Objectives:

In the slides of muscle, you need to be able to distinguish the 3 types of muscle tissue, skeletal, cardiac and smooth. You also need to be able to distinguish the different levels of organization of the muscle, endomysium, perimysium, epimysium. The sarcomeric organization of skeletal and cardiac muscle are difficult to resolve in these sections, but you nevertheless need to understand the different parts of the sarcomere, i.e. A-band, I-band, Z-disk (or Z-line), M-line and for cardiac muscle the intercalated disc.

Panels A-D are from slide14A whereas panels E-H are from 14B. Panels A & E are monocytes, panels B & F are eosinophils, panels C & G are lymphocytes and panels D & E are neutrophils. Note that characteristically lymphocytes are about the same diameter as RBCs whereas monocytes are about twice as large.

14A Human blood smear, Wright
14B Human blood smear, Giemsa
There are two groups of blood smears, one stained with Wright's stain, the other with Giemsa. You will be able to tell very little difference between the two. Giemsa stain is supposed to stain leukocytes a reddish purple but otherwise it is similar to Wright's stain.

There are several items that you need to keep in mind when identifying the different types of WBCs. The cytoplasm of both lymphocytes and monocytes stain blue and depending on the intensity of stain, you may not be able to distinguish between them. However, you can distinguish between them on the basis of size. The size of lymphocytes is very near that of RBCs, whereas monocytes are usually twice as large as RBCs. Keep in mind the color of the granules. Neutrophils have granules, but the granules do not stain intensely; they just have a fairly faint pink color in most of these slides. Eosinophils have red granules, and basophils, if you find any, will have blue granules.

Prepare a qualitative white blood cell count:

Use the 40x objective to scan quickly to learn the appearance of the most common types of WBC, the neutrophil and the lymphocyte. Since the staining varies from one slide to the next, you need to familiarize yourself with the appearance in your slide. Using your atlas, pages 211 & 213 to identify the different cells and try to obtain a relative count of the different WBCs. Scan for no more than 30 minutes, or as long as it takes to count 50 WBCs. Scan the slide systematically from side to side or front to back (meander line).

Note that blood smears may contain young, immature granulocytes with ribbon-shaped, non-segmented nuclei (band-cells). You should be aware that increased percentage of these young forms ("shift to left") is usually sign of infection or hematological disease .
 
 

CELL TYPE	NUMBER COUNTED	PERCENT COUNTED	EXPECTED PERCENT
Neutrophil
Eosinophil
Basophil
Lymphocyte
Monocyte
TOTAL

SKELETAL & VISCERAL STRIATED MUSCLE - skeletal muscle is attached to the skeleton; visceral striated muscle, which is identical to skeletal muscle, is associated with soft tissues and can be found in the tongue, pharynx, upper esophagus, diaphragm, pelvic diaphragm : anus (external anal sphincter) and urogenital diaphragm (external urethral sphincter). Striated muscle is characterized by large, multinucleate fibers with nuclei at the periphery (under the sarcolemma). Striations are visible in longitudinal sections.

Suggested slides: 15 Diaphragm; 18 Skeletal muscle; 74 Tongue;

CARDIAC MUSCLE – Only the myocardium (muscular layer of the heart) contains this special striated muscle. The cardiac muscle of the atria and ventricles are associated with, and electrically isolated from each other by fibrous rings surrounding the heart valves at the levels of the coronary sulcus of the heart. Unlike skeletal muscle, it consists of often branching CELLS containing a single, centrally located nucleus. Cells are attached end –to-end by intercalated discs thus forming long fibers.

Suggested slides: 19 Cardiac muscle; 21 Rabbit cardiac muscle, plastic, BF-MB

SMOOTH MUSCLE - found along the alimentary canal, blood vessels, genitourinary tract and respiratory tract as well as a few specialized organs such as the iris and ciliary body of the eye, in the skin of the scrotum and in the arrector pili muscles of hair follicles. Characterized by spindle shaped cells with a single, centrally located nucleus. There are NO striations, since the actin and myosine filaments are not as strictly organized as in striated muscles.

Suggested slides: 8 Transitional epithelium; 94 Small intestine composite
 

A. Skeletal Muscle

15A  Diaphragm, Masson, human

This is well preserved section of skeletal muscle. You should be able to see the striations as well as the A-band, I-band and Z-line. The fibers are arranged in bundles surrounded by a perimysium. However, you may have trouble finding the perimysium which would appear as a thin layer of connective tissue. For those who like to review, there is a very nicely preserved mesothelium on one side, which we would call the natural edge. An adventitia is found on the other side but is probably not the natural edge of this muscle. Cut edges are found on the left and right hand sides.
 

15B Diaphragm, Wilder’s, Human

Wilder’s is a reticular fiber stain so the basal lamina, the endomysium of the muscle fibers stands out clearly. The muscle striations are visible. There are some other regions that are very dense in stain that are probably smooth muscle fiber bundles. Each smooth muscle cell is also surrounded by a basal lamina. Since smooth muscle cells are much smaller than skeletal muscle cells, this would explain the high density of stain. Where would smooth muscle come from in a piece of diaphragm tissue? (a gross anatomy question…)
 

15C Diaphragm, Verhoeff, human

Verhoeff stain highlights elastic fibers as well as nuclei which stand out black. Like the Masson stained diaphragm (slide 103) there is a nicely preserved mesothelium on one edge. Take time to examine it because some regions show both transverse and longitudinal sections through the mesothelium, which is something that you have not seen previously. Because the nuclei stand out with this stain, you should have no trouble identifying that the skeletal mucle nuclei are at the edges of the fibers.
 

18A E. O. Musc., XS, Masson, human

This is a poorly preserved piece of muscle, but it reveals two important characteristics of skeletal muscle fibers. First, the nuclei are at the periphery of the cell, which is a distinguishing characteristic of skeletal muscle. Second, the poor preservation has caused the muscle cells to shrink up revealing the endomysium. The punctate appearance of the muscle fibers in cross section is due to individual myofibrils. While the endomysium surrounds the individual muscle fibers, a connective tissue capsule, the perimysium, surrounds fiber bundles. Not seen in this picture is the epimysium, which surrounds the muscle itself. It may be visible in other areas of the slide.

B. Cardiac Muscle

 19a Cardiac muscle, human

This slide is stained with iron-hematoxylin, which brings out nuclei, mitochondria and muscle striations in blue-black color. You can see that the cardiac myocytes have central nuclei and that the muscle is striated. These are the distinguishing characteristics of cardiac muscle. However, the striations are not easily visible.
 

20 Intercalated discs, heart

This slide is also stained with iron-hematoxylin reveals both striations and the intercalated discs, which show up as a black line easily distinguished from the muscle striations. The nuclei also can be seen clearly with the 40X objective. They are centrally located, a distinguishing characteristic of this muscle.
 

21 Rabbit cardiac muscle, plastic, AF-TB

This slide of plastic embedded cardiac muscle shows exceptional detail. You should identify and differentiate intercalated discs (id) and cross striations from the sarcomeres. Also clear is the location of the nucleus in the cells, and the branching aspect of the individual cells. Within the clear places of the cardiomyocyte there are small, dark spots. These are probably mitochondria.
 
 

C. Smooth Muscle

Smooth muscle can be found in all of the slides of the GI tract from the lower esophagus to the colon. If your box does not have a good example of the slides given below, look at one of the other slides of small intestine (slides 87-94) and use the pictures shown below as a guide to locate smooth muscle.
 

23 Smooth muscle, c.s & l.s.

By way of review, you should be able to identify the epithelia lining the lumen and covering the exterior surface (peritoneum’s mesothel) of this organ. Identified above are the different regions of this organ (you will have to know these eventually). The submucosa is dense, irregular connective tissue. However, the collagen fibers do not stand out in this stain (H&E). Note that the smooth muscle cells are arranged in 2 layers in the muscular layer of the intestinal wall:

There is an inner circular and an outer longitudinal layer. This arrangement provides you a perfect opportunity to observe cross- and longitudinal sections of smooth muscle cells. (See Fig. 10.13, page 230)
 
 

90D Jejunum, c.s.
92A Ileum
You looked at these slides in the lab on epithelium. The arrangement of muscle layers is identical to slide 23. Either one of these slides is a very good example of smooth muscle. Note the high density of nuclei and the lack of any other distinguishing characteristics. This is typical of smooth muscle. Smooth muscle should not be confused with dense regular connective tissue. Why?
 

8 Transitional epithelium, rabbit bladder

You should have already looked at this slide in the epithelium lab. Besides having a well preserved epithelium, it also has well preserved smooth muscle that comprises the [tunica] muscularis. Smooth muscle is found in various orientations within the [tunica] muscularis. What makes this slide useful is the demonstration of an important characteristic of smooth muscle, the serrated (postage stamp) edges. Compare these images with Fig. 10.16a in RR&K. In cross section, very few of the cells have nuclei, which occurs because of the elongated fusiform shape of the cells.
 

 Motor end organ, w.m.

HANDLE THIS SLIDE WITH EXTREME CARE!!! THERE ARE ONLY THREE COPIES (and they are real nice slides!!!). The slide will be mounted on an extra microscope at the side bench. This is a whole mount slide, not a section. Muscle fibers run left to right while a nerve fiber runs front to back. The motor end plates (MEP) are in many places and appear as a punctate patch on the muscle fiber. Typically, histochemical staining for acethyl-cholinesterase enzyme visualizes MEPs, which are hard to find in ordinary sections because there is only one per fiber. This prep was taken from the region of a muscle where most of the MEPs are concentrated. (See Fig. 10.7 in RR&K)
 

 Muscle spindle, w.m.

HANDLE THIS SLIDE WITH EXTREME CARE!!! THERE ARE ONLY TWO COPIES (and they are real nice slides!!!). You will find this slide mounted on an extra microscope on the side bench. Muscle spindles are special mechanoreceptors contributing to the afferent loop of the monosynaptic strech reflexes (like the patella=knee jerk reflex). Extensor muscles (like the quadriceps femoris muscle) very abundant in muscles spindles. Muscle spindles are involved in the regulation of muscle tone. Afferent (sensory) nerve fibers innervate the receptor and it also receives efferent (motor) fibers (axons of g -motoneurons). The spindle fibers are thin muscle fibers that are broad where the nerve fibers make contact with them. There are both afferent and efferent nerve fibers in this view but you cannot distinguish them. Around the spindle fiber are extrafusal muscle fibers. (See Fig. 10.10, page 227 in RR&K)