to the cardiac glands . The surface mucous cells cover the entire surface of the stomach and line the pits. These cells are stained pink in slide 77A, due to the presence of mucus.

Terminology for Glands of the Stomach

The terminology with regard to glands of the stomach is somewhat confusing. For example, the term gastric glands might be thought to indicate that these glands occur throughout the stomach, whereas they are absent from a narrow zone around the cardia and from the lower part of the pyloric region. A synonym (fundic glands) is even more misleading, for it indicates that these glands are limited to the fundic region, whereas they are much more widespread. We can consider that there are three types of glands: l) gastric glands , distributed throughout the greater part of the gastric mucosa; 2) cardiac glands , found in the cardiac region of the stomach near its junction with the esophagus; and 3) pyloric glands , confined to the region immediately above the pylorus.

Gland Cell Types

Chief cells are probably so-called because they are the most numerous gland cell. The significance of parietal is more obscure. The word means a wall surrounding a cavity or volume.

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ESOPHAGUS AND STOMACH

In ordinary fixation techniques, the zymogen granules of chief cells are not well preserved. Thus, these protein-rich cells have an unexpectedly empty or foamy interior, in contrast to the denser and more uniform parietal cells, where the mitochondria and other internal membranes (visible in stomach EM #5) are numerous and more readily preserved. In better preserved material, chief cells will clearly show many large, moderately dense granules and a basal basophilic region as the result of extensive rough endoplasmic reticulum (see stomach EM #5) and the nucleus. Parietal cells will show numerous, much smaller granules that prove to be acidophilic mitochondria, surrounding a central nucleus. Slide 78A is a cross section of the fundic stomach, which shows the branched tubular gastric glands which open into the gastric pits. You should be able to find the surface mucous cells, parietal cells and chief cells. The neck mucous cells occur at some distance below the surface mucous cells, and contain a distinct type of mucigen granules as can be seen in stomach EMs #l and #2. Neck mucous cells are easily seen only in the PAS-stained part of slide 80A. The lamina propria is relatively thin between the gastric glands, but contains smooth muscle cells (as well as most of the usual connective tissue elements). The muscularis mucosae consists of an inner circular and an outer longitudinal layer, and is closely applied to the lamina propria at the bottom of the gastric glands. The submucosa contains blood vessels, nerves and lymphatics. The muscularis externa consists of three muscle layers: an outer longitudinal, a middle circular, and an inner oblique. Slide 80A is an exceptionally thin preparation of mucosa from the fundus. The gray, iron hematoxylin stained section shows the mitochondria (black) of the cells. Mitochondria are particularly abundant in the parietal cells, which stand out rather nicely in this preparation (Some of these preparations contain spirochetes). The blue stained (PAS) portion of the slide makes it easy to distinguish virtually all of the cell types of the gastric glands, including the neck mucous cells (which are violet, compared to the blue of the surface mucous cells). This section was cut from plastic-embedded material with the ultramicrotome, and is extremely thin. The same is true for the section on slide 79A. This H & E section is particularly good for studying the composition of all the layers of the stomach. Several types of enteroendocrine cells are evident in this section. These cells are located in the bottom ends of the glands, and have a scattering of small eosinophilic granules in a relatively clear cytoplasm. These cells are part of the glandular epithelium and, as such, occur within the surrounding basal lamina which they face; this can be seen in stomach EM #4. Enteroendocrine cells should not be confused with mast cells, which have many large red granules and occur in the lamina propria.

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Intestine
Duodenum
Slide 83A shows the abrupt pyloroduodenal junction in longitudinal section. Note how much deeper the gastric pits are in the pyloric mucosa than in the mucosa of the fundus. The pyloric glands, like the cardiac glands, are of the mucous type. Many solitary lymph nodules are evident in this region. Note the large circular sphincter muscle at this junction. The muscularis mucosae appears to ride up over the Brunners (duodenal) glands , and comes to lie just under the crypts of Lieberkuhn. The muscularis mucosae does break up, however, and send branches down around the glandular elements. The slide also contains sections of lymph nodes and pancreas. Both the pyloric glands and the Brunners glands are mucus secreting (mucin stains pink). Also note the abundance of goblet cells in the mucosa. Low broadvilli are observed for the first time in the duodenum. The microvilli (striated border) on the absorptive epithelia are coated with PASpositive material. Neurons and nerve fibers of the myenteric (Auerbachs) plexus can be observed between the inner circular and outer longitudinal muscle layers throughout the small intestine. For a whole mount view of this plexus, see slide 93A. Note in studying the slides for this laboratory that the epithelium of the villus contains absorptive cells, goblet cells and enteroendocrine cells. In the epithelium of the crypt or gland, Paneth cells are located at the bottom of the crypt, and are most numerous in the duodenum and jejunum, less frequent in the ileum and absent in the colon. The duodenal cell types are most clearly distinguished in the plastic section87A. Above the Paneth cells are the stem cells (some of which may be seen dividing), and above them are cells in the process of differentiating into absorptive and goblet cells. Lymphocytes may be found scattered throughout the lamina propria of the villi at all levels.

Jejunum
In the jejunum (85A), goblet cells are much more abundant than in the duodenum. There are also eosinophilic Paneth cells at the bottom of the crypts of Lieberkuhn. Lymphocytes are abundant between the epithelial cells of the absorptive mucosa. The plastic section 86A is best for studying some of the finer structural details (e.g., the entire length of the goblet cell often can be seen, the striated border is well preserved, and the Paneth cell granules are nicely shown). The size of these granules can be contrasted with those of mast cells which are easily found within the muscle. Look for mitotic figures in the crypts on slide 86A. Some of the villi may have streamers of exfoliated cells at the tip, indicating the location of the extrusion zone.

Ileum
The ileum (slides 88A and 9lA) is distinguished by its few short, club-shaped villi and poorly developed plicae. As one proceeds through the small intestine, there is an increasing number of goblet cells; thus, the lining of the ileum contains a large number of them. Aggregates of lymphatic nodules (Peyers patches) are particularly abundant in slide 9lA of the ileum (Slide 9lA is from a cat and contains globular leukocytes, a cell type not found in man). These cells have small dark nuclei and large eosinophilic granules and are of uncertain function. The striated border, goblet cells, enteroendocrine cells and some Paneth cells can be seen in the plastic section 88A.

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INTESTINE

Colon
Slide 94A The colon differs histologically from the small intestine in that it lacks villi, having only tubular pits for glands (glands of Lieberkuhn). We can observe that the number of goblet cells is continuing to increase as we go down the alimentary canal. Paneth cells are rare. The submucosa contains some fat cells. There are numerous solitary nodules of lymphatic tissue. The outer longitudinal layer of the muscularis is grouped into three thick bands (taeniae coli), parts of which will be visible in your slides. This is not to imply, however, that there is no longitudinal muscle between the taeniae. Longitudinal fiber bundles from the taeniae can be observed to interrupt the circular layer.

Appendix
The appendix (96A) has a relatively small lumen. The mucosa is only slightly folded and has an epithelium which is largely goblet or mucous cells. The abundance of lymphatic nodules is the most striking feature. The muscularis mucosa is poorly developed.

Rectum
Slide 95A The glands of the rectum consist almost entirely of goblet cells. The longitudinal and circular layers of the muscularis are uninterrupted.

Recto-anal Junction
Slide 99A It has been a long way since we left the integument at the vermilion border of the lip until we pick it up again at the anus (about 9 meters actually). The epithelium changes abruptly from the simple columnar epithelium we have observed in the whole gastro-intestinal tract, to a stratified cuboidal and then, finally, cornified - stratified squamous epithelium of the anus. Goblet cells are very abundant in the long broad glands of Lieberkuhn of the rectum. The integument of the anus contains hairs with sebaceous glands, eccrine sweat glands and the circumanal glands which are similar to the apocrine sweat glands that you observed in the axilla. The circumanal glands are not present in all the slides; you may have to look at a neighbors preparation.

Electron Micrographs
The apical surface of columnar absorptive cells of the intestine have numerousmicrovilli. The very regular nature of the microvilli accounts for the striated border seen in the light microscope. The glycoprotein coat (fuzz) present on the microvilli accounts for the intense PAS staining of the apical surfaces, as seen in your light microscope preparations. Mitochondria are rather numerous in the columnar absorptive cells. Granular endoplasmic reticulum is not well developed, but agranular ER is often conspicuous in the apical cytoplasm. The apical cytoplasm of intestinal goblet cells is filled with mucigen droplets. The granular endoplasmic reticulum is extensively developed in the basal and perinuclear cytoplasm. In contrast to the absorptive cells, the apical border of goblet cells has only a few microvilli.

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Pancreas
Exocrine Pancreas
(Slides l0B-l2B)
The exocrine portion of the pancreas can be seen in all the slides. The pancreatic tissue in general is surrounded by an accumulation of fat. The human pancreas also contains much connective tissue, which separates the exocrine portions into lobules. The exocrine portion of the pancreas is a compound tubuloalveolar gland composed of serous secretory units. Note the location of the zymogen granules in the apical portion of the cell and the intense basophilia in the basal portion. This is nicely shown in the H & E stained plastic section (l2B).

Duct System
The lumen of eachpancreatic acinus is continuous with a small duct called the intercalatedduct. This duct is distinguished by its pale staining simple cuboidal cells (which often extend into the acinus itself) as individual pale-staining cells called centroacinar cells. These ducts are tributaries of larger interlobular ducts, lined by columnar epithelium containing a few goblet cells. The interlobular ducts, in turn, join the two main pancreatic ducts. Many83A slides have a large section of pancreas, in which the goblet cells of the major ducts are clearly seen.

Endocrine Pancreas
Identify the pancreatic islets. Differentiation of the alpha, beta and delta cells is not possible in H & E stained sections. In the aldehyde fuchsin stained pancreas, the beta cells (which synthesize insulin) have fine purplish stained granules. The alpha cells (which synthesize glucagon), have a yellowish appearance in these islets. This stain permits one to identify small groups of isolated beta cells in the pancreas. Delta cells cannot be identified in these preparations. The rich blood supply to the islets is difficult to see but, in an ideal section, under oil immersion, the capillaries can sometimes be distinguished.

Electron Micrographs
In your microscope slides, pancreatic exocrine cells were characterized by the zymogen granules in the apical cytoplasm while the basal cytoplasm was strongly basophilic. In the electron micrographs, numerous dense zymogen granules are present in the apical cytoplasm, while the basal cytoplasm contains extensive granular endoplasmic reticulum indicative of active protein synthesis. Centroacinar cells are readily identified by the low density of their cytoplasm, and the paucity of cell organelles, as well as by their position.

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Liver and Gallbladder

Liver
The basic functional unit of the liver is the acinus, but the classical lobule is usually the easiest structural unit to discern. The connective tissue capsule (Glissons capsule) which surrounds the liver is continuous with some of the connective tissue of the portal canals. In the human (and many other mammals) this connective tissue incompletely separates the lobules, whereas in other animals (such as the pig), it completely separates each lobule. The hepatic lobule has two main constituents: an epithelial parenchyma made up of hepatic cells arranged in anastomosing plates or sheets, and a system of blood channels. The blood supply to the liver is unusual in that a portion of it is a PORTAL SYSTEM. A portal system is one in which blood, after being collected from one set of capillaries, passes through a second set of capillary-like vessels before it returns to the systemic circulation. In the hepatic portal system, blood collected from the capillaries of the stomach, most of the intestine, the pancreas and the spleen is conducted by way of the portal vein to the liver, where it passes through the sinusoids before entering the inferior vena cava (by way of the hepatic veins). In the slides which follow, note the portal tracts (also known as portal triads or portal canals). Contents of the portal tract l Preterminal (interlobular) branches of the portal vein 2 Preterminal (interlobular) branches of the hepatic artery 3 Branch of the bile duct (lined by cuboidal epithelium) all in a common investment of connective tissue which may, in addition, contain lymphatics. From these branches of the portal vein and hepatic artery arise the terminal branches. These, in turn, empty their blood into the hepatic sinusoids which, in turn, empty into the central veins (the terminal branches of the hepatic vein). Note that preterminal branches are parallel to the central vein and terminal branches are perpendicular. In slide 5B of the liver, the blood supply of the liver is delineated by dye injected into the vascular bed. Central veins may be identified as the dense red areas from which sinusoids radiate toward the periphery of the lobules. After studying the terminal branches of the portal vein in this section, you should have little trouble discerning the acinar arrangement in a section of noninjected liver. Now study slide 2B, (human liver). Note that although a complete connective tissue capsule is lacking for each lobule, the lobules can nevertheless be defined by the presence of the central vein in the center and the portal triads at the periphery. Note also the characteristic pattern of the parenchymal cells in this, and other slides, and their relation to sinusoids . Slide 3B is from a dog injected in vivo with carbon black, and slide 4B is from a rat injected with Trypan Blue. These substances are taken up by the phagocytic Kupffer cells, which are clearly identified. Note the variation in shape of Kupffer cells from flat, squamous-like cells to large ones which bulge into the lumen. In slide 4B, tissue macrophages in the interlobular CT have also phagocytosed the dye. Slide 3B also shows good lobulation, dilated sinusoids, prominent nuclei and nucleoli of liver

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parenchyma. Compare nuclear size and number in liver cells on slide 3B. The thinner plastic section (7B) is best for studying cellular detail. In the sinusoid wall, Kupffer cells can be distinguished from endothelial cells by the abundance of granules (lysosomes) resulting from the greater phagocytic activity of the Kupffer cell. In perisinusoidal spaces, you will observe a number of cells with vacuoles which represent areas of extracted fat droplets; these are fat-storing cells which are stellate in shape. They tend to be more numerous in intermediate and peripheral portions of the lobule than in central zones. Their functional significance is not yet understood but it is known that vitamin A may accumulate in the lipid droplets. Circulating white blood cells are nicely shown; the neutrophils appear smaller because they have been sectioned (rather than spread out, as in a smear). Using oil immersion, search the membranes between adjacent liver cells for evidence of bile canaliculi, which are minute canals formed by the membranes of two adjacent cells. The cell membrane delineating the bile canaliculi can be stained with silver; look at slide lB to see areas where canaliculi surrounding individual liver cells are stained in this manner. What do you notice about the size of parenchymal cell nuclei? Slide 6B is a section of regenerating rat liver and, consequently, shows an unusually large number of mitotic figures. The variability in size of the parenchymal nuclei reflects the varying degree of polyploidy present in the mammalian liver. Although all of the parenchymal cells of the mammalian liver are diploid at birth, 80% of the nuclei are tetraploid by the time the animal has reached maturity. Note also the cytoplasmic basophilia evident in the cells.

Gallbladder
Begin your study of gallbladder with slide 9B. Note that the mucosa is thrown into numerous folds (which probably are not present when the gallbladder is distended), and has a tall columnar epithelium. This epithelium is responsible for reabsorption of much of the water from the contents of the gallbladder. A layer of smooth muscle and associated collagen and elastic fibers surround the gallbladder. The epithelium rests on a lamina propria of loose CT; there is no true muscularis mucosae. There is often a layer of fat cells between the underlying connective tissue of the serosa and the muscular layer. The plastic section of gallbladder (8B) is a circle-shaped transverse section displaying the three layers: (l) mucosa, a simple columnar epithelium situated upon a lamina propria; (2) a thin muscularis coat of interlacing bundles of smooth muscle; and (3) anadventitial outermost coat of irregular connective tissue containing nerves, blood vessels, and scattered adipose cells. Where the gallbladder is not overlain by liver tissue, a serosa is present. In the lamina propria are a conspicuous number of capillaries, mast cells, and, in these slides, a great abundance of plasma cells. Is a striated border present on the epithelial cells? Do not spend much time studying the muscularis which is difficult to discern in certain areas; its extent can be best appreciated at low magnification.

Electron Micrographs of Liver

In the electron micrographs identify the hepatic sinusoids . Note the thin lining of these vessels. The space of Disse lies between the cells lining the sinusoids and the surface of the liver parenchymal cells. Numerous irregular microvilli project from the surface of the liver cells into the space.

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Within the hepatic cells are dense accumulations of glycogen, numerous mitochondria and both granular and agranular ER. Some micrographs may also show some crystalloid-containing microbodies (peroxisomes). Also identify bile canaliculi; note that a canaliculus is merely an expansion of the intercellular space rather than a completely separate duct. The juncture of a bile canaliculus with a branch of the bile duct is shown on one of the micrographs.

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Urinary System
Kidney
The kidney is a highly-organized structure, whose morphology is more obviously related to its functional activity than in many other organs. The arrangement of the cortex of the kidney can be clearly seen in slide 43B (unilobar kidney, rabbit). Portions of the medulla extend into the cortex as medullary rays; a medullary ray surrounded by its immediate cortical substance constitutes a lobule. The cortex, with its medullarypyramid, is called a lobe. Portions of the cortex extend downward between the medullary pyramids. On most of your slides, the continuity of these medullary rays with the medullary tissue extending to a minor calyx (at the apex of a pyramid) can be seen. However, only a few of the slides will actually show the area cribrosa where the papillary ducts open into the minor calyx.

Blood Supply
The general pattern of organization of the kidney blood vascular system is not readily apparent. To simplify identification of all the blood vessels, look for them at the lowest magnification, and also try to find continuities from one type to another. Be sure to distinguish the arteries from the veins as you follow them in the kidney. Continue with slide 43B, but also turn to 46B as necessary. Find the corticomedullary junction and identify the arcuate vessels. Only rarely is the section in a plane which permits the arcuate vessels to be observed in longitudinal section. However, multiple cross sections of these vessels can be seen ranging in depth from the edge of the cortex to about a third of the distance into the cortex from the medulla. Interlobar vessels (from which the arcuate vessels arise) are larger and may be surrounded by connective tissue in contrast to the arcuate vessels; the interlobar vessels leave the connective tissue space, enter the renal columns and course towards the corticomedullary junction, where they branch into arcuate vessels. Radially directedinterlobular vessels are readily seen in a number of the slides (particularly 43B and 46B). The afferent arterioles arise from interlobular arteries and enter the renal corpuscle as the glomerular capillaries. The peritubular capillaries arise from efferent arterioles which exit from the cortical glomeruli (at some distance from the medulla), whereas the vasa recta arise from the efferent arterioles of the more juxtamedullary glomeruli. These can be seen accompanying the loops of Henle and collecting tubules in the medullary portions. Look for peritubular capillaries and vasa recta in the sections from injected animals (43B, 46B). How do peritubular capillaries differ in their fine structure from glomerular capillaries? The vasa recta have a counter current arrangement which plays an essential role in the formation of a hypertonic urine.

Renal Corpuscle & JG Cells

The detailed histology of the kidney (including glomerulus, nephron, and collecting tubules), can be seen most readily in the injected rabbit kidney (slide 46B), and in the plastic section (44B). Study the structures first in slide 46B and then more closely in slide 44B. At the vascular pole of the renal corpuscle is the juxtaglomerular apparatus. This consists of a portion of the afferent and efferent arterioles (along with associatedmesangial cells ) in intimate contact with a specialized portion of the distal tubule (the macula densa, see below). Juxtaglomerular cells are modified smooth muscle cells in the wall of the afferent arteriole and can be seen to contain granules (which are known to contain renin). These granules, visible under oil immersion, are PAS-positive (46B), but may also be stained after H & E alone (44B). You may find them more easily in slide 46B, but dont spend too much time searching for them. The parietal layer of

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Bowmans capsule is readily apparent. Mesangial cells cannot be distinguished in this type of preparation, but can be seen in appropriate EMs. The relationship of the foot process of the podocytes, the sieve areas of the endothelial cells and the basement lamina of the glomerulus should also be studied in appropriate EMs. What layers does the glomerular filtrate cross in its passage from the blood stream to the urinary space?

Tubular Nephron
The conduit pathway of the nephron consists of the space between the visceral and parietal walls of Bowmans capsule (the visceral wall of podocytes being closely applied to the glomerular capillary), the proximal convoluted tubule, the loop of Henle, and the distal convoluted tubule. The latter ends by joining a collecting tubule. The straight descending portion of the proximal tubule and the straight ascending portion of the distal tubule, together with the thin walled recurrent tubule which joins them, constitute the hairpin-shaped conduit segment known as the loop of Henle. The straight portions are sometimes calleddescending or ascending limbs of the loop. The predominant type of tubule near the glomeruli is the proximal convoluted tubule which is distinguished by greater thickness of the cells, eosinophilic cytoplasm, and PAS positive material in the brush border (46B). In scattered areas in certain slides, more distal portions of proximal convoluted tubules contain very large PAS-stained granules; these granules are presumed to be greatly enlarged lysosomes, specifically telolysosomes or brownish lipofuscin pigment granules. Electron micrographs of these cells reveal not only the brush border, but also many absorption tubules and lysosomal derivatives. PAS positive material can also be seen in the thick segments of the descending loops of Henle. The thin segments of the loops of Henle generally show only three or so nuclei in cross section, and the cells are low cuboidal to squamous in shape. These cells are not as flat as the endothelial cells of the vasa recta and, hence, their nuclei do not bulge into the lumen as those of endothelial cells do. This characteristic is particularly well shown in 44B. The ascending loop undergoes a transition from a thin to a thick segment well before it enters the distal convoluted tubule. If you carefully examine cross sections of the tubules in the medulla on slide 46B, you will note that ascending thick segments and thin segments are numerous, but you will find few descending thick segments. How can you account for this? It may help you to make a longitudinal drawing of the loops of Henle in the medulla. The wall of the distal convoluted tubule is modified in the area of apposition to the vascular pole of the glomerulus (specifically, the afferent arteriole); this modification is known as a macula densa, a region in which the nuclei of the distal convoluted tubule cells appear more crowded and elongated (46B). A characteristic feature of distal convoluted tubule cell (as well as of proximal convoluted tubule cell) fine structure is the interdigitation of basal folds containing prominent mitochondria. These infoldings (plus aligned mitochondria) account for basal striations.

Collecting Duct
The point of passage of the distal convoluted tubule into the curved collecting tubules may be difficult to find in histological preparations. Straight collecting tubules (in medullary rays) can be distinguished from the proximal and distal tubules, in part, by the visibility of the lateral cell borders and the regularity in the spacing of nuclei. The tubule wall is composed of two cell types, light and dark (44B). The light cells are more numerous and appear gray; the less numerous dark cells stain pink, presumably because of the higher number of mitochondria. Dark cells are not seen in the papillary region. On slide 46B, the region where the collecting tubules come

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together to form papillary ducts can be seen only in cross section.

Immature Kidney
Examine the kidney sample in slide l7B, which is very immature and, thus, simplified in structure. Here the nephron is shorter and the glomerulus is smaller. The metanephric nephrons of this fetal human kidney show many stages of development. Look for different stages in the development of the renal corpuscle. You will see examples where the visceral layer of the corpuscle is just beginning to invaginate and develop into podocytes. The loops of Henle are still quite short, and many have not extended down into the medulla.

Ureter & Bladder

Examine the ureter (48B) and bladder (5lB, 67B). The lining epithelium of these structures is transitional epithelium. This epithelium, unique to the urinary system, is well suited to the requirement for dramatic expansion and contraction. Most of the examples you will see in these slides are highly contracted and folded. In a distended epithelium, the cells become greatly flattened parallel to the surface, with the consequence that the layer of transitional epithelium is thinner. One of the two sections on slide 5lB is from a stretched bladder and one is from a relaxed bladder. It is this ability of transitional epithelium to change in morphology with stretching that gives it its name. Some of the superficial transitional epithelial cells contain two nuclei; it is known that there is a progression from diploidy to octaploidy as you approach the epithelial surface. The nature of the granules present is not known. There are no true glands in these passages of urinary secretion. Both the ureter and bladder are composed of three layers. Layers of ureter and bladder 1. Mucosa layer, consisting of transitional epithelium supported by a relatively prominent and dense lamina propria. There is no submucosa. 2. Muscularis layer, usually consisting of three layers of smooth muscle in a longitudinal, then circular, then longitudinal orientation. This is not always visible but this arrangement may be seen on slide 67B (In the upper 2/ 3 of the ureter, the outer longitudinal layer of smooth muscle is not present). The layers of smooth muscle differ from those of the GI tract in that they are penetrated by connective tissue and, thus, are divided into bundles.

3. Adventitial layer, external to the muscularis, composed of irregular dense connective tissue. In 67B, peritoneal mesothelium is seen at the abluminal rim; this layer of simple squamous epithelium (plus a thin layer of supporting connective tissue) is termedserosa. Serosa covers the upper part of the bladder whereas the remainder is covered by a fibrous adventitia.

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Endocrine System
Hypophysis
In all these slides, one should note the rich capillary supply and their close relationship to the secretory cells of these endocrine glands. In the EM book, do you find evidence of fenestrated capillaries in these glands? Slide 26B is a sagittal section of hypothalamus and pituitary of the cat. In this material you can see part of the third ventricle of the brain, and the infundibular stalk surrounded by pars tuberalis (notice the many colloid cysts in this region and the portal blood vessels). The posterior lobe (pars nervosa) is almost completely surrounded by intermediate lobe (pars intermedia ). A narrow cavity that represents the original lumen of Rathkes pouch can sometimes be seen between the anterior and intermediate lobes. Bothacidophilic and basophilic secretory cells can be identified in the anterior lobe (pars distalis). The blood vascular bed in the anterior lobe is of the sinusoidal type. Slide 23B This human pituitary sample contains anterior, intermediate and a small part of the posterior lobe. Note the colloid cysts in the intermediate lobe, and acidophilic and basophilic cells in the anterior lobe. This material was probably obtained at autopsy and, consequently, it shows considerable shrinkage and fixation artifact. Slide 24B Human hypophysis Massons trichrome stain (acid fuchsin, aniline blue, light green). Note the thin capsule of connective tissue that surrounds the gland. Basophils stain a deep lavender. Note the number and organization of these cells; most of them synthesize TSH or gonadotropins. The acidophils, which stain reddish-brown, are smaller than the basophils and have relatively large nuclei. These cells synthesize growth hormone. The chromophobes (the small number of cells that are unstained) synthesize ACTH or represent degranulated acidophils or basophils.

Thyroid
The thyroid slides show sections of thyroid follicles with their central, homogeneously stained colloid surrounded by epithelial cells. Follicles are variable in size. Within the scant interfollicular connective tissue are found the usual vascular elements, fibroblasts, mast cells and macrophages. The abundance of colloid and size (especially the height) of the thyroid follicular cells vary with the functional state of the gland. The C-cells are difficult to identify without special staining (e.g., silver or antibody to calcitonin). In routine stains, the C-cells generally exhibit more lightlystained cytoplasm, larger size (both whole cell and nucleus), eccentric nuclei, and are characteristically located in connective tissue spaces or in abluminal positions within the follicular epithelium. The latter situation is not always clear at the LM level. The best procedure to recognize the C-cells is to first register the staining and size of the numerically dominant follicular cell nuclei and cytoplasm. Then, proceed to identify cells with larger nuclei and less clumped chromatin surrounded by lighter cytoplasm (relative to the thyroid follicular cells). Find them first within the more interfollicular spaces, then in the follicular epithelium. Slide l3B shows a slightly more active gland. The slight basophilia of the follicular epithelium is apparent. The C-cell cytoplasm here does not exhibit the same basophilia. In some follicles, there are follicular cells with pyknotic nuclei. These are found even within normal glands. One of your EM pictures shows such a degenerating cell among other healthy follicular cells. Slide l4B contains both a section of the thyroid gland and the closely applied parathyroid gland. The parathyroid gland is surrounded by a thin layer of connective tissue that constitutes

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its only separation from the thyroid gland. The cells of the parathyroid are relatively uniform in size, with nuclei smaller and more basophilic than those of the thyroid cells. There are no oxyphils in the dog parathyroid gland. The follicles are lined by cuboidal to low columnar cells. Careful examination may reveal some phagocytosed colloid within these cells. The vacuoles in the pink colloid are fixation artifacts. Occasionally, C or calcitonin cells can be found in the interfollicular spaces or wedged between two follicular cells. With the slightly different stain combination used on this slide, the cytoplasm of some C-cells are slightly more orangish pink than other C-cells and the follicular cells. If you apply the criteria of size, nuclear staining and location suggested above for identifying C- cells, you will successfully distinguish them from the follicular cells. Slide l6B shows a highly active thyroid. Much of the colloid has been mobilized; some has been lost during fixation. As the result of both colloid depletion and hypertrophy (and hyperplasia) of the follicular cells, the epithelium shows infolding. There is also vascular engorgement accompanying the increased glandular activity.

Parathyroid
The section of human parathyroid on slide 22B shows many oxyphils scattered throughout the gland and much fatty tissue. What does that tell you about the age of the ex-owner of the gland? The oxyphils are easy to spot, due to their smaller nuclei and their more abundant and eosinophilic cytoplasm. The increased acidophilic staining is due to what organelle within their cytoplasm? Sometimes similarly stained cells can be seen in pathological thyroid glands. These also exhibit greatly increased numbers of the same cytoplasmic organelle. They are referred to as oxyphils also, or as Hurthle cells.

Adrenal Gland
Slides l9B and 20B demonstrate the histologic organization typical of the adrenal in most mammals. The gland is covered by a connective tissue capsule, which contains many branching arteries and nerve fiber bundles. Some sections may contain cross sections or oblique sections of the preganglionic nerve fibers coursing through the cortex to innervate adrenal medulla cells. The cortex is not innervated. Most of the capsular arteries branch and enter the cortex in the form of fenestrated capillaries. Some of these capillaries may be collapsed; others distended with red cells. The capillaries extend from the zona glomerulosa to the cortico-medullary junction, and enter into the medullary bed via a few small vessels. In slide 20B, you may find an example of a medullary artery that goes directly from the capsule to the medulla without branching. The medulla receives blood indirectly from the cortical capillaries and directly from the medullary arteries. Note the large branches of veins which eventually anastomose with the large central veins in the medulla. The cortex is divided into three zones: glomerulosa, fasciculata and reticularis. Note the size, shape, and organization of the cells in the three zones. Which zone is the largest? Are the zones easily identified?

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Adrenal Medulla
The adrenal medulla in the central portion of the gland (slides l9B and 20B) contains large polyhedral-shaped cells (often referred to aspheochromocytes because they react with chrome salts to give the chromaffin reaction). The cells are arranged in cords or aggregates which are surrounded by capillary networks. Preganglionic sympathetic nerve fibers (which penetrate the medulla) synapse with the pheochromocytes, which are thus homologous with sympathetic ganglion cells. On careful inspection, one can see a small number of sympathetic ganglion cells scattered among the adrenal medullary cells.

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Male Reproductive System

Testis
Slide 54B, of the monkey testis is particularly useful for studying the organization of the testis. Under low power, the parenchyma of the testis is seen to consist of coiledseminiferous tubules, separated in groups from one another to form lobules bounded by septa of connective tissue. These lobules appear to radiate from the mediastinum testis, a region of vascular connective tissue, which lies close to and parallels the epididymis (However, since your section is not in a precise mid-sagittal section, the mediastinum will be internal and to one side). Within the mediastinum is the rete testis, a labyrinthine system of large spaces lined by a simple cuboidal epithelium. The rete is continuous with the seminiferous tubules via straight tubules which course within the septa. The junctions of the straight tubules with the seminiferous tubules are difficult to find. The straight tubules are lined by a simple cuboidal epithelium. Note the plexus of venules which is also located within the mediastinum.

The rete testis is drained by ductuli efferentes. These are not present on your slides. They lead to the tortuous epididymis (a highly-coiled tubule lined with a very tall pseudo-stratified columnar epithelium, whose cells have the long microvilli classically called stereocilia). Find also the ductus deferens characterized by a larger lumen, a lower epithelium which shows a festooned outline and an appreciable amount of smooth muscle. Masses of spermatozoa and desquamated cells of the seminiferous epithelium will be seen within the lumina of the epididymis and ductus deferens.

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Interstital Tissue
The relative and absolute amounts of interstital tissue show wide variations among mammals. In slide 55B, from an adult monkey, interstitial cells are present in small clusters. The foamy appearance of the cytoplasm of the inerstitial cells results from the extraction of lipid during histological preparation.

Seminiferous Tubules
The seminiferous tubules (slides 54B and 55B), because they are coiled, will be seen cut at variuos angles to their long axes. Enveloping the basal surface of the tubules are cells with elongated cork-screw shaped nuclei which belong to the myoid layer. In many instances, however, these nuclei cannot be distinguished from those of immediately adjacent fibroblasts. The plastic section (55B) is especially good for studying spermatogenesis. The seminiferous epithelium varies considerable in appearance depending upon the particular stage of the spermatogenic cycle as well as the plane of section of the specific tubule. Begin by identifying cells in various stages of spermatogenesis and then consider the entire epithelium with respect tothe grouping of cells undergoing spermatogenesis in various segments of the seminiferous tubules. In this type of material, it is not possible to identify every cell, but it is possible to identify typical expamples of most cell types.

Sertoli Cells
The nuclei of Sertoli cells tend to be elongated and folded and to contain a large prominent nucleolus. The scanty heterochromatin of Sertoli nuclei is frequently seen as elongated threads parallel to the nuclear long axis. The highly branches and attenuated cytoplasm is not usually discernible but when seen appears as tufts of acidophilic cytoplasm. The cell nucleus generally lies adjacent but perpendicular to the boundary layer or among the primary spermatocytes. Look for Sertoli cells in slides 54B, and 55B.

Spermatogonia
Virtually all nuclei located at the base of the epithelium which are not in Sertoli cells are spermatogonial. Typically, these nuclei are large and contained either distinctly stained chromatin or large chromatin masses representing poor fixation of mitotically dividing cells. You may be able to distinguish light and dark spermatogonia.

Primary Spermatocytes
These cells are located in 2 or3 layers central (luminal) to the spermatogonia and contain much larger nuclei than those of the spermatogonia. The nuclei exhibit various stages of the prophase of the first meiotic division.

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Secondary Spermatocytes
These cells are appreciably smaller than primary spermatocytes. They are much less frequently seen because of the relatively short duration of this stage compared to others. It is not worthwhile searching for these cells unless you have a special interest in spermatogenesis.

Spermatids
Most tubules contain two generations of spermatids. The younger generation lies adjacent to the spermatocytes, and consists of cells with rather small nuclei containing a uniform dispersion of chromatin. The second generation of spermatids consists of maturing spermatozoa (recognized by the dense chromatin of the nucleus) which forms the head of the spermatozoan. intermediate stages are easily identified. Note that for any given stage of spermiogenesis, there is the same set of stages in that portion of the tubule. Locate a region of the tubule that contains early spermatids adjacent to primary spermatocytes near the basal side. Find the acrosomal granule, a small eosinophilic organelle beside the nucleus. Does the granule face the basal or adluminal side? Which way does the acrosome face in the mature spermatids? The structure of sperm per se is better appreicated in electron micrographs, as is the association of the acrosome with the Golgi region in spermatids, and the mitochondrial differences between Sertoli cels and the germ cell line.

Accessory Reproductive Structures Prostate

Slides 64B and 63B. The prostate consists of 30 to 50 compound tubuloalveolar glands about the prostatic portion of the urethra (64B), in a bed of dense fibroelastic connective tissue containing appreciable amounts of smooth muscle. Each gland is lines by a simple cuboidal or columnar epithelium in some regions and more often by pseudostratified epithelium in others. It is continous with the urethra at the sides of the colliculus seminalis (verumontanum), a longitudinal eminence in the posterior wall of the p rostatic urethra. The secretion of the prostate frequently (and normally) collects in the lumen of the gland as an eosinophilic onion-like mass termed a concretion. Such concretions are often found in seminal fluid and may calcify in situ. The relationships of the prostate are best send in slide 64B. The ejaculatory ducts are not present on your slides. EMS of the prostatic cells show the abundant RER of typical protein exporting cells. Slides 65B is a plastic section of the prostate of a young monkey, which is a much more active-looking gland than the older specimens we hve seen. In particular, it lacks the distended alveoli and concentrations which seem to accompany aging. The tall columnar shape of the secretory cells and their numerous apical secretory granules suggest that this is an actively secreting gland. Surrounding the tube-alveoli is a fibroelastic stroma containing smooth muscle cells (which is a characteristic feature of this gland). The smooth muscle cells stain bright red while the elastin is conspicous by its lack of stain.

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Prostate Gland: Slide 64B

Spermatic Cord
This slide (59B) shows the contents (not the coats) of the spermatic cord (the blood vessels are extraordinarily thick-walled in this sample). In this slide the three layers of muscle in the ductus deferences may be seen.

Seminal Vesicle
The seminal vesicles (slide 61B) are saccular outgrowths of the ductus deferens. The individual saccules have a highly folded mucosa which further subdivides the continous lumen of these organs. However, since all of the pockets of the lumen thus formed communicate directly, the folding consitutes an increase of surface of the mucosa rather than separate glands. The epithelium of the mucosa of the seminal vesicles is generally considered to be columnar to pseudostratified columnar. Pigment can occasionally be seen in the epithelial cells. Note the abundance of smooth muscle beneath the mucosa. Slide 60B of monkey seminal vesicles is a plastic section which shows the well preserved (and highly secretory) columnar epithelium of this gland. Note the numerous sperm that have foiund their way into the labyrinthine lumens of this gland.

Bulbo Urethral Gland

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Examine briefly this compound tubuloalveolar gland (62B). The tubular nature of some at the glandular endings is apparent, but that it secretes a polysaccharide-rich secretion is less obvious. This section does not contain the urethra.

Penis
Slide 66B. The following structures can be seen on this slide: corpora cavernosa, septum, tunica albuginea, corpus spongiosum, dorsal vein, central arteries, helicine arteries, numerous nerves, and the penile urethra.

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Female Reproductive System

Ovary
In slide 68B of a monkey ovary, numerous oocytes are seen at the periphery of the ovary. The covering epithelium can be seen on the surface of the ovary, underlain by a substantial tunica albuginea. When the granulosa cell layer is flattened and only one cell layer thick, the follicles are primordial follicles; when the granulosa cells become cuboidal and prolierate rapidly to form a stratified epithelium, the follicles are calledprimary follicles. Secondary follicles(containing a fluid-filled cavity -- the antrum) are numerous in this ovary. The larges follicles which approach ovulation are termed Graafian follicles. If there are advanced follicles on your slide, the large, vacuolated, estrogen-secreting cells of the theca interna will be nicely shown in this plastic section. You may also be able to discern the basement membrane separating the follicle cells from the theca interna. The zonal pellucida is prominent in this preparation; it is seen in both developing and atretic follicles. Some of the very large atretic follicles (because they appear mainly acellular) may at first appear to be corpora albacantia, but of course the corpora would not contain residual zona pellucida (because the zona would have left with the egg at ovulation). Purple strands in the primary oocyte nuclei are the somewhat condensed chromosomes, arrested in the meiotic prophase stage. Large lymphatic vessels are clearly distinguishable from the erythrocyte-loaded blood vessels in the ovarian medulla; in particular, distinguish between venules and lymphatic vessels by content and frequency of endothelial nuclei. The section of corpus luteum of the 25th day of menstrual cycle (slide 69B), illustrates the folding that occurs in the folidng that occurs in the human corpus luteum and the way in which the lutein cells reflect the streaming of the granulosa cells at ovulation. Note also the hyaloid nature of the corpus albicans from a previous cycle. The antral follicles present at this stage are all undergoing atresia. Compare the size of the lutein cells with the granulosa cells of the atretic follicles. In pregnancy (slide 70B), the lutein cells increase further in size andtheca lutein cells (derived fro the theca interna) can be distinguished in clusters at the periphery of the corpus luteum. The follicles (both in the ovary with the active corpus luteum and in the contralateral ovary) are in various stages of atresia, except for the primordial and primary follicles. Many of the stages of follicular development and atresia can be seen in slide 73B. you may find it useful to exchange slides with your neighbors to see various stages. Pick a moderate sized antral follicle and examine it closely. Note that a basement membrane separates the peripheral granulosa from the theca interna. Note the vascularity of the theca interna, and the presence of glandular thecal cells in this layer (but not in the theca externa). in favorable sections, the stalk of granulosa cells and the group of granulosa cells surrounding the oocyte (cumulus oophorous) will also be seen. At various places in the slide, the hypertrophy of the theca interna as it converts to interstitial tissue is apparent. Although a similar hypertrophy occurs in the human ovary (especially during pregnancy), the interstitial tissue formed is more transitory than in most other species.

Uterus
Slide 80B is a plastic section of monkey uterus in the resting stage of its estrous cycle. The myometrium comprises a thick layer of interlacing smooth muscle bundles. Many examples of the rather thick-walled intramural branches of the uterine artery may be seen. The endometrium contains numerous simple tubular uterine glands in the midst of a very cellular stroma. Scattered mitotic figures are found among the stromal cells as well as in the simple columnar epithelium

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of the uterine glands.

Proliferative Phase
On the left side of slide 78B, you will find a portion of a uterus from the proliferative phase of the cycle. Note that the glands are simple tubular structures, are relatively short, and have few branches. The stroma is dense. The epithelial cells are crowded columnar, and the lumina of the glands are relatively devoid of secretion.

Luteal (Secretory) Phase

On the right side of slide 77B is a portion of a uterus from the early luteal phase. Note that the glands are becoming coiled. Many of the nuclei are displaced from the base of the cells, which contain a basal vacuole where glycogen has been washed from the cell. Note also that there is secretion in the lumina of the glands, and the stroma is not quite as dense as in the previous stage. At the left of slide 77B, can be found a late luteal stage. Note that the glands are now highly irregular. The glandular epithelial cells are ballooned and vacuolated. In many regions the stroma is edematous and is highly vascular. Beneath the luminal epithelium the stroma shows the hypertrophy of the fibroblasts typical of the pseudodecidual reation and some leukocytic infiltration.

Menstrual Phase
On the right side of slide 78B is endometrium from early in the menstrual period. Note the extravasation of blood into the stroma, and the general loosening up and disintegration of much of the functionalis of the endometrium. Note, however, that some of the glands in the basalis are still relatively compact. It is from these twists of gland that regneration occurs. Slide 79B shows the endometrium in the first trimester of pregnancy. The conversion of stromal cells into large spherical decidual cells is clearly seen here. Note also that the glands have become relatively inconsequential.

Immature Uterus
Slide 81B is a section of the uterus of a term fetus. It includes both the body and cervix of the uterus. The transition from a stratified squamous epithelium to a mucous-secreting epithelium can be seen where the exocervix and vaginal epithelium come together. Following the epithelium into the endocervix, one eventually comes to a simple columnar mucous epithelium. The glands of the cervix are relatively simple infoldings. In the body of the uterus, more complex infoldings give an indication of the rich glandularity that will eventually characterize the endometrium. The muscle layers of the uterus are divided (for pedagogical purposes) into an inner oblique, middle circular, and outer longitudinal layer, but the fiber directions are not so clear cut as the designationn of layers would suggest. Between the circular layer and longtudinal layer, is a region through which the major vessels pass, the area vasculosa.

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Slide 82B is of one wall of the adult cervix. At this stage, stratified squamous epithelium extends into the external OS where it changes abruptly to the simple columnar epithelium of the cervical canal. The endocervical glands are well developed. In the vaginal portion of the cervix (portico vaginalis), the stratified squamous epithelium may occasionally obstruct the opening of the cervical glands, resulting in so-called Nabothian cysts. You may find a few places on your slide where the cervical glands open through the stratified squamous of the portico vaginalis.

Vagina
Slide 83B of monkey vagina shows that the more superficial epithelial cells appear empty (except for nuclei), because the high glycogen content (which is characteristic of this type of epithelium) has been washed out by the histological technique.

Oviduct
On slide 75B, you can see the typical folds of the mucosa of the oviduct. Note that the mucosa runs directly down to the muscularis, without the intervention of a submucosa. Note also the dense stroma. Both the complex folding of the mucosa and the numerous cilia indicate that this section is in the ampullary portion of the oviduct. Slide 74B includes sections of the isthmic and ampullary portions of the oviduct. The muscular organization and the well-developed ciliated columnar epithelium of the monkey oviduct are shown by slide 88A.

Placenta
Nowhere in the class Mammalia is there an organ more variable in structure than is the placenta. Within genera (and in most cases families) the variation is not very great, but the difference between orders and, in some instances, suborders is pronounced. One of the bases by which the different types of placenta are characterized is the relationship of the maternal tissues to the Female Reproductive System

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fetal trophoblast or chorion. When the fetal trophoblast is in direct contact with maternal blood, the placenta is called hemochorial. The human is an example of villous hemochorial chorioallantoic placenta. An understanding of the complex interactions that take place in the establishment of the placenta cannot be achieved by examining a few representative sections. It should be possible, however, for you to see some of the changes with age that occur in the human placenta.

First Trimester Placenta

Slide 90B passes through part of the myometrium, the endometrium and into the intervillous space. There is a spongy zone of dilated endometrial glands beneath the compact region of enlarged stromal cells (decidual cells). At the surface of the basal plate, you can see a discontinuous layer of red fibrin and many giant cells (clumps of syncytial trophoblast). In addition, there are regions where anchoring villi form portions of the basal plate. Syncytial trophoblast is not interposed between cytotrophoblast and maternal tissue at the bases of the anchoring villi. The cytotrophoblast cells nearest the connective tissue stroma of the anchoring villus are basophilic and compact, but as the cells extend toward the maternal side of the basal plate, they become vacuolated and are more loosely arranged. Numerous free villi are seen in the intervillous space, but the section does not extend through the entire thickness of the placenta to the chorionic plate . Slide 86B is useful for studying the free villi of the early placenta. The characteristics of the early placenta shown include a nearly complete cytotrophoblast layer (Langhans layer) underlying the syncytial trophoblast, numerous Hofbauer cells (vacuolated macrophages characteristic of the early placenta), and many nucleated erythrocytes.

Midtrimester and Term Placenta

The general topography of the placenta may be appreciated by examining slide 9lB of the midtrimester, and 89B of the term placenta. The narrow margin of both of these sections is the edge of the placental disc where the chorion frondosum becomes chorion laevae, (and where consequently, the basal and chorionic plates are closely apposed). Note the increased fibrin in the chorionic plate of the term placenta, and that the residual anchoring villi at the basal plate are fibrotic and largely devoid of cytotrophoblast. Examine the free (terminal) villi of the term placentas in slide 87B. Note that there are relatively few cytotrophoblast cells, and the nuclei of the syncytium are clumped. Blood vessels are close to (and even indenting) the syncytium. There is an increased density of the connective tissue, and in many regions fibrotic areas can be seen. Slide 93B dramatically demonstrates the functioning of the 3rd trimester placenta in counter-current exchange between fetal and maternal circulations. Careful examination of the epithelium will reveal only occasional cytotrophoblast cells (lighter cytoplasm), and many syncytial knots.

The Umbilical Cord

The umbilical cord (slide 96B) carries two muscular arteries and one muscular vein. The vessels lack elastica internae and adventitia, their muscular coats being embedded directly in the thick cylinder of mucous connective tissue in which they are carried. This tissue consists of stellate fibroblasts, scattered through a gelatinous ground substance known as Whartons jelly. The

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mechanical properties of the ground substance and the vessel walls permit the umbilical to be twisted and, occasionally, even knotted without occlusion of the circulation.

The Female Breast

Nonlactating Mammary Gland
Slides 92B, 94B, and 95B include three different functional states of the human mammary gland. The gland of a nonpregnant adult (92B) consists largely of a connective tissue stroma and fatty lobules. Sections through the tubular epithelium of the ducts appear blue, because of the close proximity of nuclei in these areas. During this stage, the non-functional mammary gland consists of islands of ducts in a loose connective tissue stroma surrounded by dense interlobular connective tissue stroma. There is substantial lymphocyte infiltration in this preparation. Slide 94B is an involuted gland(postmenopausal), consisting of scattered large ducts and connective tissue with very little fat. Both the columnar epithelium and myoepithelial cells remain healthy.

Lactating Mammary Gland

The lactating gland (95B) shows the expanded alveoli filled with a pink-staining secretory material. The dense connective tissue is considerably reduced between the glandular lobes. The interlobular ducts are large, and have stratified columnar epithelium. Some of these sections contain a portion of the base of the nipple, showing several lactiferous sinuses surrounded by a dense connective tissue matrix. Slides 99B and l00B are from monkey, and provide another opportunity to compare the resting and functional state of the mammary gland. The alveoli in slide l00B are congested with milk. What hormone could you give to cause emptying of these alveolar spaces? What types of stimulation cause this hormone to be secreted in the nursing mother? Do you see evidence of mitotic activity in the alveolar cells in the lactating gland? How might this be related to reports of lower incidence of breast cancer in women who have experienced extended periods of lactation? Locate the myoepithelial cells. Would you expect to find nerve endings on these cells in studying electron micrographs of these regions?

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Glossary
A
Alcian blue - Stains complex carbohydrates rich in free acidic groups. Connective tissue mucins, most epithelial mucins and mast cell granules are stained a turquoise blue color. Aldehyde fuchsin - Stains elastic fibers violet to purple. Secretory granules in mast cells, gastric chief cells, pancreatic B cells, beta cells of the pituitary, and mucins also stain purple.

B
Bodian silver - A technique for impregnating nerve axons with reduced silver (black). The nuclei and Golgi of most cells also stain with this method.

C
Carmine - A basic dye used as a counterstain for vital dyes, in the Mucicarmine technique, and for coloring gelatin used to inject blood vessels. Copper-chrome hematoxylin (CCH) - Stains mitochondria deep blue against a yellowish background.

F
Fast green - An acid dye used as a counter stain (rarely used alone) that stains connective tissue a light green. Formalin - The most common fixative for routine light microscopy, consisting of an aqueous solution of formaldehyde. Works by binding to certain side groups of amino acids to form methylene bridges between protein molecules. Aldehydes allow lipid extraction but penetrate tissues quickly.

H
Hematoxylin and eosin (H & E) - The most common histologic stain used for routine study of general morphology. Stains nuclei blue and practically all cytoplasmic structures red. Those constituents staining blue are commonly called basophilic and those staining red, acidophilic. Pronounced basophilia in the cytoplasm of cells usually indicates a high level of RNA and protein synthesis (such as is observed in developing organs in the embryo, or in cells of the adult organism) e.g., the pancreatic acinar cells. As a general rule, the basic components of a tissue stain with acidic dyes and so are called acidophilic whereas the acidic components stain with basicdyes and are called basophilic. Hematoxylin and orange G-erythrosin (H & OGE) - A general purpose stain for morphology. The hematoxylin primarily stains nuclei and other basophilic constituents of the cell, if any. Orange G is a rather strongly acid dye which stains acidophilic components (such as the cytoplasm) an orange-red color. The erythrosin is also an acid dye, but stains some structures (such as smooth muscle) a light pink.

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GLOSSARY

I
Iron hematoxylin (IH) - This stain is particularly useful for secretory granules, but in addition stains chromatin, nuclei, mitochondria and cross striations of voluntary muscle an intense blue. The stain is not specific for anything other than dense protein in any tissue. The iron (iron ammonium sulfate) actually serves as a mordant, to which the hematoxylin can bind.

M
Mallory trichrome (M) - This stain consists of three dyes: acid fuchsin, aniline blue and orange G. Stains collagen an intense blue, mucus and amyloid various shades of blue; and nuclei, cytoplasm, elastic fibers, neuroglia, myoglia and fibrin red. Erythrocytes and myelin sheaths stain yellow to orange. Metachromasia - A shift in the absorption spectrum of a basic dye, such as thionin or toluidine blue. This results from interaction between dye molecules when they are bound close enough together (within .5-.7 nm) to a series of uniformly-spaced negative sites such as exist in heparin, other acid mucopolysaccharides and nucleoproteins. Tissue substances that are stained metachromatically do not appear to be the same color as the dye used but, rather, acquire a reddish shade. Methylene blue - A common basic dye and counterstain. Mucicarmine (Muci) - An aluminum carmine compound that stains mucins a bright red.

P
PAS-PTH - A combination stain of periodic acid Schiff and phosphotungstic acid hematoxylin. It allows the simultaneous staining of mitochondria, muscle fibers, etc., with basement membrane, glycogen, etc. Periodic acid Schiff (PAS) - A histochemical technique which stains structures rich in polysaccharides, mucopolysaccharides, glycoproteins and glycolipids. The periodic acid selectively oxidizes l,2 glycols and l,2- amino alcohols, thus splitting off free aldehydes (which are then detected in situ by forming a stable complex with the Schiff reagent to form a reddish-purple color). Tissue constituents which are stained various shades of red by this technique include glycogen, basement membranes of epithelia, mucins, and colloids of thyroid and anterior pituitary. It is most frequently used in your slide collection to show basement membranes and the glycocalyx. Phosphotungstic acid hematoxylin (PTAH) - A common technique for staining mitochondria a deep blue. Collagen and ground substance of cartilage and bone stain yellow to brownishred. Many fibrous elements of tissue (such as fibroelastic fibroglial, myoglial and neuroglial fibrils, striated muscle fibers and fibrin) stain blue. Coarse elastic fibers may stain purple.

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GLOSSARY

R
Resorcin-fuchsin (RF: Weigerts resorcin fuchsin) - A stain for elastic fibers, deep purple to violet. Nuclei stain paler red, cartilage matrix stain violet and cytoplasm yellow.

S
Silver methods - All of the silver techniques entail the impregnation of blocks or sections of tissue with silver compounds (AgNO3) and a subsequent reduction of the so-called argyrophilic tissue elements to free silver. The various techniques are used to demonstrate nerve axons, reticular fibers and melanin.

T
Tetrachrome - A mixture of hematoxylin, orange G, fast green, and chromotrope 2R, an acid dye that stains the cytoplasm mildly pinkish. The orange G stains the erythrocytes orange in this preparation.

V
Verhoeffs hematoxylin (VH) - An empirical stain which stains elastic fibers a clear blue-black or black. Fibroglia, myoglia, neuroglia and fibrin may stain pink.

W
Wrights stain - A mixed stain containing eosin and a series of methylene blue derivatives, especially azure B. Used almost exclusively for blood smears, marrow smears, etc. When handled properly, this mixture stains myloid cells as follows: Erythrocytes - pink; nuclei - deep blue; basophilic granules - deep purple. Eosinophils - granules red to red-orange, bluish cytoplasm. Neutrophils - granules - reddish brown, pale pink cytoplasm. Monocytes -azure granules. Lymphocytes - granules more reddish than monocyte granules, sky blue cytoplasm. Platelets - violet to purple.