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.
Reading: Ross and Romrell: Chapter 10, pp. 214-255 (for MUSCLE
Gartner and Hiatt: Chapter 6, pp. 100-121 (for
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
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.
5. Usually appears in
6. Well vascularized by
7. High glycogen and
mitochondrial content (EM analysis).
Images to introduce skeletal
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
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
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
Glass slides in your collection
on which to view skeletal muscle.
- (skeletal muscle stained with H & E)
- (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
- tongue: Here you can find a lot of skeletal muscle fibers, cut in many
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.
- 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
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.
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.
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.
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
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
- cardiac muscle: Stained with PTAH as we saw earlier, this specimen shows
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
- 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.
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
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.
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
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
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.
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.
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?
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).
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
- 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.)
- jejunum: Find both the outer longitudinal and the inner circular layers
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
- 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?
- uterine smooth muscle: Muscle bundles here are traversing the slide in
directions. The surrounding and interdigitating dense CT is less deeply
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.
- bladder: This is a plastic section, showing a high resolution view of
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.
ADDITIONAL 35MM SLIDES FOR PRACTICE
Use images E15
(uterus) and E16 to practice telling smooth muscle from dense CT
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
Practice, practice, practice!
Smooth muscle in
TEM of dense