2007

Table of Contents for all Laboratories

Laboratoy # 1-A. Check out your microscope and get familiar with it !

Laboratory 1-B. Look at a slide and some elelctron micrographs and become familiar with cells and organelles.

Look at the drawing which illustrates how various three dimensional structures look after they are sectioned!!!!!

Laboratory # 2. Study the various types of epithelium as indicated.

Laboratory # 3. Laboratory on Connective tissue.

Laboratory # 4. Laboratory on Nervous Tissue

Laboratory # 5. Laboratory on Muscle, Cartilage, Bone, and Bone Development

Laboratory # 6. Laboratory on Vascular System and Blood

Laboratory # 7. Laboratory on Eye, Ear, and Lymphatics

Laboratory #8. Laboratory on Respiratory System and Gastrointestinal System - 1

Laboratory # 9. Laboratory on Gastrointestinal System - 2 and Urinary System

Laboratory # 10. Laboratory on Female and Male Reproductive Systems

Laboratory # 11. Laboratory on Endocrine System and Integument

 

 

Table of Contents for Laboratory Demonstrations

HistoLab Demos, - The Cell

HistoLab Demos, - Epithelium

HistoLab Demos, - Connective Tissue

HistoLab Demos, - Nervous Tissue

HistoLab Demos, - Muscle Tissue

HistoLab Demos, - Cartilage and Bone

HistoLab Demos, Eye and Ear

HistoLab Demos, Lymphatics

HistoLab Demos, GI - ONE

HistoLab Demos, Respiratory Sys.

HistoLab Demos, Endocrine Sys.

HistoLab Demos, Female Reproductive Sys.

HistoLab Demos, Male Reproductive Sys.

HistoLab Demos, Urinary Sys.

 

 

 

 

 

 

NOTICE TO CLASS of 2008:

Three years ago this manual was typed into a new word processor because it was easier than trying to convert it. During the typing the inevitable mistakes occurred. Most of those were caught by the spell checker, but there are others such as wrong slide numbers that cannot be fixed by spell checkers. Many of these mistakes have been brought to the attention of the author by students during the course, and many have been fixed.

Even though many of the errors were eliminated during the last two years we are going to put a copy of the lab manual on a chain in the lab specifically for the purpose of inserting corrections and/or suggestions for the next edition. We would appreciate your cooperation in finding any remaining errors. We are also open to suggestions for change if they are CONSTRUCTIVE!

 

 

 

HISTOLOGY LAB I-A

HISTOLOGY LABORATORY PROCEDURE

 

1. No materials may be taken out of the multipurpose laboratory!

2. Microscopes:

a) Two students are assigned to one microscope, therefore, cooperation is paramount! Please keep your scope clean and in proper working condition. At the end of each laboratory session, return your microscope to its appropriate place.

b) Carefully read the instructions in this manual on proper care and handling of the microscope. If you don't remember anything else remember to CLEAN ANY OIL OFF THE 100X OBJECTIVE AND CHECK TO SEE THAT THE CONDENSER IS ALL THE WAY UP!

3. Slide Boxes:

a) You are sharing slide boxes with other students. please cooperate by leaving them in good order!

b) Before each lab, check for damage and/or lost slides. Report any damage or loss immediately. The instructors will check slide boxes periodically, and you will be charged $10.00 per damaged/lost slide.

c) After viewing slides, clean off any oil & put them back into the proper slots. The students sharing slides with you don't have time to search for misplaced slides.

d) At the end of each lab, return your slide boxes to the to the cabinet with your microscope.

4. Any demonstration slides and electron micrographs the instructors set out may be used for examination purposes. If any of this material is to be used, you will be notified either verbally or by notes written on the blackboard.

5. Each laboratory session is scheduled for ONEAND ONE HALF HOURS; it will be necessary for you to use this time efficiently. This will greatly decrease the time you will need for review.

6. Keep the lab tables clean! This laboratory is used for Histology and and other courses; we all need to cooperate. If any extraneous materials (not

belonging to the Anatomy Department) are scattered about your space, please inform the instructors at once.

7. The instructors are available any time during lab. Do not hesitate asking for help when you need it. No question is "stupid" or "dumb". Initially, we expect a lot of questions; this is normal and will gradually decrease as you become more comfortable with your microscope and tissues. Please have your questions ready before calling on an instructorand while we want you to get the help you need there may be a discrepancy between what you think you need and what you really need.

8. Microscopes and slide boxes are available in the UNECOM Library for use outside of regular lab hours. Ask at the Circulation desk since slides are on reserve.

9. There are computer/videodisc tutorials and quizzes available in the laboratory . These are NOT meant to be a SUBSTITUTE for histology laboratory work, JUST AN ADDITIONAL AID. You will need A CODE NUMBER TO USE THEM WHICH WILL BE GIVEN TO YOU.

10. Students are strongly advised to bring both their ATLAS and their TEXT to the laboratory! These books are selected for their excellent illustrations. Bring your text to lab! Bring your text to lab! Bring your text to lab! What should you bring to lab? YES! Bring your text and your atlas to lab.

 

 

HISTOLOGY LAB 1-A

PROPER USE OF THE MICROSCOPE

 

BACK TO TOP

 

I. GET FAMILIAR WITH THE CONTROLS

 

A. FOCUS CONTROL: This is located beneath and lateral to the stage and consists of two knobs mounted concentrically. The larger of the two knobs is the Coarse Focus Control and the smaller knob is the Fine Focus Control. When the microscope is in proper adjustment, it should be impossible to damage an objective lens with the Coarse Focus Control for it will stop before the objective can contact the slide. Do not depend on this device, especially with high power objectives. Watch the objective as you bring it close to the slide to be certain it does not contact the specimen.

B. MECHANICAL STAGE: Controls are mounted beneath the stage on the right rear corner of Olympus scopes.

C. EYE PIECES: The interpupillary distance can be adjusted with the knob between and beneath the two eye pieces on the AO scopes. On the other scopes, simply move the eye pieces directly. To adjust for differences in your eyes focal length, focus first for the right eye using the focus control at the base of the microscope and then bring the left eyepiece into focus using the knurled ring on the eye piece tube so that the left eye sees the image the same as the right eye.

D. CONDENSER: The condenser can be focused up and down with the control located below the stage on the left hand side on the Olympus. The iris diaphragm is adjusted with the lever protruding from the front of the condenser. THE BEST LOCATION FOR THE CONDENSER IS ALL THE WAY UP + or - a small amount! This will give you the best image!

E. ILLUMINATION: The illuminator is turned on and adjusted with the small knob on the front of the microscope base plate or a lever on the side of the base.

 

II. PROPER ADJUSTMENT OF ILLUMINATION

A. Turn on the illuminator and turn light up to about the middle of its range.

B. Turn to the 10X objective and focus on a stained slide.

C. Focus the CONDENSER until the illumination is bright and even. This will be near the uppermost limit of the condenser focusing range. (Don't change microscope focus!) The condenser is now focused properly. You may want to

move it up or down very slightly from optimum focus to avoid seeing dust on the glass of the illuminator or the ground glass.

D. Proper adjustment of the iris diaphragm of the condenser is very critical if one wants to get the best resolution from any microscope. The condenser diaphragm is not intended for adjusting the intensity of the light but rather the exit size of the cone of light.

The condenser diaphragm should be readjusted for each objective lens used. It is done easily and quickly by removing one eye piece and closing the diaphragm until it cuts off about 1/4 of the area of the field of vision f the objective being used.

Figure 2 (right) > shows you how to do this. After you have done it this way a few times you become familiar with about how much dimming of the illumination takes place during this process and you will no longer have to remove the eyepiece to accomplish the ideal setting of the condenser.

 

Figure 2

 

NOTE:

There may be times with difficult slides AT LOW MAGNIFICATION when the rule needs to be broken and you might have to close down the condenser diaphragm a bit more. Closing the diaphragm down more doesn't actually make the image sharper, but causes diffraction rings around objects in the image giving the illusion of sharpness. However, TO GET THE SHARPEST IMAGE POSSIBLE FOR SEEING FINE DETAIL AT HIGH MAGNIFICATION THE CONDENSER DIAPHRAGM SHOULD BE SET AS DESCRIBED ABOVE. NEVER USE IT TO ADJUST THE INTENSITY OF THE LIGHT!! USE THE LIGHT INTENSITY CONTROL ON THE BASE OF THE MICROSCOPE!

III. STUDYING THE SPECIMENS WITH THE MICROSCOPE

A. Generally, inexperienced people tend to use a magnification which is too high for the subject being studied. Always start with the lowest magnification for orientation and then work up to the magnification best suited to the structures you are examining.

B. The oil immersion (100X) lens is rarely used in Histology but is necessary for some situations. This (100X) objective lens is both the most expensive and most easily damaged lens on the microscope. Grinding it into the slide could cost you a lot of money. The 10X and 43X objectives are adequate for most of the work in this course. The 4X objective is used to find your way around the slide before you use a higher magnification,

IV. TAKE GOOD CARE OF THE MICROSCOPE (OR YOU WILL RUE THE DAY)

A. Clean the delicate optics frequently and only with lens paper. Breath moisture may be used to aid in polishing the lenses.

B. Remove oil from the oil immersion objective lens as soon as you finish using it (with lens paper & alcohol), and there will be few problems. If oil dries on the lens or other accumulations of stuff will not come off, ask your instructor for help. USE ONLY THE ALCOHOL PROVIDED to clean objective lenses. Other organic solvents can dissolve the cement holding the lenses together.

 

BACK TO TOP

 

HISTOLOGY LAB I-B METHODS & CYTOLOGY

BE ABLE TO IDENTIFY:

Light Microscope:basement membrane--Look for this on slide # 49 -Tracheaeuchromatin and heterochromatinnucleus and nucleolus

Electron Micrographs:basal bodybasal lamina -Same as Basement Membrane which is the light microscope term. Basal Lamina is the electron microscope term.

centriolecilium and flagellummitochondrion and cristae mitochondrialeseuchromatin and heterochromatinglycocalyxgolgi apparatuslysosomemicrovillusnuclear membrane and porenucleus and nucleolusplasma membranerough and smooth endoplasmic reticulumvacuole

 

Most of the tissues you will be examining with the microscope are thin slices (called sections) of the tissues and organs being studied. The drawings on the next page are from an old Ham's Histology text no longer in print and are very helpful in visualizing what various three dimensional structures look like when sliced (sectioned).

1. Use of Microscope: Follow the instructions above on the

"Proper Use of the Microscope"

Get an instructor to check you out when you have mastered the information.

2. Slide #1 is a whole mount of mesentery stained with silver to bring out cell boundaries. Remember, you are looking through two cell layers and connective

tissue. Focus up and down to see different cell (epithelial) layers. A section perpendicular to the surface of this mesentery would look like this:

The image on the left demonstrates what you will see in the microscope when it is focussed on the upper layer of the mesentery shown in the drawing. Note that the cell boundries are stained black with silver. If you focussed down through this specimen you would see another similar layer on the other side. (see diagram above). This is an example of two layers of simple squamous epithelium separated by some connective tissue.

 

 

 

Examine the electron micrographs which are available in the laboratory.

There are also electron micrographs in your text! Bring your text to lab! 

 

 

 

Key to labels on EMs in lab: Be sure you can identify these cell organelles!

 

Lysosomes are sometimes called "suicide bags" because they are filled with enzymes that can degrade proteins, lipids, carbohydrates, and nucleic acids. Inability to break down certain compounds because of inherited enzyme deficencies can cause illness and death. Tay-Sachs Disease is an example.

 

 

Nuclear pores are regions of the nuclear (NOT NUCULAR) envelope that are thinned in order to allow messenger RNA to move from the nucleus to the cytoplasm.

SER = smooth endoplasmic ret.

RER = rough endoplasmic ret.

 

 

 

Peroxisome is a specialized type of Lysosome that breaks down H2O2 among other things.

 

 

Very High magnification of one end of the Golgi apparatus associated with a Pigment granule or Lysosome and contributing vesicles to it. The trilaminar (another more accurate word for "bilayer") membrane is shown where indicated.

Actually all of the cell membranes are somewhat globular.

 

 

Mitochondrion Shown on the right at high magnification in the transmission EM

 

 

 

 

 

 

C= Cristae Mitochodriales = Shelf like evaginations of the inner mitochondrial membrane.

RER= Rough Endoplasmic Reticulum

DNA = DNA containing granules found within the mitochondrion that is involved in mitochondrial reproduction.

 

 

 

 

M= mitochondria

GLY = glycogen stored in this liver cell

RER= Rough Endoplasmic Reticulum

 

 

Units of measurement

mm = millimeter = 1/1000 of a meter

µm = micrometer (micron) = 1/1000 of a millimeter

nm = nanometer = 1/1000 of a micrometer

å = angstrom unit = 1/10 of a nanometer or 10 to the minus 7 mm

 

 

DO NOT REMOVE ELECTRON MICROGRAPHS FROM THE LABORATORY!

BACK TO TOP

 

HISTOLOGY LAB II - EPITHELIAL TISSUE AND GLANDS

BE ABLE TO IDENTIFY:

brush (or striated) borderciliaepithelium endothelium and mesothelium (simple squamous epithelium)pseudostratified columnar (+ or - cilia)simple columnar (+ or - cilia), cuboidal, and squamous epitheliastratified columnar and cuboidal stratified squamous (keratinizing and nonkeratinizing) transitional epitheliumgoblet cells

Observe the different types of epithelia on the slides listed below. You should know from reading your text, they functionally significant specializations of these various types of epithelia, at both the light and electron microscope levels. Use the electron microphotographs in your text for further study of fine structure of epithelia.

 

Simple squamous epithelium, (endothelium, mesothelium)

Remember that endothelium and mesothelium are just names for simple squamous epithelium that are found in specific locations.

 

 

 

Slide #1. You looked at this slide before you went through the Electron Micrographs! Cat mesentery has been treated with a silver stain bringing about a blackening at the edges of the mesothelial cells. Note double layer of mesothelial cells. This may be done by adjusting the fine focus. Review the definition of mesothelium.

 

 

 

 

 

 

 

 

 

 

Examine Slide #58 (monkey eye). Look at the posterior surface of the cornea at 400X (Which will be your 40X objective) This inner or posterior surface is lined with simple squamous epithelium. (endothelium). You may also look at any blood vessel such as the two arterioles illustrated below and see that they have an internal lining of endothelium.

 

 

 

 

 

 

 

 

 

 

Endothelium lining the lumen of an arteriole in the light microscope.

 

 

 

 

 

 

 

 

 

 

 

Arteriole as seen in transverse section in the transmission electron microscope. Note the Endothelial Cells indicated by the arrows. Smooth Muscle Cells (SM) are found external to

the endothelium. 

 

 

 

 

 

 

 

 

 

 

 

Simple Columnar Epithelium

Slides #179 & 180. Human small intestine. The epithelium lining the small intestine is Simple Columnar (tall columnar). At high magnification (40X objective) you will notice that the luminal surface of these epithelial cells looks "fuzzy" (or like a double line). This appearance is caused by microvilli which collectively are called a brush border when seen in the light microscope. Cilia are much longer than the microvilli making up the

brush border in this light micrograph.. There is a demonstration on the right showing how much longer Cilia (arrow) are than the microvilli in the brush border.

 

 

 

 

 

 

 

 

 

 

 

Simple Columnar Ciliated Epithelium

Slide #89. Human ampulla of oviduct. This simple columnar epithelium is ciliated in places. Compare this "ciliated" columnar to the columnar with a "brush border". Be sure you know the difference between the two.

 

 

 

 

 

 

 

Simple Cuboidal Epithelium

Slides #85,#126,109, & 160. Sections through the human kidney. What is frequently called a simple cuboidal epithelium lines the collecting tubules. The cells found in cuboidal epithelium are shorter than those found in columnar epithelium. Their profile in sections resembles a "Square". Usually the cells are just a bit taller than they are wide.

 

 

 

 

 

 

 

Stratified Squamous Epithelium

Slides #28 & 29. Human esophagus cross section. The esophagus is lined with non-keratinizing stratified squamous epithelium. Notice that all the squamous cells out to the surface have nuclei.

 

 

 

 

 

 

 

 

Slide #177 . Stratified Squamous Epithelium, Keratinized, in human skin. Notice the difference in thickness between these two types of skin. Also be aware that the cells at the surface do not exhibit nuclei and are dead! Look at the deepest cell layers in these tissues and notice that they are not all squamous. Remember that an epithelium is named by

the cells at its surface. Other examples of Keratinized Stratified Squamous Epithelium can be found on Slides #59,#62, &#92

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pseudostratified Columnar Epithelium

Slide #49. Section through the monkey trachea. The internal surface is lined by a pseudostratified ciliated columnar epithelium. The sections may vary in the quality with which the cilia are shown.

 

 

 

 

 

 

 

Transitional Epithelium

Slide #116, #109. Transitional epithelium in the human ureter and renal pelvis. Two diagnostic characteristics of transitional epithelium are: luminal cells (1) are sometimes binucleate and (2) "balloon" out into the lumen giving an uneven appearance to the luminal surface. Transitional Epithelial cells can slide by one another allowing the epithelium to get thinner (as in a full bladder) without losing the ability to form a strong layer.

 

 

 

 

 

 

 

BACK TO TOP

 

 

 

 

Some locations (not all) of Epithelial Tissues

Simple squamous

inner surface of membr. labr. ear

tympanic membrane

Bowman's capsule

amnion and chorion

 

 Simple columnar

uterus (endometrium)

lines digestive tube

secretory ducts of glands

some kidney tubules

 Endothelium

lines blood vessels,

heart, and lymphatics

also found in canal of Schlemm

and trabecular meshwork

 

Simple columnar (ciliated)

oviduct

small bronchioles

central canal spinal cord

epididymis (stereo-cilia which are really long microvilli)

 Mesotheliumlines serous cavities (peritoneum, pericardium, pleura)

 

Stratified columnar

fornix of conjunctiva

ducts of salivary glands

ductus deferens

 

 Simple cuboidal

thyroid follicles

kidney tubules

choroid plexus

capsule of lens

pigmented layer of retina

liver parenchyma (epithelioid form)

germinal surface of ovary (low cuboidal)

Pseudo-stratified columnar

parotid gland ducts

male urethra

 

Pseudo-stratified ciliated columnar

respiratory passages

 

Stratified cuboidal

ducts sweat glands

urethra

covering of iris and ciliary body

ducts of salivary glands

 TransitionalRenal pelvis, ureter & bladderupper part of male urethra

 

BACK TO TOP

 

HISTOLOGY LAB III , CONNECTIVE TISSUE PROPER

BE ABLE TO IDENTIFY:

adipose tissuecells: fibroblast, macrophage, and plasma cellCT (dense regular and irregular; loose)fibers (collagen, elastic, reticular)lamina propria (a loose CT in a specific location)

 

Connective tissue (C.T.) is broadly classified according to the relative amounts of fibers and ground substance. C.T. with few fibers and an abundance of ground substance is designated as a loose C.T. conversely; lots of fibers and a small amount of ground substance is designated as dense C.T. Another classification called medium C.T. would be handy since there is a continuum of densities in C.T. from very loose (areolar C.T.) up to very dense (tendon). However, we are stuck by tradition with

calling C.T. either loose or dense. So --- if it isn't loose, it's dense!

Loose C.T., Adipose cells & tissue, and Macrophages (have been called Histiocytes)

Slide #4 is a whole mount (not a section) of some loose areolar C.T. At low power you will see some dark blue/purple squiggly fibers (elastic fibers) some larger straight pink fibers (collagen) and some pale blue staining cells. The cell nuclei may be in either fibroblasts or

macrophages. It is difficult to tell them apart on this slide. You can see that the fibers make up a relatively small portion of the total volume of this tissue. You may be able to locate loose connective tissue directly beneath the columnar epithelium in any sections of the gut. Here the loose C.T. is called lamina propria. Try slide #179 (jejunum). The lamina propria layer just beneath the epithelium has lots of cells but few fibers. You may be able to find a PLASMA CELL in the lamina propria.

 

 

 

 

 

 

 

 

At higher magnification the fiber types and cells are seen clearly.

Fibroblasts - - Magenta arrow

Elastic fibers - - Red/Orange arrow

Macrophages - - Green arrow

Collagen - - Blue arrow

 

 

 

 

 

 

 

 

 

 

 

Fibroblasts - - Magenta arrow

Elastic fibers - - Red/Orange arrow

Macrophages - - Green arrow

Collagen - The pink fibers labeled as such

 

 

 

 

 

 

 

 

Slide #160 & #141 are sections of rat kidney and intestine. Hold the kidney slide (#160) up to the light. Notice it has an indentation on the lower side. In this area at low magnification, you will find a blood vessel and some adipose (fatty) tissue. This adipose tissue looks sort of like a piece of chicken wire at low power (a beat up looking chain link

fence to those of you from the big city). The open spaces contained fat droplets which were dissolved away during fixation andembedding. At higher magnification you will see a nucleus and a thin strip of cytoplasm surrounding the space where the lipid (fat) droplet used to be (the holes in the fence). There is also adipose tissue on slide #141. Look at adipose tissue on SLIDE #5 also---faintly stained---a demonstration will be available showing adipose tissue stained with osmium which keeps the fat droplet intact and stains it black.

 

 

 

 

 

 

The rat who donated his kidney for SLIDE #160, was injected with a blue particulate dye called Trypan Blue. The dye particles are ingested by macrophages and you will see some of these cells between the fat cells of the adipose tissue . Use the 40X objective and look for cells with blue particles in their cytoplasm (indicated by red arrows). There are some macrophages on slide #141 as well (same donor). Later in the course we will see macrophages in thin sections that show a lot more detail and do not need trypan blue for identification.

Look for adipose in other slides of organs like gut and skin. You should feel confident that you can recognize adipose when you are finished. Well---fairly confident!

 

 

Dense Connective Tissue: Regular and Irregular

Dense connective tissue is classified as regular or irregular depending upon the arrangement of the collagen fibers. If the fibers are all lined up parallel to one another, the C.T. is regular and if the fibers are randomly oriented, the C.T. is irregular. Thefibroblasts are usually squeezed into a flattened condition in dense C.T.. Between the fibers ground substance is not readily apparent cause there isn't much there!

 

 

 

 

 

The dermis of skin beneath the epithelium is Dense Irregular C.T. (SLIDES #60, #61, #62, & 177)

 

 

 

 

 

Dense Regular C.T. is found in SLIDE #6 mammalian tendon. Look at the slide without the microscope!

 

 

 

 

 

There is a longitudinal section (upper image here) and cross section (lower image) of tendon. Look at the longitudinal section to the left Note the parallel bundles of collagen, fibroblasts, and the dark flattened nuclei.of the fibroblasts. It is impossible to visually separate the collagen fibers and the fibroblast cytoplasm in this slide. or the next slide below.

 

 

 

 

 

 

 

 

Now look at the cross-section to the left. What do you see? Can you correlate these structures in this image with those in the image above?

 

 

 

 

 

 

 

Reticular Fibers are anastomosing bundles of small collagen fibers which are stainable with silver. A lot of reticular fibers are found in lymph nodes. Slide #3 is a lymph node stained for reticular fibers.

 

 

Elastic Connective Tissue

We used to tell students to look at slide #7 which was elastic tissue. It looked like a pink blob of nothing! We don't bother to do that now. You have seen some elastic fibers in the loose areolar C.T. spread (slide #4) You will see more in other tissues. Elastic fibers are particularly useful for structures which must contract and expand, like arteries, bladders and lungs. When we study these organs, look for the elastic components. Elastin stains pink like collagen in H&E strained sections. It can only be differentiated from collagen when treated with special stains which stain it dark purple to black. Elastin is also made by fibroblasts.

Remember! Study the formation of collagen fibers in your text! Reticular fibers, collagen fibers, and elastic fibers are all produced by fibroblasts.

 

 

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