Histology Lab Manual

55-357: Animal cells and tissues Lab Manual 1 ______

55-357 Animal Cells and Tissues

Laboratory Manual

Department of Biological Sciences University of Windsor Windsor, Ontario. N9B 3P4 Aliya U. Zaidi, Dora Cavallo, Barbara S. Zielinski

Updated: 08/2007 – Alyson J. Laframboise & Barbara S. Zielinski 55-357: Animal cells and tissues Lab Manual 2 ______

Autumn 2007

Labs start on Sept. 12th

Laboratory Date

1. Sept. 12/13: Lab 1: a. Alignment of the brightfield light microscope b. Epithelium: simple, pseudostratified, transitional, stratified

2. Sept. 19/20: Lab 2: a. Epithelium - glands b. Connective Tissue: loose, dense, adipose,

3. Sept. 26/27: Lab 3: a. Connective Tissue: i. cartilage, ii. bone

4. Oct.3/4: Lab 4: Quiz 1 a. Blood b. Muscle Tissue

5. Oct. 10/11: Lab 5: a. Nervous Tissue b. Eye and ear

6. Oct. 17/18: Lab 6 a. Quiz 2 b. Digestive System: I. Oral cavity, esophagus, stomach

7. Oct. 24/25 Lab 7: a. Digestive System II. Esophagus and G-I Tract

8. Oct. 31/Nov.1 Lab 8 a. Quiz 3 b. Digestive System III. Compound Glands

9. Nov. 7/8Lab 9: a. Circulatory System b. Endocrine System

10. Nov. 14/15 Lab 10: a. Quiz 4 b. Reproductive System

11. Nov. 21/22 Lab 11: a. Integumentary System b. Respiratory System

12. Nov. 28 during the lecture period Final Lab Test

2 55-357: Animal cells and tissues Lab Manual 3 ______

This lab manual is to be used only as guide during the laboratory sections of 55-357. Progress in this course depends entirely on knowledge and understanding of the structure of the histological preparations on the microscope slides. Students are encouraged to spend the full laboratory periods examining the microscope slides provided.

. There will be question for locating a cell or tissue on a slide during each quiz. Each student must write the quiz for his or her assigned lab section (no exceptions). There will be 4 quizzes. The top three quiz grades will be used for the lab grade assignment. Quizzes must be written in the assigned lab period. Any missed quiz will be assigned a grade of 0. Professionalism – treatment of microscope, interaction with fellow students and TA

Grade distribution for the lab (30% of the final grade).

Each quiz is 3% (3 x 3 = 9)………….. 9 % Lab book……………………………… 3% “Professionalism”…………………….. 3% Final Lab Test………………………...15%

Laboratory 1

Overview of components requiring ID:  Simple epithelia – squamous, cuboidal and columnar  Psuedostratified columnar epithelium  Transitional epithelium  Stratified squamous epithelia – nonkeratinizing and keratinizing

Part 1. Intro to Light Microscopy

There are several things that you need to keep in mind when working with light microscopes and slides:  Handle microscopes and slides with extreme caution –carry microscopes with two hands, supporting the scope by the base and the arm  When you are finished with the scope, make sure you turn down the light intensity and/or turn off the light source  Put the scope back on the lowest objective before removing a slide  All the microscopes should have a number. At the end of the lab, put your microscope back in the locker with the same number. Remember to wrap the cord around the scope and to cover it with a dustcover  Never change objective by grabbing the objective lens – use the rotating nosepiece  Replace slides in the boxes as you find them; that means right-side-up and facing forward Failure to adhere to these rules will result in a reduction of participation/good scientist marks

A note on histological preparations: most of the slides we use in this lab are stained with hematoxylin and eosin, otherwise known as H&E. Stained cells and tissues are either basophilic or acidophilic. Basophilic cells/tissues are stained by hematoxylin and appear as blue or purple. Acidophilic cells/tissues are stained by eosin and appear as pink or red.

Part 2. Alignment of the Light Microscope

Purpose: To direct the light from the microscope’s light source onto the specimen. If the microscope is not properly aligned, you will be unable to achieve the sharpest image possible. The best image is always in the centre of the field of view where the light is most focused. It is important to align your scope at the start of EVERY LAB  Put a microscope slide onto the stage of the microscope  Swing blue filter out of the light path  Focus specimen with 10X objective, compensation lever at M, transformer at 5-6 (on older scopes)  Close both iris diaphragm of the condenser and field stop of the illuminator  Bring field stop into focus by adjusting the condenser  Set small circle of light in center of field by manipulating the two condenser centering screws  Open field stop slowly so that the entire field can be seen  Remove eyepiece and open iris diaphragm slowly so that the entire field is illuminated  By adjusting the lamp centering screws, bring light to maximum intensity and center it (only on older models)  Replace eyepiece and swing blue filter back into place  View tissue on slide

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Part 3. Epithelium

Epithelium is a basic tissue that covers the body surfaces, lines body cavities and constitutes glands. It is avascular and is classified on the basis of cell shape of the uppermost layer of cells and by the number of cell layers.

Simple epithelium consists of a single layer of cells whereas stratified epithelium is at least two cell layers thick. A pseudostratified epithelium is only one cell thick but gives a false impression of layering. 4 55-357: Animal cells and tissues Lab Manual 5 ______

Modifications

 Simple Squamous Epithelium

The width of these cells is greater than their height, they give a scale-like appearance. Location: Luminal lining of blood vessels (e.g. slide 15).

 Simple Cuboidal Epithelium

Cells have the same height, width and depth and appear square in cross section. Nuclei located in the centre of each cell.. Location: Covers the surface of the ovaries (slide 66), collecting tubules of the kidney (slide 56)

 Simple Columnar Epithelium

The height of these cells exceeds their width and they appear column shaped with basal nuclei. This epithelium is usually modified for secretion or absorption, and may contain mucus-producing goblet cells. This is called simple secretory epithelium. A thin, refractive layer present on the free border of these cells is the striated or brush border Location: Luminal surface of the stomach and intestine (slides 34-43).

 Pseudostratified Ciliated Columnar Epithelium (with Goblet Cells)

All the cells touch the basement membrane but not all cells extend as far up as the free surface. The nuclei are visible at more than one level creating a false impression of layering. This epithelium contains many goblet cells in most areas. Location: Luminal surface of trachea (slide 24)

 A pseudostratified columnar epithelium that is not ciliated and does not secrete mucus: Location: Luminal surface of the ductus epididymis (slide 60 or 61)

 Transitional Epithelium

An intermediate between squamous and cuboidal. The presence of this epithelium allows for stretching. The cells appear bigger at the free surface and tend to decrease in size as they approach the basement membrane. Location: Luminal surface of the urinary tract (slides 57,58, or 59)

 Stratified Squamous Nonkeratinizing Epithelium

The word squamous describes the superficial cells, the basal cells are columnar and the cells in between these cells are polyhedral. This tissue does not produce keratin. Location: Lines the surface of the tongue (slide 28) , the esophagus (slide 31), the skin (slide 20), and the vagina.

 Stratified Squamous Keratinizing Epithelium

The cells at the apical surface (top) are small and cells closer to the basement membrane are larger. The cells at the free surface (top) are scales of soft keratin. This epithelium provides protection to surfaces that are constantly exposed to air, abrasion, microbial infection and resists water loss and uptake. Location: Lines the outer surface the skin (slides 21-22).

Laboratory #2: Epithelium: glands. Connective Tissue: loose, dense, adipose ______

Overview of components requiring ID:  Mucous glands  LCT – collagen and elastic  Goblet cells fibers  Serous glands  LCT – fibroblasts and mast  Mixed glands cells  Loose connective tissue  Dense connective tissue – regular and irregular  Adipose tissue – adipocytes

Part 1: Glands

 Glands

Glands are epithelial cells that are specialized to synthesize and secrete a specific product. The two types of glands are classified according to the types of secretion they produce: mucous and serous; however it is important to remember that these are TYPES of glands, not NAMES of glands. The entire secretory unit of a gland is called an acinus (plural: acini) and each acini is made up of several secretory cells. Mucous glands There are two types of mucous glands: multicellular glands (containing acini) and goblet cells, which are unicellular mucous-producing glands (1) Multicellular – mucous is washed out during processing, leading to a pale-staining secretory unit with peripherally located, flattened nuclei (e.g. slides 34, 46) (2) Goblet cells – found in simple and pseudostratified columnar epithelia in the respiratory and digestive systems (slides 41-143)

Serous glands Enzymes and proteins produced by these glands remain after processing, thus the secretory unit is triangular and dark-staining with central, spherical nuclei Parotid gland (slide 51)

Mixed Glands Secretory units are a combination of mucous and serous units Usually contains serous demilunes, half-moon shaped caps sitting on top of mucous acini Submandibular (49) and Sublingual glands (slide 50) ______

Part 2: Connective Tissue

Connective tissue is another basic tissue which supports, interconnects and nourishes other tissues.

Modifications

 Loose Connective Tissue (Areolar Connective Tissue, slide 1)

Collagen Fiber Wavy, unbranched, wide structures, light staining

Elastic Fiber Straight, narrow, branching structures, dark staining

Fibroblast Spindle-shaped with wide cytoplasmic processes; oval nuclei which are paler and larger than those of macrophage; nucleoli prominent due to active protein synthesis Produce all fibers

Fibrocyte Inactive fibroblasts – appear rounder Appear bipolar with no processes visible

Mast Cell Produce heparin and histamine Large cell with a spherical nucleus Mast cell granules are eosinophilic (red)

Macrophage Eliminate infectious microorganisms, cell debris Eccentric nucleus shaped like a kidney bean Large cells which may contain ingested materials ______

 Dense Connective Tissue

Fewer cells than loose connective tissue and is less vascular. There are two modifications of this tissue based on the arrangement of the fibers.

Dense Regular CT (slide 2) Parallel arrangement of collagen fibers (i.e.) tendons and aponeuroses

Dense Irregular CT (slide 57) Collagen fibers arranged in many different planes Locations: organ capsules, dermis of the skin (slide 20)

 Adipose Tissue (slide 70)

Fat cells (adipocytes) are seen in loose connective tissue. Lipid becomes extracted from tissue sections and therefore cells appear clear with a flattened nucleus located at the cell periphery.

Laboratory #3 Connective Tissue: cartilage, bone.

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Overview of components requiring ID:  Hyaline and elastic cartilage – chondrocytes, lacunae  Intramembranous ossification – trabeulae, osteons, osteocytes, lacunae, osteoblasts  Endochondral ossification – including all zones  Smooth muscle  Skeletal muscle  Cardiac muscle

 Cartilage Supporting, flexible tissue which is avascular. Much of the cartilage that develops prenatally is replaced by bone tissue. The cells embedded within the matrix are termed chondrocytes. There are three types of cartilage but for the purposes of this lab, only hyaline and elastic cartilage will be examined.

Hyaline Cartilage (slide 24) Characterized by a homogeneous matrix Chondrocytes are located in lacunae and contain capsules Cell nests (many chondrocytes in one lacuna) are present

Elastic Cartilage (slide 25) Resilient form of cartilage adapted to withstand repeated bending Similar to hyaline except its matrix contains elastic fibers

Bone Bone matrix is heavily calcified and the embedded bone cells which are called osteocytes also occupy lacunae. Canaliculi interconnect lacunae. Bone is a vascular tissue as opposed to cartilage. There are two types of bone development, intramembranous and endochondral ossification which give rise to the same kind of bone tissue.

Components of bone: The osteon (AKA Haversian system) is the basic histological unit of compact bone; consisting of osteocytes organized around a central canal (AKA Haversian canal) and separated by concentric lamellae Osteoblasts produce bone matrix

Intramembranous Ossification (slide 5) Flat bones that form the cranium and facial bones Slender, bony trabeculae composed of osteons Osteoblasts are located on the periphery of the trabeculae

Endochondral Ossification (slide 4) Long bones form in this manner where each long bone is preceded by a temporary cartilage model. The cartilage is then gradually replaced by bone. Four zones are seen during this bone growth.

 Zone of Resting Cartilage – Chondrocytes are not actively contributing to bone growth and are seen distributed singly or in small groups in their lacunae.

Zone of Proliferating Cartilage – Chondrocytes are actively undergoing division. These proliferating cells rapidly become arranged into columns.

Zone of Maturing Cartilage – The chondrocytes remain arranged in columns but have hypertrophied and are filled with accumulated glycogen and lipid; the cells are therefore pale-staining.

Zone of Calcifying Cartilage – This is the zone where insoluble calcium salts are deposited into the cartilaginous matrix. Capillaries with associated osteogenic cells can grow into this zone thus providing a vascular environment in which osteoblasts can differentiate.

Laboratory 4

Blood, Muscle Tissue,

Blood (slides will be provided by TA) Blood contains red blood cells or erythrocytes, white blood cells or leukocytes and cytoplasmic fragments known as blood platelets.

Erythrocytes (Red Blood Cells) Anucleate, biconcave, hemoglobin content causes their pink colour ~7m in diameter Reticulocyte Immature erythrocytes with residual rRNA Slightly larger than erythrocytes with a blue tinge

Leukocytes (White Blood Cells) Agranular (mononuclear) Leukocytes Lymphocyte - round or slightly indented nucleus very little cytoplasm second most abundant leukocyte

Monocyte - largest leukocyte nucleus is indented or horseshoe-shaped

Granular (polymorphic nuclear) Leukocytes Neutrophil - Most abundant leukocyte Segmented nucleus with 2-5 lobes Mauve granules

Eosinophil - Larger than neutrophils with a 2 lobed nucleus Acidophilic granules that stain red

Basophil - Rarest blood cell and therefore difficult to find 2 or more lobes Bluish green granules

Platelets Cytoplasmic fragments which appear purplish in colour

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Muscle Tissue

Muscle cells are also known as muscle fibers which produce contractions. Three distinct muscle types are recognized: smooth (involuntary) and the striated muscle types: skeletal (voluntary) and cardiac muscle.

Smooth Muscle (slide 16, wall of the artery; 38, 39) These fibers contain one centrally located nucleus per cell with no cross-striations. It is quite difficult to differentiate between smooth muscle and dense regular connective tissue.

Striated Muscle types: Skeletal Muscle (slide 6) These muscle fibers can be identified on the basis of their peripherally located nuclei, multi- nucleated cells and cross-striations. Be sure that you view an H&E stained slide.

Cardiac Muscle (side 8) These muscle fibers contain centrally located nuclei. Their most distinctive feature is the presence of intercalated discs distributed amongst many cross-striations. The fibers undergo tremendous branching Part 1: Nervous Tissue Muscle Tissue

Overview of components requiring ID:  White matter (neurons/somata) & grey matter  Ganglion – satellite cells  Myenteric Plexus  Meissner’s corpuscle & Pacinian corpuscle  Red blood cells  All white blood cells

**Note on this lab: this lab contains a huge amount of information, but there is very little actual identification required. You are, however, responsible for knowing all of the details included. It is all testable material.

Lab 5 Nervous Tissue, Eye and Ear

The Nervous System: The nervous system is the system that controls the actions and reactions that the body undergoes in response to stimuli produced by its internal and external environment. The nervous system can be divided into the central nervous system (CNS) which includes the brain and the spinal cord, and the peripheral nervous system (PNS) which incorporates nerve fibers and groups of neurons outside of the CNS. The PNS is also subdivided into the somatic (voluntary) and autonomic (involuntary) nervous system. Neurons are the main cell type in the nervous system. Their function is to sense a stimulus and respond to it. A neuron is composed of a cell body termed a soma (plural: somata) which contains the nucleus, and multiple cytoplasmic processes termed dendrites and axons. Dendrites receive information from a stimulus and axons transmit that information to other cells. Nissl bodies are termed for the rough endoplasmic recticulum and polysomes found in the cytoplasm of the soma. Nissl bodies are basophilic and easily detected against the pale staining of the soma. Neurofibrils are termed for other organelles and microfilaments.

Dendrites A neuron may have one or more dendrites. Sensory neurons contain one dendrite per neuron and motor neurons contain two or more. Dentrites are generally shorter in length than axons, and transmit information towards the soma.

Axons All neurons contain only ONE axon and it may vary in length. Axons transmit information away from the soma. The area which connects the soma to the axon is called the axon hillock. The axon hillock is the initial segment of the axon which is unmyelinated and contains no Nissl bodies. The cytoplasm of the axons is called the axoplasma. Axons may or may not contain a myelin sheath made up of lipids. The myelin is produced by Schwann cells that wrap the myelin around the axon. During sectioning of the tissue the myelin is dissolved and thus appears as a clear space under the microscope though the Schwann cells are visible. Nodes of Ranvier are points on the axon where the myelin pinches in toward the axon. There is no myelin sheath at the Nodes of Ranvier, and electrical impulses travel down the axon by jumping from one Node of Ranvier to another. This method of transmission – termed saltatory conduction – is a faster method of transmitting information down the axon

Central Nervous System (slide 73 or 74) The CNS encompasses both the brain and spinal cord. Here we will focus only on the spinal cord of the CNS. The spinal cord is composed of gray and white matter

Gray Matter In a cross section of the spinal cord the gray matter encompasses the central region of the spinal cord that is H shape and contains the central canal. The gray matter is composed of motor neurons that elicit responses. The axons of the neurons in the gray matter are unmyelinated and together with the dendrites they constitiute the neuropil. The term neuropil is Greek for felt-like and thus the reason for the gray appearance under the microscope.

White Matter The area that surround the gray matter. It does not contain any neuron cell bodies. The axons it does contain are myelinated, and thus appear clear under the microscope, hence white matter.

The cells that produce myelin in the CNS are called oligodendrocytes as opposed to Schwann cells in the PNS. The white matter also contains astrocytes and microglial cells.

Neuroglia are found in the CNS. The four types of neuroglial cells are oligodendrocytes, astrocytes, microglia and ependymal cells.  Oligodendrocytes are analogous to Schwann cells in that they produce myelin around the axons of the white matter.  Astrocytes are cells that provide mechanical and nutritional support.

 Microgial cells are resting macrophages and become active when there is damage to the CNS.  Ependymal cells are epthelial cells which line and support the central canal. All these cells except for the epedymal cells contain fine cytoplasmic processes that are visible under silver or gold staining.

Peripheral Nervous System. The PNS is the division of the nervous system that includes nerve fascicles and ganglions. Ganglions are groups of nerve cell bodies and nerve fascicles are bundles of nerve fibers

Ganglia (slide 72): Ganglia (singular: ganglion) are collections of sensory neuron cell bodies. Surrounding the cell bodies is a layer of flat satellite cells (capsule cells) which provide support.

Myenteric Plexus(slides 38-42) – nerves found in-between the circular and longitudinal muscle fibers in the muscularis externa of the digestive tract.

Nerve Fascicles (slide 75): Nerve fascicles are nerve fibers (axons) grouped into bundles. There are three membrane layers around the nerve fibers. The three layers are as follows: i) Endoneurium - loose connective tissue around each individual nerve fiber ii) Perineurium - dense irregular connective tissue around a group of nerve fibers (fascicle) iii) Epineurium - dense irregular connective tissue surrounding all groups of nerve fibers

Neurolemma is the myelin sheath produced by the Schwann cells around each axon. It takes many Schwann cells to myelinate a single axon. The Schwann cells may also be visible in the cross sections of the fascicles.

Specialized Sensory Receptors (slide 19) These encapsulated sensory receptors transmit specialized impulses from the PNS to the CNS. Three specialized sensory receptors are as follows: i) Meissner’s Corpuscle – mechanoreceptor sensitive for touch and found in the papillary layer of the dermis (epithelium layer) of the skin. It somewhat resembles a light bulb.

ii) Pacinian Corpuscle – pressure receptor found in the dermis and subcutaneous level of the skin. It is the largest of the encapsulated receptors and it resembles an onion bulb in cross section

The eye and the ear.

Overview of components requiring ID:  Eye – 8 layers of the retina  Parts of the cochlea (see list below)

The Eye (slide 76) The eyes are the elaborate paired sensory organs necessary for the sense of sight.

Retina The retina is the image-sensing component of the eye and is also known as the neural retina. It is the light- sensitive portion of the eye and contains photoreceptors called cone and rod cells. There are 8 layers in the retina

In the order from which light enters the eye, they are:

 Ganglion Cell Layer o This layer contains large, ganglion neuron cell bodies and scattered neuroglia cells.  Inner Plexiform Layer o The axons of bipolar cells synapse with the dendrites of ganglion and amacrine cells.  Inner Nuclear Layer o This layer contains the nuclei of bipolar cells, horizontal cells and amacrine cells. 12 55-357: Animal cells and tissues Lab Manual 13 ______

 Outer Plexiform Layer o The axons of the photoreceptors synapse with the dendrites of bipolar and horizontal cells. Bipolar cells relay impulses to the ganglion cells and horizontal cells interconnect photoreceptors laterally. This layer is more lightly stained  Outer Nuclear Layer o This darkly-stained layer contains the nuclei of rod and cone cells.  Inner Segment (aka External limiting membrane) o This layer contains the inner segments of the photoreceptor cells which contains the cell’s metabolic machinery.  Outer Segment (aka lamina of rods and cones) o This layer appears feathery and contains the outer segment of the rods and cone cells which is composed of membranous disks that contains the photosensitive visual pigment, rhodopsin.  Pigment Epithelium o This outermost layer absorbs the light that has passed through the retina thus preventing reflection back into the eye and maintains the photoreceptor cells. These cells are cuboidal and contain pigment in the inner apical parts of the cells; processes with pigment granules extend into the rod and cone layer. This layer is thin and dark.

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The Ear (slide 77)

The ears are paired sensory organs that are responsible for the sense of hearing and enable the body to maintain its sense of balance. Hearing involves receptor cells found in the inner ear within the Organ of Corti that is housed within the cochlea.

Cochlea

The outer wall of the cochlear duct is formed by the stria vascularis which is a stratified columnar epithelium (unusually in that this epithelium contains blood vessels). The spiral ligament is the lamina propria which consists of collagenous fibers and fibroblasts. The roof of the cochlear duct is formed by vestibular membrane which also partitions it from the vestibular duct. The vestibular membrane extends from the stria vascularis to the spiral limbus and is two cell layers of squamous epithelium. The spiral limbus, which is connective tissue, forms part of the floor of the cochlear duct and is covered by a columnar epithelium. The tectorial membrane is a lateral extension of this epithelium. This membrane overlies the hair cells. The basilar membrane is a plate of vascularized connective tissue. The organ of Corti which includes the sensory hair cells, rests on this membrane and extends from the spiral limbus to the spiral ligament. Bipolar cells of the spiral ganglion terminate on the hair cells.

The parts you are expected to identify (all are shown in the atlas):  Spiral ligament  Limbus spiralis  Stria vascularis  Scala vestibuli  Scala tympani  Organ of Corti  Vestibular membrane  Cochlear duct

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Lab 6 and 7 Digestive System: I. Oral cavity and pharynx

The digestive system is made up of several organs and glands working together to digest and absorb food and eliminate waste products.

Oral Cavity The oral cavity is the initial point of the digestive system. It is composed of the lips, the tongue, the palate, the tonsils and the salivary glands.

Tongue (slide 28-30) The dorsal surface of the tongue is stratified squamous keratinized epithelium, and the ventral surface is stratified squamous non-keratinized epithelium. The connective tissue beneath the epithelium is loose connective tissue that contains lingual glands (mixed), nerve, blood and fat. The interior portion of the tongue is made up of skeletal muscle arranged in three different planes. This allows for the complex movements performed by the tongue. The anterior keratinized dorsal surface of the tongue also contains three types of lingual papillae (raised small projections of the tongue).  Fungiform papillae which resemble mushroom caps are found on the tip of the tongue.  Filiform papillae which resemble thread-like flames of fire are found on the sides of the tongue.  Vallate papillae which are the biggest of the papillae are found in a V shape arrangement towards the back of the tongue and ressemble large fungiform papillae surrounded by moat-like troughs. The papillae are extensions of the epithelium and connective tissue of the tongue. In addition, nerve endings are found in filiformn papillae whereas taste buds are found on the epithelium of the fungiform and vallate papillae. Taste buds are modified columnar cells that comprise sustentacular (round, light stain) cells and receptor (flat, dark stain) cells. On the tip of the taste bud facing away from the tongue is the taste pores and the microvilli.

Pharynx and Epiglottis (slide 25)

The pharynx which is part of the respiratory system also plays a role in the digestive system. In addition to conducting air between the nasal cavity to the larynx, it alos guides food from the oral cavity to the esophagus. Therefore, the function of the epiglottis is to prevent food from going to the larynx. The digestive portions of the phayrnx and epiglottis are both lined with stratified sqamous nonkeratinizing epithelium. The epiglottis is supported by a plate of elastic cartilage which allows for its function as well.

Lab # 6 and 7. Digestive System: II. Esophagus and G-I Tract For Lab 6, complete esophagus and stomach For Lab 7, complete small intestine, large intestine, Overview of components requiring ID:  Three different divisions of the stomach  Three different divisions of the small intestines  Myenteric (Auerbach’s) plexus

Esophagus - Anus (slides 31-48) The four main layers utilized to differentiate these sections of the digestive tract are the mucosa, the submucosa, the muscularis externa and the adventitia/serosa.

i) Mucosa: the innermost layer  divided into three layers: o epithelium lining and glands o underlying loose connective tissue termed the lamina propria with mucosal glands o muscularis mucosa with basal smooth muscle

ii) Submucosa: beneath the mucosa  consists of loose connective tissue with blood vessels and may contain submucosal glands  autonomic ganglion cells and nerve fibers may be found on the border between the submucosa and the muscularis externa termed the submucosal plexus

iii) Muscularis Externa: beneath the submucosa – thick muscle layers (2-3 layers thick)  a longitundinal layer is muscle fibers arranged up and down the tract  a circular layer is muscle fibers arranged around the tract  an oblique layer is muscle fibers arranged in a diagonal pattern around the tract  autonomic ganglion and nerve endings found in-between the muscle layers are termed Myenteric (Auerbach’s) plexus

iv) Serosa/Adventitia: outermost layer – loose connective tissue containing blood vessels, nerve and adipose  it is termed a serosa if the region of the digestive tract is covered by visceral peritoneum  if the tract is not covered by visceral peritonium it is termed the adventitia

ESOPHAGUS STOMACH SMALL INTESTINES APPENDIX LARGE INTESTINES

LAYERS Cardiac Fundic Pyloric Duodenum Jejunum Ileum Colon Rectum Anus Mucosa Epithelium Stratified Simple Simple columnar Simple -Simple Simple columnar Simple Simple Tall simple Tall simple Simple columnar squamous columnar columnar columnar brush border columnar columnar columnar columnar (upper half) non-keratinized Pits ~1/4 mucosa brush border goblet cells brush brush border brush brush border - stratified depth Pits ~ ½ few goblet cells border goblet cells border goblet cells Squamous non mucosa depth many goblet cells keratinized (lower goblet cells half)

L. propria Cardiac gland Gastric Gastric glands & Gastric glands Crypts of Crypts of Crypts of LCT LCT LCT Dense irregular (top & bottom glands & pits: & pits: Lieberkühn Lieberkühn Lieberkühn Big lymph Lymph Lymph CT thirds) pits: (straight & (branched nodes Intestinal Intestinal -Lymph LCT (straight branched) tubes) Paneth cells Paneth cells Paneth cells glands glands No glands tubes) Parietal, chief, Parietal, chief, Parietal, mucus and mucus and Peyer’s- mucus & enteroendocrine entero- Patches entero- cells endocrine endocrine cells cells

M. mucosa Smooth muscle 3 layers of 3 layers of 3 layers of Discontinuous Continuous NONE Discontinuous Smooth Smooth muscle Dscontinuous smooth smooth muscle smooth smooth muscle smooth muscle smooth muscle (upper half) muscle muscle muscle NONE (lower half)

Submucosa LCT LCT LCT LCT Brunner’s NO glands NO glands Lymph LCT LCT LCT Esophageal No glands No glands No glands Glands NO glands NO lymph or NO lymph or NO lymph or glands glands glands glands

Muscularis Skeletal (top 3 layers of 3 layers of 3 layers of 2 layers of 2 layers of 2 layers of 2 layers of 3 layers of 2 layers of 2 layers Externa third) smooth smooth smooth smooth: smooth: smooth smooth: smooth: smooth: smooth (upper Mixed (mid. inner - inner - oblique inner - oblique inner - circular inner - circular inner - inner - circular inner - inner - circular half); third) oblique middle - circular middle - outer – outer - circular outer - circular outer - skeletal (lower Smooth (lower middle - outer - circular longitudinal longitudinal outer - longitudinal outer - longitudinal half); third) circular longitudinal outer - longitudinal longitudinal NO Tenae coli inner - circular outer – longitudinal Auerbach’s Auerbach’s Tenae coli outer - longitudinal longitudinal Plexus Plexus Auerbach’s Plexus

Adventitia/ Adventitia Serosa Serosa Serosa Adventitia Serosa Serosa Serosa Serosa Adventitia Adventitia Serosa 55-357: Animal cells and tissues Lab Manual 18 ______

Characteristics of Cell Types Found in the Digestive System

Cardiac glands - mucous glands found in the L. propria of the top and bottom thirds of the esophagus

Esophageal Glands - mucous glands found in the submucosa of the esophagus

Gastric Glands - found in the epithelial lining of the stomach and form gastric pits in to the L. propria - consists of four types of cells: i) mucous neck cells - found in the top portion of the pits  secrete mucous  pale-staining ii) parietal cells - found throuhgout the pits  secrete HCl and intrinsic factors  acidophilic iii) chief cells - found at the base of the pits  secrete digestive enzymes basophilic iv) enteroendocrine cells - found at the base of the pits  secret hormones  also called APUD cells

Brunner’s Glands - mucous secreting glands found in the submucosa of the duodenum of the small intestines

Crypts of Lieberkühn - mucosal glands located in the L. Propria of the small intestines

Paneth Cells - enzyme secreting cells found at the base of the Crypts of Luberkuhn - acidophilic (granular)

Peyer’s Patches - lymph nodules found in the L. Propria of the ileum of the small intestines - the lymph nodules are composed of lymphocytes thus appear highly basophilic

Tenae Coli - the longitudinal muscle of the musculais externa is arranged in three longitunal bands - appears as an extra layer of muscle only in the colon of the large intestines 55-357: Animal cells and tissues Lab Manual 19 ______

Digestive System III. Salivary Glands, Pancreas Liver, Gall bladder

Salivary Glands (slides 49-51) There are three exocrine salivary glands associated with the digestive system. They are the submandibular (aka submaxillary), sublingual, and the parotid glands.

i) Submandibular - mixed serous and mucous - compound tubuloalveolar - septa and capsule - serous demilunes

ii) Sublingual - mucous (pale stain, nuclei flat & on side of cells) - with serous demilunes - well developed septa and thin capsule

iii) Parotid - serous (dark stain, nuclei round & in center of cells) - compound tubuloalveolar glands - well developed septa and capsule

Overview of components requiring ID:  Pancreas – Islets of Langerhans  Liver – portal area o central vein, portal vein, hepatic (portal) artery, lymphatic vessel, bile duct

Pancreas (slide 52): The pancreas which is part of the digestive and endocrine system is a gland that has both exocrine and endocrine functions. The exocrine portion of the pancreas is comprised of a compound acinar gland that produces a variety of digestive enzymes. Clusters of cells called Islets of Langerhan make up the endocrine portion of the pancreas and its function is to produce hormones required for the regulation of digestion. The Islets of Langerhan are found throughout the acinar gland though they only comprise 1% to 2% of the total pancreatic volume. The acinar gland resembles the parotid gland in that the secretory units are serous. In the middle of the acinar unit there are cells termed centroacinar cells. They are the initial portion of the ducts that transport the digestive enzymes away from the pancreas. The Islets of Langerhan however are groups of cells that are lighter in stain than the acinar gland and do not have any ducts. The capsule surrounding the pancreas is loose connective tissue instead dense irregular connective tissue.

Liver (slides 53, 54): The liver is a gland that produces bile and secretes it in an exocrine manner. In addition it produces plasma proteins and glucose and secretes them into the bloodstream in an endocrine manner. The liver also functions as a producer of cholesterol and somatomedin, detoxifier of drugs and a converter of T4 into T3. The main cell types of the liver are hepatocytes. Hepatocytes are cuboidal in shape and are arranged in cords (rows) around a central vein. This arrangement is hexagonal in shape and is termed the hepatic lobule. Many hepatic lobules arranged next to one another comprise the liver. Between hepatic lobules is an area termed the portal area that is comprised of connective tissue, a portal vein, an hepatic (portal) artery, a lymphatic vessel and a bile duct. In between the cords of hepatocytes are sinusoids that run into the central vein. Blood flows from the vessels in the portal area through the sinusoids into the central vein. The bile produced in the liver flows in the opposite direction. It flows from the hepatocytes through the bile canuliculi into the bile duct of the portal area. The bile is transported out of the liver and into the gall bladder. In addition, the liver contains fixed macrophages termed Kupffer cells which are localized on the surface of the sinusoids. The liver is encapsulated by dense connective tissue. 19 55-357: Animal cells and tissues Lab Manual 20 ______

Gall Bladder (slide 55): The gall bladder is an organ that concentrates and stores bile produced by the liver. During the digestion of fat, the gall bladder expels the bile into a duct system which will transport the bile to the small intestines. The mucosa of the gall bladder is composed of tall simple columnar epithelium with microvilli. The underlying lamina propria is made up of loose connective tissue containing no glands. It does not have a muscularis mucosa nor a submucosa. The muscularis externa is comprised of three smooth muscle layers containing elastic fibers. The exterior coat of the gall bladder is termed the perimuscular coat and contains loose connective tissue and smooth muscle.

Lab 9 Circulatory System Endocrine System

Circulatory System (slides 13-16) ______Overview of components requiring ID:  Tunica intima, tunica media, tunica adventitia of all vessels  Muscular vs. elastic arteries vs. arterioles  Veins vs. venules  Vasa vasorum

The main function of the circulatory system is to transport blood throughout the body using the heart as a muscular pump and the vessels connected to it. There are three main layers associated with the heart and blood vessels. The three layers of the heart are the endocardium, which is adjacent to the lumen, the myocardium, which is composed of muscle and the epicardium, which covers the heart. In the blood vessels these layers are termed the tunica intima, tunica media and tunica adventitia, respectively.

I. Composition of the Layers

 Endocardium / Tunica intima: i) endothelium ii) connective tissue (with a layer of elastin)

 Myocardium / Tunica media: i) muscle (cardiac in heart/smooth in vessels)

 Epicardium / Tunica adventitia: i) mainly loose connective tissue ii) vasa vasorum (small blood vessels)

II. Blood Vessels There are two main types of blood vessels, arteries and veins. Arteries transport oxygenated blood away from the heart to the rest of the body, and veins transport deoxygenated blood from the body to the heart. Two differentiating characteristics are as follows: one is that arteries have thick walls and therefore do not collapse after death whereas veins do, and second veins contain valves whereas arteries don’t. Blood vessels however are distinguishable by their three tunica layers.

LAYERS ARTERIES VEINS TUNICA INTIMA Thin Thin TUNICA MEDIA Thick muscle layer Thin muscle layer TUNICA ADVENTITIA Thin Thick

IIa. Arteries There are three types of arteries: elastic arteries, muscular arteries and arterioles. Elastic arteries transport blood out of the heart (aorta), muscular arteries transport blood to the organs and arterioles regulate blood pressure. The three types of arteries are separated based on the characteristics of the T. intima, T. media and T. adventitia.

LAYERS Elastic Arteries Muscular Arteries Arterioles LUMEN ~ 12 or more RBCs across ~ 12 or more RBCs across ~ 3-12 RBCs across T. INTIMA Thin Thin Thin T. MEDIA Thickest layer; > 2 layers smooth Thick layer; > 2 layers of Thick; ~ 1-2 layers muscle with elastic fibres smooth muscle smooth muscle T. ADVENTITA < ½ thickness of T. Media; > ½ thickness of T. Media; Thin; no vasa vasorum contains vasa vasorum contains vasa vasorum

Note: The lumen refers to the open space in the middle of the blood vessels.

The vasa vasorum is the collection of blood vessels supplying the walls of the larger arteries and veins

IIb. Veins: There are two types of veins: veins and venules. These two blood vessels can also be distinguished by their three tunica layers.

LAYERS Veins Venules Lumen ~12 or more RBCs across ~3-12 RBCs across T. INTIMA thin thin T. MEDIA Thin; smooth muscle Thin; ~1 layer of smooth muscle T. ADVENTITIA Thickest; contains vasa vasorum very thin; no vasa vasorum

IIc. Capillaries : thin tunica intima, no tunica media, thin tunica adventitia Capillaries are another type of blood vessel that connect both arteries and veins. Capillaries function in groups to provide tissue fluid, oxygen and nutrients for the cells of tissues. Capillaries have a very thin T. intima and may contain a very thin T. adventitia, however, it DOES NOT contain a T. media. Capillary are small in diameter and can hold only one RBC across. Sinusoids

Sinusoids are capillary-like vessels that are larger in diameter than capillaries and serve as passage ways for the movement of cells (i.e. macrophages) and large molecules.

Endocrine System

Overview of components requiring ID:  Pituitary gland – chromophils (acidophils and basophils)  Thyroid gland – follicular and parafollicular cells; colloid  Parathyroid gland  Adrenal gland – cortex (zona glomerulosa), medulla,  Islets of Langerhans  Epidermal layers (thin & thick skin)  Dermal layers  Sebaceous glands and arrector pili muscle  Hair cross section – cuticle and cortex

Part 1: The Endocrine System

The Endocrine System involves dissemination of signal molecules over long distances by way of the bloodstream. Signal molecules produced by end cells are hormones.

Pituitary Gland (slide 81) This small but important gland is made of two parts, a glandular anterior lobe (AKA pars anterior) and a neural posterior lobe (AKA pars nervosa), separated by the pars intermedia which contains colloid-filled vesicles. The anterior lobe stains brightly and consists of large epithelial secretory cells. Some of these cells are chromophils which stain brightly because of their stored secretory granules. Other cells with less hormone appear smaller and on’t stain brightly. These are known as chromophobes and are believed to be in a quiescent stage. The chromophils can then be further classified as acidophils or basophils based on their affinity for acid/basic stains. The posterior lobe is composed of nerve fibers and processes and is therefore very pale-staining.

Thyroid Gland (slide 79) Composed of follicles which have a large lumen and are lined with simple columnar/cuboidal epithelium (aka follicular cells). Follicles are filled with colloid which takes on an acidophilic stain. Parafollicular cells (aka C-cells or light cells) are sparsely distributed throughout the gland between follicles.

Parathyroid Gland (slide 79) This gland is so named because of its anatomical position beside the thyroid. This gland consists of two cell types:

Chief cells (aka Principal cells) Abundant cell type which is small, round, pale-staining with a centrally located spherical nucleus. Oxyphil cells Strongly acidophilic and appear to be larger than chief cells.

Adrenal Gland (slide 80) This gland consists of a capsule, cortex and medulla. The cortex is made up of three zones: zona glomerulosa, zona fasciculata and zona reticularis.

Zona glomerulosa Cells are arranged in ovoid groups. The cytoplasm contains sparse lipid droplets which appear as vacuoles.

Zona fasciculata Cells are arranged in columns oriented in a radial direction. Lipid droplets give a vacuolated appearance.

Zona reticularis Cells in this zone form anastomosing cords which run in various directions.

Adrenal medulla Simpler in organization than the cortex. Grouped ovoid secretory cells are visible.

Islets of Langerhans (slide 52) These are the pancreatic islets, called Islets of Langerhans. They are seen as small, irregularly shaped, light staining areas, sparsely distributed throughout the pancreas. These islets are responsible for the production of insulin and glucagon.

Lab #10 The Male and Female Reproductive Systems

Overview of components requiring ID:  Testes: leydig, sertoli, spermatogonia, primary spermatocytes, spermatids  Ovary: all types of follicles  Corpus luteum  Uterus  diestrous vs. estrous  Mammary gland lactating phase

Male: The major functions of the male reproductive system are to produce spermatozoa, androgens and to facilitate fertilization.

Testes (slide 60, 61) Each testis is a compact ovoid organ with an epididymis extending around it. They are composed of seminiferous tubules.  Leydig Cells: are located between the seminiferous tubules and produce testosterone. Their cytoplasm appears pale because of their lipid content. .  Sertoli Cells: are the supporting cells; they are tall columnar cells that span the entire epithelium from the basement membrane to the lumen and are found at the base of the tubule; they are pale-staining and contain triangular nuclei.  Spermatogonia: found at the base of the tubules. These cells are large and contain round nuclei.  Primary spermatocytes: contain condensed chromatin which gives them a spidery-appearance.  Spermatids  Spermatozoa: are elongated and have a tail; they also have a dark nucleus.

Epididymis This duct is lined with pseudostratified columnar epithelium with stereocilia (really microvilli). Smooth muscle circumscribes the duct.

The Female Reproductive System Some important functions of the female reproductive system are to produce ova, to facilitate the meeting of ova and sperm promoting the process of fertilization, to maintain implanted eggs throughout their gestation period and to nurture infants postnatally through lactation.

Ovary (slides 64, 65, 66) The capsule is composed of cuboidal epithelium and is sometimes called the germinal epithelium. Beneath the epithelium is the tunica albuginea, which is a few layers of dense fibrous connective tissue.

 Primordial follicle o The oocyte is enclosed by only a single layer of flat squamous follicular cells known as granulosa cells.  Early Primary Follicle o The follicular cells now are cuboidal and a zona pellucida (refractile extracellular glycoprotein) is discernible. 1 layer of follicular cells  Late Primary Follicle o Follicular cells are stratified. Theca folliculi or connective tissue cells surrounding the follicle is visible.  Secondary Follicle o The theca has layers: theca interna which is cuboidal and the theca externa which is connective tissue and smooth muscle. A large space, the antrum, surrounds the oocyte (This space may be so large that the oocyte may be absent from sections).  Mature (Graafian) Follicle o The oocyte sits on top of a stack of cells that is called the cumulus oophorus. The cells that surround the oocyte are called the corona radiata

Corpus luteum 24 55-357: Animal cells and tissues Lab Manual 25 ______

A highly folded structure in which the remains of blood clots persist in the interior of the corpus luteum. Granulosa cells of the follicle hypertrophy and become transformed into granulosa lutein cells; the theca interna cells likewise become theca lutein cells. The lumen is filled with coagulated fluid and connective tissue.

Uterus The uterine wall consists of endometrium (mucosa), myometrium (muscularis) with smooth muscle and connective tissue.  Uterus diestrous (slide 67) o The endometrium is in an early proliferative phase and is thick. The top layer of cells are not yet simple columnar epithelium but appear small and compacted. Long glands that are growing are visible but contain no mucous secretory product.  Uterus estrous (slide 68) o Secretory glands with secretion in the lumen of the glands is seen. Simple columnar epithelium covers the surface of the endometrium.

Mammary Gland Each mammary gland (breast) is a group of up to 20 compound alveolar glands that open onto a surface elevation known as a nipple. Until pregnancy, these are in the resting or non-lactating phase and during pregnancy, they are in the active or lactating phase.

 Resting (Non-lactating) Phase (slide 70) o During this phase, connective tissue is abundant and glandular tissue is minimal. Lobules contain groups of small tubules lined with simple cuboidal or columnar epithelium; dense interlobular connective tissue and fat surround this. The resting breast is the only gland that exhibits ducts but no secretory units.  Active (Lactating) Phase (slide 71) o Well-established lobules with secretory alveoli, which are buds forming at the end of ducts, as well as ducts are evident. As lobules continue to grow the interlobular connective tissue becomes reduced to thin partitions. Alveoli are made of simple columnar secretory cells surrounded by myoepithelial cells.

Lab 11 Integumentary System Respiratory System

Overview of components requiring ID:  Epidermal layers  Papillary layer and reticular layer of the dermis  Eccrine sweat glands  Sebaceous glands  Arrector pili muscle  Hair cross-section – cuticle and cortex

The skin is highly resistant to abrasion and infection. It is waterproof and prevents against desiccation. It is also a sensory organ which provides for perception of the environment: touch and temperature.

Thick skin is found on palms of the hand and the bottom of feet.

Layers: epidermis, dermis, subcutaneous (hypodermis) Ridges: primary epidermal ridges, primary dermal ridges

Epidermal layers(slide 21) 1. Stratum germinativum is a single deepest layer. cells are attached to each other by desmosomes and to the basement membrane by hemidesomosomes 2. Stratum spinosum. Several layers thick. cells have spine-like processes 25 55-357: Animal cells and tissues Lab Manual 26 ______

3. Stratum granulosum. Only a few cells thick. Basophilic (keratohyalin) granules. 4. Stratum lucidum. thin transparent layer, seldom evident in H and /E preparations. 5. Stratum corneum flat scales of keratin.

Dermis: Papillary Layer: Loose CT merges with an underlying layer of irregular dense CT. Supplied with capillaries and extends up into the epidermis as small projections called dermal papillae Reticular Layer: below the papillary layer, bundles of collagen. Not as vascular and fatty as the papillary layer.

Eccrine sweat glands: abundant in thick skin secretory portion of the gland is in subcutaneous tissue, myoepithelial cells that contact during secretion

Thin skin: (slide 22) substantial dermis hair growing out of follicles

Epidermis is thinner than in thick skin 1. Stratum germinativum, same 2. Stratum spinosum, thinner 3. Stratum granulosum, indistinct 4. Stratum lucidum absent 5. Stratum corneum, thin

Apocrine sweat glands Sebaceous glands, oily secretion (sebum) Arrector Pili muscles contract and hair becomes errect.

Hair: (slide 22) Cuticle, cortex contain hard keratin pigment Medulla in the center of the hair shaft

RESPIRATORY SYSTEM

Overview of components requiring ID:  Nasal region  Larynx  Vocal cords  Epiglottis  Trachea  Bronchi and Bronchioles  Tongue – papillae  Taste buds  Salivary glands

The Respiratory System consists of two lungs and a branching system of airways which allow for gas exchange between the air and blood. The basic cell type in the respiratory system is pseudostratified ciliated columnar epithelium with goblet cells.

Nasal Region (slide 23) This region can be identified by the presence of hyaline cartilage and pseudostratified ciliated columnar epithelium with goblet cells.

Larynx (slide 26) This is the sound producing organ which prevents access to the respiratory tract of liquids and foods. It is supported by hyaline cartilage and a transition occurs from stratified squamous non-keratinizing to pseudostratified ciliated columnar epithelium with goblet cells. Mucous and serous glands are also present.

 Vocal Cords Skeletal muscle, elastic fibers and stratified squamous non-keratinizing epithelium

Epiglottis (slide 25)

This flap-like structure is the uppermost part of the larynx and is a part of both the respiratory and digestive systems. One side is lined with stratified squamous non-keratinizing and the other with pseudostratified ciliated columnar epithelium. Submucousal glands are also present and taste buds may be seen *Note – the epiglottis slides do not actually show the two different types of epithelium

Trachea (slide 24) Arrangement of hyaline cartilage in a C-shape, lumen lined with pseudostratified ciliated columnar epithelium with goblet cells. The smooth muscle located between the cartilage (at the tips of the “c”) is called the trachealis muscle.

Bronchi (slide 27) (Single – bronchus) Bronchi are the result of the first division of the trachea. They are distinguished by islands of cartilage. Pseudostratified ciliated columnar epithelium lines the lumen. Smooth muscle is arranged helically around the lumen and submucousal mixed glands are also present.

Bronchiole (slide 27) Bronchioles are lined with simple epithelium. Mucosal folds are prominent but cartilage and glands are lacking.

Terminal Bronchiole Lumen lined with simple columnar ciliated epithelium; goblet cells are absent and the smooth muscle layer is well-developed.

Respiratory Bronchiole Lumen lined with simple cuboidal epithelium ; alveoli extend from their walls. Smooth muscle forms a layer close to the epithelium.

The Urinary System (omitted in 2007)

The urinary system consists of the kidneys, ureters, urinary bladder and urethra. It produces, stores and intermittently voids urine, the excretory product.

Kidney (slide 56) This organ consists of a capsule, cortex and medulla and is multi-lobar. The functional unit of the kidney is the nephron.

Nephron Renal corpuscle (glomerulus), proximal convoluted tubule (PCT) and distal convoluted tubules (DCT) lie in the cortical region of the kidney. The loop of Henle (LH) and the collecting tubules (CT) are located in medullary region of the kidney.

Proximal Convoluted Tubule (PCT) The PCT has an extremely small lumen (sometimes not even visible) which is lined with large puffy cells.

Loop of Henle (LH) The descending portion of the LH is lined with simple squamous epithelial cells (Difficult to distinguish at times from capillaries that surround it).

Distal Convoluted Tubule (DCT) The DCT is lined with somewhat smaller cuboidal cells than the PCT and appears as a wider-looking lumen.

Collecting Tubules (CT) They are the largest tubules and therefore have wide lumens. They are lined with simple cuboidal to columnar epithelium.

Ureters (slides 57, 58)

Its lumen is characteristically stellate shaped and is lined with transitional epithelium. Mucosal, submucosal glands and the submucosa are lacking. The muscular coat consists of an inner longitudinal layer and an outer circular layer (opposite to the digestive system).

Urinary Bladder (slide 59)

This sac’s expansive lumen is composed of many folds lined with transitional epithelium. The thick muscular coat of the bladder is distinctive.