Histology of Muscle Tissue
Laboratory 7












FIRST: Concentrate on the histology of muscle and then proceed to the study of bone structure. We will continue with study of bone development and remodeling in the next lab session.

Required Reading: Ross and Romrell: Chapter 10, pp. 214-255 (for MUSCLE only)

Gartner and Hiatt: Chapter 6, pp. 100-121 (for MUSCLE only)

YOUR JOB DURING THIS LAB: Following your histology lab studies of muscle you should be able to (a) identify each of the three types of muscle tissue in routine H & E stained sections; (b) understand the criteria used to distinguish each of these types and how the differences in structure relate to the particular function of smooth, skeletal and cardiac muscle; and (c) appreciate the structural differences at the EM level. Finally, it is important that you be able to distinguish muscle, particularly smooth muscle, from both dense connective tissue and nerve in routine histological specimens.


Criteria for identifying skeletal muscle:

1. Tissue is very eosinophilic due to the exceedingly high cytoplasmic content of protein.

2. Very dense with relatively few nuclei compared to cytoplasmic volume.

3. Large, but varying sized, polygonal cross-sectional regions (cells) with peripheral nuclei.

4. Striated.

5. Usually appears in large bundles.

6. Well vascularized by capillaries.

7. High glycogen and mitochondrial content (EM analysis).

Images to introduce skeletal (voluntary) muscle.

E1: This is a typical example of skeletal muscle stained with H & E. You should note the large polygonal cross-sections which dominate much of the image. Find the very peripherally placed nuclei, visible in many but not all of the muscle cells. (Why are not all of the nuclei seen?) Examine the connective tissue surrounding the muscle fibers. Find the perimysium and the endomysium. Careful observation of some of the cross-sectioned fibers will reveal a punctate pattern, representing end-on views of the myofibrils. Examine the interstices between the muscle fibers and find capillaries, many of which remain filled with an erythrocyte.

In one corner of the image you will see skeletal muscle fibers cut longitudinally. Here, the peripheral nuclei and the striations are quite obvious.

E2: A longitudinal section of skeletal muscle, illustrating the peripheral nuclei and cross-striations. Note that these muscle fibers are unbranched and extend the length of the muscle cell. The fibers are multi-nucleated.

E3: A high magnification view of skeletal muscle showing the histological components of the sarcomere. Review your textbook on pp. 217-220 and identify each of the labeled parts. The bracket spans a distance of 10 sarcomeres. Remember that the sarcomere is defined as the area between two adjacent Z discs. This image also shows the very close apposition of the nuclei to the sarcolemma (muscle cell plasma membrane).

E4: Another review of sarcomere structure. The H band is labeled here, where portions of the myosin thick filaments are no longer overlapped by actin thin filaments. This is a low magnification electron micrograph.

Glass slides in your collection on which to view skeletal muscle.

Slides #35/35B - (skeletal muscle stained with H & E)

Slide #36 - (skeletal muscle stained with phosphotungstic acid/hematoxylin)

These are the best slides for studying skeletal muscle in both longitudinal and oblique section. Scan the slides with the 10X and 40X objectives first. Find the nuclei (obscured by the stain on slide #36), capillaries and look at the CT present. Identify the relative positions of endomysium and perimysium. At high power, select fibers on which the cross-striations are prominent and examine the sarcomeric structure. NOTE THAT THE APPARENT SPACE BETWEEN MUSCLE FIBERS IS NOT REAL, BUT AN ARTIFACT OF SHRINKAGE.

1. The A and I bands and occasional Z discs may be visualized using oil immersion at 100X. In both slides #35 and 36 the I band appears pale and is transected by the darkly stained Z disc. In slide #35 the A band is pink and in slide #36, it is dark blue. The H band is difficult to detect, but would be a lighter region in the middle of the A region.

2. Slide #36 shows parts of the endomysium between individual muscle cells. Some perimysium is also evident. With this stain the CT stains an orange-brown color. Why do you not usually see the epimysium in histological sections?

3. Slide #35B has two pieces of skeletal muscle. That piece cut obliquely or in cross-section is good for looking at nuclei. The piece cut longitudinally is good for looking at striations. Be sure to look at both samples.

Slide #101 - tongue: Here you can find a lot of skeletal muscle fibers, cut in many different
directions. Why are the muscle bundles not all aligned in this sample? You can also find excellent examples of both serous and mucous glands, occasional nerves, and an epithelium (classify it). Compare the appearance of the skeletal muscle with the CT immediately underlying the epithelium.

Slide #106 - esophagus: Depending upon exactly where along the length of the esophagus you section, you will find both skeletal and smooth muscle. This slide shows both types, so use it to distinguish them. The smooth muscle is found in small bundles, close beneath the epithelium and is cut primarily in cross-section. (Why do you think it is there?) Compare the muscle histology with the nearby dense CT. Examine the endomysium and associated capillaries closely in the skeletal muscle bundles. Note again that many slides will exhibit a shrinkage artifact as the protein rich muscle cells have pulled away from each other during fixation. This is not representative of the living condition (but often does help you to identify this tissue as skeletal muscle!).

Slides 18 (but not 18B); 91; 13; and 103 Find skeletal muscle in each of these slides. Study its relation to the other tissues seen on the slides and pay particular attention to the differences in appearance between the muscle tissues and any dense connective tissues which you may find. Practice your ability to distinguish these tissues.

ULTRASTRUCTURE OF SKELETAL MUSCLE - In ADDITION to the electron micrographs shown in your textbook (Ross & Romrell) and atlas (G&H), study the following figures in your loan copy of FAWCETT:

Figs. 10-12; 10-16; 10-17; 10-19; 10-20; 10-21; 10-22; 10-23; 10-24; 10-25


Criteria for identifying cardiac muscle:

1. Striated, "dense", eosinophilic
2. Branching fibers.
3. Central nuclei.
4. Intercalated discs.
5. Well vascularized.
6. Abundant and large mitochondria seen with the EM.

Images to introduce cardiac muscle.

E5: An excellent longitudinal section of cardiac muscle. Striations are evident, although in many common examples of cardiac muscle they would be more difficult to see, compared to skeletal muscle. Find the large central nuclei and compare these with those you saw earlier in skeletal muscle. The endomysium is shown by (a) and can be seen elsewhere with numerous associated capillaries (find some individual RBCs). The arrowheads show intercalated discs. Can you find others? The arrows indicate regions of branching; you would not see these in skeletal muscle which is unbranched. (b) shows lipochrome granules which appear in the cytoplasm near the nuclei of cardiac muscle.

E6: A good image on which to study intercalated discs (arrowheads). Find more of them on the image. Note that they sometimes seem to cross the muscle fiber step-wise, not in a straight line. This is because the terminal Z-discs of all myofilaments are not in perfect transverse alignment at the end of each cardiac muscle fiber.

E7: An EM of the intercalated disc. 1 = gap junction; 2 = fascia adherens; 3 = desmosome. Compare this with Fig 10-44 in Fawcett (page 303) and with Plate 32 in Ross & Romrell (p. 246-247). Do you understand the function of each of these constituents?

Glass slides to study cardiac muscle.

Slide #34 - cardiac muscle: This slide is excellent to locate the central nuclei and neighboring capillaries (see the RBCs nicely lined up, even if the endothelium is not easily visible) typical of cardiac muscle tissue. Follow individual fibers to find branching points of the cardiac muscle cells. Focus carefully up and down with your fine control to help yourself locate the intercalated discs.  Note: you will find some nuclei between the muscle fibers; these are from the CT cells and endothelial cells. Do not confuse them with muscle cell nuclei. Observe the much smaller cell diameter in relation to the nuclei that is typical of cardiac, compared to skeletal, muscle.

Slide #37 - cardiac muscle: Stained with PTAH as we saw earlier, this specimen shows the
 cardiac muscle striations to better advantage. Intercalated discs are stained darkly and can
 be easily seen in those areas sectioned longitudinally. Muscle nuclei are harder to see using this stain, but some may be detected in the center of the fibers if you look closely. Find branch points. Compare this slide directly with slide #36 of skeletal muscle.

Slide #39 - cardiac muscle: This is a high resolution 1.5 um, plastic embedded section stained with H & E. Study it carefully to identify all of the characteristic features of cardiac muscle. You will probably have to over/under focus slightly in order to see the intercalated discs which are stained lightly here.

Ultrastructure of Cardiac Muscle

In ADDITION to the figures and plates in your textbook (Ross & Romrell) and atlas (G&H) you are also responsible for studying following figures in FAWCETT:

Fig. 10-37; 10-38; 10-39; 10-40; 10-41; 10-42; 10-43; 10-44



Images to examine

E8: This shows a MUSCLE SPINDLE, directly in the center of the image; compare with figure 10-32 of Fawcett (page 294) and figure 10.10 of Ross & Romrell (page 227). This image is courtesy of Dr. Odor.

E9: This image is dominated by the large, lightly pink stained PURKINJE FIBERS of the heart. Note their size in relation to the cardiac muscle fibers cut in cross-section and visible in one corner of the slide. The Purkinje fibers are surrounded by dense CT and nerves and are also illustrated in Plate 33 (pp. 248-249 in Ross & Romrell).



Criteria to distinguish smooth muscle

1. Eosinophilic
2. Usually bundled in smaller units than skeletal or cardiac muscle, except when found in very well-organized layers or sheets.
3. Small, elongated, tapered cells.
4. Not multi nucleated.
5. Central nuclei, not visible in each cell when cross-sectioned.
6. No branching.
7. Not usually well cordoned off from surrounding CT when the smooth muscle is present as smaller bundles (important for comparisons with peripheral nerves).
8. When contracted, nuclei often assume a cork-screw appearance.

Images to introduce smooth muscle histology.

E10: Smooth muscle from the wall of the bladder. Asterisks (*) show areas of smooth muscle cut in longitudinal section, where the elongated shape of the nuclei can be viewed. Remember, however, that overall nuclei shape here will depend greatly upon the state of smooth muscle contraction. Many fibers are shown in cross-section; find the central nuclei and note that most cells do not seem to have one (why)? Muscle fascicles are indicated by the brackets. Note the relative high degree of interdigitation of dense CT and muscle fibers.

E11: Smooth muscle cut in cross-section, at high magnification, and stained with a special trichrome stain. The individual muscle cells stain various shades of purple and some have clearly visible centrally placed nuclei. The endomysium surrounding each muscle cell is stained blue here; usually the endomysium is difficult to see in H & E samples. The bright red cells are RBCs in capillaries.

E12: A low power view of the intestinal wall, showing two highly organized smooth muscle layers. This is a cross-section of the gut. Towards the outside (serosal side) one sees the outer, longitudinal layer of smooth muscle (here cut in cross-section). Progressing inward, one then finds the broad band of inner, circularly arranged smooth muscle (here cut longitudinally). Compare these two layers carefully. Note how well organized the individual muscle cells are and think about why they need assume this configuration. Can you see the simple squamous epithelium on this slide?

E13: An electron micrograph of smooth muscle cell cytoplasm showing dense bodies (arrow). Compare this appearance with the organized thick/thin filaments of striated muscle (both skeletal and cardiac).

E14: Another EM view of smooth muscle cytoplasm, taken after detergent extraction of most cytoplasmic proteins. What remains visible are the cytoplasmic filaments and dense bodies.

Glass slides on which to view smooth muscle.

Slide #135 - gall bladder: Smooth muscle bundles are found in the dense CT under the
 simple columnar epithelium. Also, find the smooth muscle in the walls of the larger blood vessels.

IMPORTANT HISTOLOGICAL HINT: Large blood vessels (actually most blood vessels) have smooth muscle making up most of their walls. Since slides of almost any tissue at low magnification will reveal some blood vessels, use the smooth muscle found there to establish your slide-by-slide appearance of smooth muscle. As a general rule assume the following: (a) relative staining, shrinkage etc. will vary from slide to slide, but (b) within any one slide, staining and artifacts will be consistent. For example, if you find an obvious blood vessel, having bright green smooth muscle, assume that smooth muscle elsewhere on the slide will also be bright green. (A good example of why you should NOT over-emphasize color in making your histological diagnoses.)

Slide #120 - jejunum: Find both the outer longitudinal and the inner circular layers of smooth
 muscle. Can you identify these as smooth muscle? What criteria have used used? Why is
this tissue not identifiable as dense connective tissue? Remember that these two muscle
layers are named with reference to the long axis of the intestine, not any particular way this
 tube is sectioned. Compare with image E12 for reference.

Slide #108/108A - esophagus: Distinguish the smooth muscle from the skeletal muscle.
Compare the smooth muscle with dense CT; could you tell the difference in a black-and-white photograph of this slide? Remember that depending upon where in the esophagus your individual slide is from, the relative proportions of striated/smooth muscle will vary. Why?

Slide 38 - uterine smooth muscle: Muscle bundles here are traversing the slide in all
directions. The surrounding and interdigitating dense CT is less deeply stained, often
 the situation. This slide is an important one to review later, when you are self-testing on
 your ability to distinguish dense irregular connective tissue, smooth muscle and nerves.

Slide 149 - bladder: This is a plastic section, showing a high resolution view of smooth muscle.

Ultrastructure of Smooth Muscle

In ADDITION to the figures in Ross & Romrell and G&H, study the following figures in FAWCETT:

Figs. 10-3; 10-4; 10-5; 10-6.


Use images E15 (uterus) and E16 to practice telling smooth muscle from dense CT and
 from nerve (I promise we will talk about nerve soon!) Image E15 might be very helpful
since it is not stained in the usual manner. Many of the slides in your collection will show
 similar microscopic fields; pick some at random and see what you can find.
Practice, practice, practice!



























Image E1
Skeletal muscle

Image E2
Skeletal muscle

Image E3
Sarcomere histology

Image E4
Sarcomere TEM







































Image E5
Cardiac muscle

Image E6
Intercalated discs

Image E7
TEM of intercalated disc














Image E8
Muscle spindle

Image E9
Purkinje fibers










Image E10
Smooth muscle bundles

Image E11
Smooth muscle
Trichrome stain

Image E12
Smooth muscle in intestine

Image E13
TEM of dense bodies

Image E14
Detergent-extracted smooth muscle












Image E15
Fast green stained tissue

Image E16
Smooth muscle and CT