LABORATORY GUIDE FOR CONNECTIVE TISSUE PROPER

EMRYONIC CONNECTIVE TISSUE

MESENCHYME
Slide 2 (fetal hand) shows the appearance of primitive c.t. in various stages of differentiation. Central areas can be appreciated where cartilage and bone are forming, while epithelium is developing on the surface. Look underneath the epithelium for plump or spindly mesenchymal cells randomly scattered in an abundant ground substance. Many of these are in the process of becoming fibroblasts.

MUCOUS
Look for an example of Wharton's jelly in the umbilical cord, Slide 71. On this slide is both a piece of placenta, which you will not cover until the end of the course, and umbilical cord, which can be identified by the presence of 3 large vessels which may contain red blood cells. Distinguish the placenta from the cord with your naked eye before putting them under the microscope! All the tissue surrounding the vessels in the cord is Wharton's jelly, or mucous connective tissue. It contains large amounts of ground substance and collagen, with scattered, rounded, immature fibroblasts.

ADULT CONNECTIVE TISSUE

LOOSE (AREOLAR) C.T.
Good examples of loose c.t. are to be found in the gut lamina propria, underneath the columnar epithelium lining the gastrointestinal tract. Find this area on slide 35 (colon) and slide 43 (gallbladder). The lamina propria represents a highly cellular form of loose c.t. It is conspicuous by its heterogeneous cell population set amidst a background of eosinophilic collagen fibers and clear spaces. After you are oriented with the 10X objective lens, begin to identify the cells typical of connective tissue proper with 40X. Do not use oil immersion yet.

Lymphocytes Usually only the dense, black, round nucleus of this cell is seen. In slide 35, large collections of these cells can be most easily and definitively identified in large clusters called lymph nodules. These nodules appear at low magnifications as a ball of black dots that distorts the surrounding tissue.

Plasma cells These may be easier to find in slide 43. They are plump, oval-shaped cells with an abundant, easily visible basophilic cytoplasm and an eccentric nucleus with a clockface distribution of heterochromatin.

Eosinophils These are found normally and in abundance in slide 35, colon, and stand out easily due to their bright orange-red cytoplasm. They are not to be confused with erythrocytes (red blood cells) which are found in vessels and are the only other cell type displaying this unique eosinophilia in an H&E section. Also, eosinophils will have a dark nucleus with one or two lobes visible, while erythrocytes have no nucleus and appear disk-like.

Fibroblasts Looking at the ileum, move deeper into the tissue where the organization is looser and there are alot of what appear to be vacant spaces with wisps of collagen and scattered cells. This is the other variant appearance of loose connective tissue. It follows the rule that the collagen fibers are widely separated and oriented in all directions, but is much less cellular than the loose connective tissue of the lamina propria. This area is called the submucosa. This is an excellent place to find fibroblasts since there are few other cell types around. Look at the collagen fibers, and in association with them will often be elongated, cigar-shaped nuclei, without visible cytoplasm. This is the typical appearance of the fibroblast, which, like the macrophage, doesn't easily show its cytoplasm in connective tissue.

Macrophages The cytoplasm of the connective tissue macrophage is difficult to resolve against the surrounding ground substance. Thus tissue histiocytes often appear only as bare nuclei and are difficult to distinguish from fibroblasts without alot of experience. You will not be required to identify tissue histiocytes in connective tissue proper!

However, in other locations, macrophages are easy to find. Lymph node macrophages are easily identified in slide 87 (lymph node). Large black particles are found clumped in the cytoplasm, sometimes totally obscuring the nucleus. The cytoplasm otherwise doesn't show up, thus the outline of the particles is the only indication of the cell' outline. Alveolar macrophages are seen in the alveoli of slide 50 (lung) even without cytoplasmic inclusions, due to the fact that their cytoplasm shows up easily against the white alveolar space. Any large cell in these spaces with an oval to round, pale nucleus and abundant pink cytoplasm, sometimes appearing foamy, is an alveolar macrophage. Finally, if you think you've aced these, go to the liver (slide 42) to look for Kupfer cells. With the 40X objective, look in the spaces or sinusoids for a similar cell type, again, a mononuclear cell with abundant pink cytoplasm, which may be elongated. There are other cell types in these spaces, since there is blood here, so identification of Kupfer cells is optional at this time. You will not be required to find them until the liver is covered later.

Mast cells This cell type can't be seen with H&E stained paraffin sections. However, with toluidine blue stain and plastic sections, these cells show clearly. Look at slide 100 (ear) and slide 21 (testes, epididymus, vas deferens) for near capillaries in the connective tissue where mast cells tend to congregate. These cells contain large dark blue granules filling the cytoplasm. What component is responsible for this basophilia? Frequently the nucleus is not seen because of the overlying granules; in other cases the nucleus appears as a central light zone in the otherwise darkly staining cell.

DENSE CONNECTIVE TISSUE
DENSE IRREGULAR C.T. This type of c.t. can be found in such locations as scalp (slide 7), thick skin (slide 9), and vagina (slide 70) beneath the epithelium. Note many closely packed and randomly oriented collagen bundles. Why are there so few wandering cells in this type of c.t. as opposed to the c.t. of the lamina propria of the gut?

Dense irregular c.t. takes on a more organized appearance in the perichondrium, periosteum, and perineurium. Look at slide 47 (trachea) for an example of perichondrium surrounding a C-shaped ring of cartilage which you may need assistance today in identifying. Collagen here appears regularly oriented, but in fact runs in two directions as a meshwork covering the surface of the cartilage. Further from the cartilage, and forming the cut surface of the trachea, the collagen becomes more randomly-oriented. It is classified as dense irregular c.t. in both locations, thus you can appreciate that categorization of this type of c.t. requires (1) a sufficiently low magnification to see relationships, and (2) an understanding of the appearance of bone, cartilage and nerve with its associated periosteum, perichondrium, and perineureum, respectively.

DENSE REGULAR C.T. This c.t. consists primarily of collagen fibers oriented in one direction only, as exemplified by tendons. A good example may be seen in slide 6 (finger joint). Tendon appears as regularly and closely packed parallel fibers inserting near the articular surface of the bone. The fibroblast nuclei appear as black, elongated wavey lines between collagen bundles. Also you can use this slide to see more dense irregular c.t. in the form of periosteum. Have your lab instructor verify your identification of periosteum and tendon, as they appear very similar and require experience to distinguish.

FIBERS
As you now know, collagen can be seen easily with H&E as eosinophilic fibers and bundles.

Elastic is most easily identified when a specific stin such as aldehyde fuchsin is used. Look for elastic laminae or plates as darkly staining, circumferentially-priented lines composing much of the inner wall of the aorta (slide 96). Elastic also occurs as fibers, seen as in the matrix of elastic cartilage of the external ear (slide 100).

Reticular fibers are revelaed most clearly with silver stain in slide 191 (spleen). Look for very think black fibers of varying lengths throughout the tissue.

LABORATORY GUIDE FOR ADIPOSE TISSUE

UNILOCULAR FAT
Slide 9 (skin from sole of foot) displays large accumulations of fat in the subcutaneous tissue under the skin. Smaller groups of fat cells are seen dispersed throughout the deeper dermis. Try to find nuclei of these cells.

MULTILOCULAR FAT
Slide 84 (monkey aorta) contains clumps of brown fat, which you must find amidst other structures you have not studied yet. some of these slides do not contain multilocular fat, necessitating a glance at your neighbor's slide. Why are you not likely to find this type of fat elsewhere in your slide box?

LABORATORY GUIDE FOR CARTILAGE

HYALINE CARTILAGE
Examine Slide 47 (human trachea) for the following features:

  1. Cartilage matrix
  2. Lacunae with chondrocyte remnants
  3. Capsules (capsular matrix)
  4. Isogenic cell nexts
  5. Perichondrium: consists of an ill-defined outer fibrous and inner chondrogenic layer, merging imperceptibly into the cartilage matrix. All cell types in the perichondrium (fibroblasts, osteoprogenitor cells, chondroblasts) are flat and can't be distinguished from one another.
Identify these same features now on Slide 98 (monkey trachea). Articular cartilage is seen in Slide 6 (finger joint). Observe the lack of a perichondrium, typical of articular cartilage. Finally, look at cartilage as it forms within mesenchyme in parts of the fetal hand, Slide 2.

ELASTIC CARTILAGE
Examine Slide 99 (human ear). Identify the cartilaginous core of this organ, noting it has findamentally the same features as hyaline cartilage, which you should be able to identify as above. In elastic cartilage, however, the matrix is not as amorphous as hyaline cartilage, being much more fibrous even without an elastic stain. Slide 100, monkey ear, has a special stain which turns elastic fibers blue-black. Note their extensive network in the matrix.

FIBROCARTILAGE
This type of cartilage is most obvious in intervertebral discs (Slide 5) and in joints where it forms some attachment for ligaments and tendons to bones (finger joint, Slide 6).

Orient yourself to the topography of the tissue in Slide 5 by examining the slide grossly first to determine exactly where you will look when you put it under the microscope. On either end of the tissue are marrow spaces and bone, since this is a longitudinal section through vertebrae. In the middle is an amorphous, pale purple zone, which is the nucleus pulposus. Between the bone and the nucleus pulposus, on either side, is a thin zone of fibrocartilage, evidenced by the presence of pink Type I collagen fibers coursing through it. You can distinguish this from connective tissue proper by the presence of lacunae.

Did you find a perichondrium with any of these examples of fibrocartilage?