.

 

	1.	to understand how tissues appear under the microscope.
	2.	when given a section of mammalian tissue under a microscope or a magnified picture of a tissue (including electron micrographs), to identify the tissue, the cells that it contains and other visible structures of that tissue.
	3.	to describe the major tissues and cells that comprise the organs of the body.
	4.	to relate the functions of those tissues and cells to their structures.
	5.	to understand how the main functions of cells relate to subcellular structure.

---

Grading

The grade for this course depends upon the cumulative score for all tests as follows:

		0-70 %		NP	(not pass)
		71-73 %		M	(marginal pass)
		74-90 %		P	(pass)
		>90 %		H	(exceptional performance)

The relative importance of the various exams is:

		10%	Lab quizzes (each counts 10%, one may be dropped)
		30%	Midterm, written
		10%	Midterm, laboratory (counts as a lab quiz)
		30%	Final, written
		20%	Final, laboratory (tissue identifications)

---

Reviews

Three on line resources are available for you to use for review:

		Blue Histology
		Geoff's Review by Topic
		Short Answer Practise Quiz

 

---

Use of the microscope

Pointers In Eyepieces It is essential to have a pointer in one eyepiece of your microscope so that both you and an instructor (or classmate) can agree on what you are talking about. There is little difficulty in doing this in eyepieces of conventional design. Remove the ocular piece, turn it over, unscrew the basal element and observe a shelf placed approximately halfway down the barrel. This shelf is in the focal plane of the ocular lens, so that anything put in that location will be silhouetted when you look down the eyepiece. Put a small drop of white glue on the ring, then take an eyelash and insert its basal end in the glue. An eyelash is especially good for a pointer because it comes to a very fine point. Since these hairs are not straight, make sure that you place yours so that it bends within the plane of focus; otherwise you will get a "floating pointer." After the glue has dried, reassemble the eyepiece and return it to the microscope. If the pointer is fuzzy (especially at the tip), it is not in the exact plane of focus. Yank out another eyelash and try again. The tip of the pointer should be somewhat off center so that you can change its position in the field by simply rotating the ocular. Most wide-field eyepieces are also provided with pointers. These eyepieces usually have only a single (upper) lens. If you look into the open (lower) end, you will see a ring or shelf about 1-1 cm up. As in ordinary eyepieces, this is located at the focal plane. In this case, insert glue and hair from below, check for focus and make adjustments if necessary before the glue is completely dry.

Condenser Lens Locate the condenser under the stage. Its function is to focus light onto your slide. The condenser should be set so that the top of the lens is 1-2mm below the slide (using the knob located below the stage on the right hand side). A proper setting is unimportant at 25x, marginally so at 100x, important at x400 and indispensable for oil immersion. One problem with your microscope is that the condenser focuses the light onto a smaller area of your slides than you view at lowest power. Consequently, you see a black ring or are due to the lack of illumination around the edge of the field. However, your microscope has a nifty switch to handle the situation (also located below the stage on the right). Rotating it slides the top lens of the condenser system out of the line of vision. This illuminates the entire field at low power (at which the condenser is not needed) without changing the height of the condenser. When you go to high power be sure to always flip the condenser lens back in.

Omitting the condenser has an interesting consequence. Your slides have two components on them. One is a thin section of tissue with the various molecules of the meat, brain, or toe nail. The other is dye molecules absorbed onto the tissue components. By and large, you will want to examine the colored dye molecules instead of the tissue itself A properly set condenser allows you to do this. If the condenser is way out of position, then you will see influences from the differences in refractive index of the tissue parts superimposed on the colors. It is generally best to avoid this extra complication. A few specialized tissues are exceptional. In looking at the ground, unstained sections of bone and teeth, you will want to see variations in tissue density instead of just dye. These cases are few, however, and except where mentioned in your manual, keep your condenser positioned right for routine viewing at x450.

Your condenser also has a diaphragm or iris to cut down the area of the light beam at that point. Its purpose is to eliminate stray light. However, this is about as useful for your old machine as a no spitting sign in front of the ocean. Leave the iris wide open all of the time. Narrowing its aperture degrades your image in a way similar to misadjusting your condenser. In particular, do not use your iris to cut down the brightness. Most of your light sources have a rheostat for that purpose. If yours does not and the light streaming out of the microscope would fry a retina, drape a piece of lens paper over the light source or call over an instructor (who will probably do just that for you).

Oil Immersion Your highest power objective (100X) needs to be used with immersion oil. A thin meniscus of oil must fill in between the top of the glass cover slip and the lens of the objective. This oil has the same refractive index as glass so that the light traveling up from your slide does not get jolted across interfaces of air and glass. WITHOUT OIL, THE RESOLVING POWER OF THIS OBJECTIVE IS -> WORSE <- THAN YOUR 45X OBJECTIVE.

Using immersion oil is mildly tricky for two reasons. One is that the plane of focus of the 100x objective is very close to the surface of the slide and is very narrow. Thus, getting the slide in focus can be a problem. It is necessary to focus first with the "high dry" 45x objective. Then rotate that lens a bit out of the way and place a small drop of immersion oil on the spot of the slide illuminated by the condenser beam. Finish rotating the oil immersion objective into the oil. Because your lenses should be parfocal, as we erudites like to say, if your slide is in focus with one objective, it should be at the right height for the other objectives.

What is the other tricky bit of using oil immersion? It is keeping from making a mess. You need only a tiny drop of oil on your slide. Slobbering gobs of the stuff all over does not let you see any better (as many people- including me- have found out). If the top of your stage is sticky to the touch, the guy who had your microscope last year was a slob. The same goes for your slides. If they are oily at the end of this quarter..... When you are finished using oil, wipe the lens off with lens paper or a Kleenex. Wetting the paper first with Windex is a good idea. One other tip: Avoid rotating the 45x objective across a slide with a drop of oil in it. You will probably get oil on the objective and have to carefully clean it with Windex. You will not use oil very often and you use very little at a time so a bunch of you people can go together to buy a bottle of oil. One of the others of your group can buy some Windex and someone else can buy the apple (or better yet, a donut) for the teacher. All of you probably should have your own supply of lens paper.

By the way, resolving power is the ability of an optical instrument to form distinguishable images of objects separated by small distances.

1. The maximum resolving power of the optical microscope is about 0.2 microns (2,000 Å).

2. The maximum resolving power of the electron microscope is about 0.0005 microns (5 Å).

 

---

 

A couple of hints for the laboratory

Remembering two particular things discussed below will make your labwork easier.

One is to get perfectly straight the way hematoxylin-eosin (H+E) stain works, including the terms basophilic and acidophilic. H&E is the work horse stain for histology. If its label does not mention the stain, then the slide was stained in this manner.

The other hint is to get out all of the slides listed in your syllabus for a particular tissue before putting any under the microscope. See what slides you have first. For many organs, the kidney as an example, these will include both human and animal tissues. If the label does not mention what species the tissue came from it will be human. The animal specimens are well-preserved. The human tissues were extracted from a diseased patient or cadaver. I would begin my examinations with the well-preserved animal slide, even if the human slide was listed first in the syllabus. Then, I would look at the human slide to see idiosyncrasies of our particular species.

Also, it is a good idea to begin with a slide that is stained with H+E (or Masson stain which you will also become familiar with soon) and not with some highly specialized stain. Most of the slides with specialized stains are included to show one particular thing, such as reticular fibers, or glycogen, or elastica, or whatever. Usually, this one thing is all that is worth looking at on the slide. Glancing in advance at the label of a slide and figuring out why it was stained as it was can save the frustration of calling over the instructor (hey, I'm talking about instructor frustration, not yours.)

---

 

Preparation of Microscope Slides

You should be generally familiar with the following background information about the preparation of your microscope slides and stains. When possibly tissues are obtained (for example, as surgical specimens, or at autopsy), they must be fixed immediately. A tissue fixative should prevent evaporation, autolysis by enzymes, swelling or shrinkage, and attack by bacteria or molds. It should also modify tissue components so that they retain their form when the tissue is subjected to the succeeding procedures of embedding, sectioning and staining. No fixative is perfect. Routine fixatives (such as 4% aqueous formaldehyde) do not, for example, retain small molecular weight tissue constituents, nor do they fully protect some large molecular weight components, such as lipids, from extraction during subsequent routine steps. Furthermore, aqueous fixatives always yield some ultimate tissue shrinkage and distortion. Consequently, the fixative must be selected to suit the particular tissue or tissue component, and its limitations be kept in mind.

Fixed-tissue specimens are too fragile to section directly. First they must be infiltrated with a supporting medium, and then embedded within a block of the same medium, which can then be attached to the specimen holder of the microtome, the instrument used to cut thin sections. The more common embedding media are paraffin and celloidin. After sections have been cut and mounted on glass slides, the supporting medium is removed, and the tissues are stained.

In brief, the "routine" sequence of events is as follows:

	1.	Obtain normal human tissue.
	2.	Fix for 24 hours or more in an appropriate fixative.
	3.	Dehydrate through ascending (increasingly higher concentration) alcohols overnight.
	4.	Replace alcohol ("clear") with xylol or chloroform.
	5.	Infiltrate with paraffin (or celloidin).
	6.	Embed in a block of paraffin (or celloidin).
	7.	Cut thin sections on the microtome (6 to 10 _ thick).
	8.	Mount the sections on glass slides.
	9.	Remove (dissolve) the embedding medium.
	10.	Rehydrate the sections in descending alcohols.
	11.	Stain the sections with an appropriate staining sequence.
	12.	Dehydrate the sections in ascending alcohols.
	13.	Clear the sections in xylol.
	14.	Attach protective coverslip with a mounting medium.
	15.	Label the slide.

---

 

Stains

1. Theory behind staining

Cells and intercellular material are colorless for the most part, and consequently difficult to distinguish from one another in the light microscope. This difficulty is surmounted by exposing the tissue sections to dyes which are organic compounds and selectively bound to certain constituents of the tissue, depending on the nature of the dye and the chemical composition of the tissue. If the dye molecule is cationic (net positive charge), it is called a basic dye. If it is anionic, it is an acid dye. Acid and basic dyes may have the same color. However, they have very different staining reactions.

Dyes react directly with the tissues. If a tissue component is acidic, it will have an affinity for basic dye-ions. The component is therefore said to be basophilic. If the tissue component is basic, it will have an affinity for acidic dye-ions, and is called acidophilic. By the way, philic means loving in Greek.

Typically,  the basophilic tissue components are DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and cartilage matrix. Typically, the acidophilic constituents are collagen, cytoplasm of red blood cells, and the granules of eosinophilic leukocytes. Some substances are amphoteric, that is, they are easily swayed toward acidity or basicity and can be stained variably with either acid or basic dyes.

Some dyes are indirectly associated with tissue component. The intermediaries are salts of certain metals and are called "mordants." The combination of mordant and dye is basic in action. The most important dye used with mordants is hematoxylin. Routinely, an aluminum salt is used as mordant. Sometimes iron, chromium and other metals are employed.

After tissues have been allowed to react with one dye, a second dye of contrasting color and staining characteristics is often used to yield additional information. This is called "counterstaining." Eosin, a red acid dye, is a common counterstain for blue or purple hematoxylin. Hematoxylin-eosin is the most frequently employed staining sequence in histology and pathology. It provides a good general distinction between nuclei, cytoplasm, and most extracellular constituents. However, for improved rendition of many tissue structures or components, "special stains" are required. Several such stains have been utilized in preparation of the class slide sets.

2. Abbreviations for stains

Abbreviation	Stain	Function
B	Bodian	Stains neurofibrils, nerve fibers, extensions of osteocytes and nuclei black
Carm	Carmine	Stains nuclei and other basophilic substances red
Decal		Mineral salts have been removed or "decalcified"
Elast	Elastic	Stains elastin black
FeH	Iron hematoxylin	Iron salt used as mordant for chromatin and other cytologic details
Kl	Klüver	Myelinated fibers, greenish-blue; Cellular elements, red-violet
Masson	Masson's trichrome	General connective tissue stain:       nuclei, muscle,erythrocytes are red;       collagen, is blue;       elastin, is light pink, yellowish or unstained
Nred	Neutral Red	Demonstrates mast cell granules
PAS	Periodic Acid Schiff	Detects 1:2-glycol groups or the equivalent alkyamino derivatives. Stains basement membranes, glycogen, mucus and other polysaccharides red
PASH		(PAS counterstained with hematoxylin)
Perf		(Organ was perfused with saline solution before fixation)
PTAH	Phosphotungstic Acid	Demonstrates intercalated discs
Polychr	Polychrome	Demonstrates pituitary cell types
Reticulum Silver		Demonstrates reticular fibers and nerve cell processes
Tol B1	Toluidine blue	Shows metachromasia (change in color towards red when adsorbed onto ionic polysaccharides)
Trichr	Trichrome (Masson's or Mallory's)	See Masson stain
Tryp Blue	Trypan Blue	Colloidal dye, injected intravitally, to demonstrate phagocytosis
Wright's	Wright's	Blood stain:      nuclei purple-violet;      basophil granules, dark violet blue;      neutrophil granules, purple to  violet;      eosinophil granules, orange-pink

 

---

 

Checklist for final tissue identification exam

For the laboratory part of the final you will be given a set of 25 unlabeled slides. These will have sections on them comparable to those in your slide sets, but cut from different blocks. The possible tissues and organs are listed below. This list contains all of the acceptable possible answers. All preparations are stained with H&E. Note that a few of these sections are more restricted than those in your sets (e.g. cornea and dentin).

adipose tissue loose connective tissue tendon smooth muscle cardiac muscle skeletal muscle muscular artery elastic artery vein vena cava heart wall heart valve blood (smear) bone marrow lacrimal gland mammary gland, resting mammary gland, lactating thin skin thick skin scalp peripheral nerve optic nerve spinal ganglion sympathetic ganglion spinal cord cerebral cortex cerebellar cortex choroid plexus lymph node palatine tonsil spleen thymus

hyaline cartilage elastic cartilage fibrocartilage articular cartilage intervertebral disc bone fetal skull fetal knee finger joint suture nose larynx epiglottis trachea bronchus lung eye cornea retina pinna of ear external ear canal internal ear/cochlea kidney ureter bladder urethra pituitary gland thyroid gland parathyroid gland adrenal gland

ovary ovary with corpus luteum oviduct uterus, proliferative uterus, secretory cervix vagina clitoris testes epididymis spermatic cord seminal vesicle prostate gland penis esophagus stomach duodenal-pyloric junction small intestine large intestine appendix anorectal junction pancreas liver gall bladder lip hard palate anterior tongue posterior tongue parotid gland submandibular gland sublingual gland tooth dentin

---

List of slides in the dental slide set

D- 1   Buccal mucosa scraping, mouth (Pap) D- 2  Adipose tissue (H&E) D- 3  Umbilical cord (toluidine blue) D- 4  Connective tissue spread (H&E, neutral red, trypan blue) D- 5  Tendon (H&E) D- 7  External ear (elastic stain, H&E) D- 8  Intervertebral disk (H&E) D- 9  Fibrocartilage (H&E) D-11  Abdominal wall, rat (Masson and PAS) D-12   Skeletal muscle stretched (H&E) D-13   Muscle spindle, cross-section (H&E) D-16   Heart muscle (H&E) D-17   Cardiac muscle (PASH) D-18   Left atrium (elastic stain) D-19  The heart (H&E) D-20   Purkinje fibers (H&E) D-22   Aorta (elastic stain) D-23   Aorta (H&E) D-24   Carotid sheath (elastic stain) D-25   Brachial artery & vein (Masson) D-26   Subclavian artery (H&E) D-27   Vena cava (Masson) D-29   Blood smear (Wright stain) D-30   Bone marrow smear (Wright stain) D-31   Bone marrow section, monkey (Giemsa) D-32   Bone marrow section, rabbit (carbon injection and Giemsa) D-33   Lymph node (reticulum stain) D-34   Lymph node, child (H&E) D-35   Palatine tonsil (H&E) D-36   Thymus, involuted (H&E) D-37   Thymus, active (H&E) D-38   Spleen, dog perfused (H&E) D-39   Spleen, human, at autopsy (H&E) D-42   Kidney, monkey (FeH) D-43   Kidney (simple cuboidal and squamous epithelia). D-44   Kidney (H&E) D-45   Kidney (PASH) D-47   Ureter (H&E) D-48   Urinary bladder (transitional epithelium) (H&E) D-49   Urinary, bladder (H&E) D-50   Female urethra (H&E) D-51   Uterus, follicular phase (H&E) D-52   Uterus (H&E) D-53   Uterus, luteal phase (H&E) D-54   Corpus luteum (H&E) D-55   Ovary (H&E) D-56   Ovary and oviduct, monkey (H&E) D-57   Ovary, cat (preg) (H&E) D-58   Ovary, monkey (H&E) D-59   Cervix (H&E) D-60   Oviduct, child (H&E) D-61   Vagina, (H&E) D-62   Clitoris (H&E) D-63   Breast, nullipar (H&E) D-64   Breast, lactating (H&E) D-65   Spermatic cord (H&E) D-66   Testis (FeH) D-67   Testis (H&E) D-68   Vas deferens (H&E) D-69   Epididymis (H&E) D-70   Prostate gland (H&E) D-71   Penis (H&E) D-72   Seminal vesicle (H&E) D-73   Ovary, monkey (H&E) D-74   Ovary, monkey (H&E) D-75   Bronchial tree (H&E) D-76   Bronchus (H&E) D-77   Lung monkey (H&E) D-78   Lung, 5 1/2; month fetus (H&E) D-79   Lung (H&E) D-80   Trachea (H&E) D-81   Oviduct and fimbriae, monkey (H&E) D-83   Nose, monkey (H&E) D-85   Cheek, fetus (H&E) D-86   Lip (H&E) D-87   Fetal palate, woven and lamellar bone (H&E) D-89   Neonatal palate (H&E) D-90   Hard palate (H&E) D-91   Hard palate (H&E) D-92   Tongue, rabbit (Masson stain) D-93   Anterior tongue (Masson stain) D-94   Tongue (H&E) D-95   Tongue (H&E) D-96   Posterior tongue (H&E) D-98   Epiglottis (elastic stain) D-99   Larynx, with epiglottis (H&E) D-100  Vocal Cord (H&E) D-101  Esophagus (H&E) D-103  Fundic stomach, monkey (H&E) D-104  Stomach monkey (PASH) D-105  Pyloric-duodenal junction (H&E) D-106  Upper duodenum and pylorus, monkey (H&E) D-107   Duodenum (H&E) D-108   Duodenum, monkey (Masson stain) D-109   Duodenum rabbit (FeH) D-111   Appendix (H&E) D-113   Colon ,monkey (Muc. Carm.) D-114   Colon (H&E) D-115   Ano-rectal junction (H&E) D-116   Gallbladder with liver (H&E) D-117   Liver (Masson) D-118   Liver (Trypan blue + Carmine) D-119   Liver, monkey (H&E) D-120   Liver, rabbit (H&E) D-121   Pancreas, monkey (Masson stain) D-122   Pancreas (H&E) D-123   Pancreas (Gomori-Masson stain) D-124   Gallbladder (Van Giessen) D-125   Adrenal Gland (Chromaffin stain) D-126   Parotid gland (H&E) D-127   Submaxillary gland (PAS stain) D-128   Submaxillary = submandibular gland (H&E) D-129   Sublingual gland (H&E) D-130   Sublingual gland (H&E) D-131   Adrenal, monkey (H&E) D-132   Adrenal, cat (H&E) D-133   Adrenal (Masson stain) D-134   Thyroid (Masson stain) D-135   Thyroid, monkey (PASH) D-136   Thyroid (H&E) D-137   Parathyroid (H&E) D-138   Thyroid (H&E) D-139   Pituitary, cat (H&E) D-140   Pituitary, cat (polychrome stain). D-141   Pituitary, rat (H&E) D-142   Suture of a newborn (synarthrosis) (H&E) D-143   Fetal skull (H&E) D-144   Toe joint of Monkey (H&E) D-145   Adult finger joint (H&E) D-146   Newborn knee (endochondral osteogenesis) (PASH) D-147   Lamellar bone in a decalcified femur (H&E) D-148   Lamellar bone, newborn (Bodian) D-149   Compact bone (Ground) D-151   Deciduous tooth, monkey (PASHE) D-152   Deciduous tooth, monkey (H&E) D-153   Molars, monkey (H&E) D-154   Tooth, adult, posterior (ground) D-155   Tooth, adult, anterior (ground) D-156   Tooth, adult, anterior, XS (ground) D-157   Gingiva, monkey (H&E) D-158   Maxillary tooth, monkey (H&E) D-159   Mandibular tooth, monkey (H&E) D-161   Thick skin (H&E) D-162   Scalp (H&E) D-163   Thin skin (H&E) D-164   Toenail (H&E) D-165   Fingertip (H&E) D-166   Fingertip (silver) D-170   Lacrimal glands (H&E) D-171   Eye lid (H&E) D-174   Eye, rabbit (H&E) D-175   Eye (Masson) D-176   External ear canal (H&E) D-177   Internal ear, Guinea pit (H&E) D-181   Optic nerve, transverse section (H&E) D-182   Peripheral nerve (Bodian) D-183   Peripheral nerve (Masson) D-184   Sympathetic ganglion D-185   Spinal ganglion, cat (B) D-188   Spinal cord (H&E) D-189   Trigeminal ganglion (FeH) D-191   Spinal cord (Kluever stain) D-192   Spinal cord (silver stain) D-193   Spinal cord (Bodian) D-194   Spinal cord (Kluever stain) D-195   Cerebral cortex (stained for astrocytes) D-196   Cerebellar cortex (H&E) D-197   Cerebellum (Bodian stain) D-198   Cerebrum (Kluever stain) D-199   Choroid plexus (H&E)

 

* * * * *