The goals of this lab are to identify the main cell types and matrix elements of the various types of connective tissue. Blood conveys cells around the circulation in a fluid medium. Connective tissue proper contains cells surrounded by the fibers and ground substance of the extracellular matrix.
We will look at a blood smear first, to see a variety of cells in simple isolation. Then we'll look for the same cells as they reside in the extracellular matrix. Finally, we'll pay attention to the fibers and more permanent cells of the connective tissue. Please spend no more than 30 minutes on the blood smear and use the remaining time on the connective tissue section.
While some varieties of the "connective tissue proper" are obvious in their form (tendon, organ capsules, fascias), others are easily confused with other tissues and cell types (for example smooth muscle, which may stain similarly to fibrous connective tissue). The more hidden features and arrangements of connective tissue may at first seem confusing or subtle, but you will meet or recall them repeatedly: the lamina propria beneath moist epithelia, the fine scant sheaths of connective tissue around small vessels and ducts, the intimately penetrating stroma (of connective tissue, often reticular fibers) which supports and helps knit together the cells to form organs. In time you will be able to identify connective tissue comfortably and quickly, wherever or however you encounter it, and to appreciate its functional significance in the local and general operations of the living body.
Some of the slides were chosen to illustrate particular classifications of connective tissue. Use 5X and 10X objective settings for a few minutes on each slide, to survey the patterns of fibers and cells. Notice how fibers vary in thickness and in direction (parallel, random, woven, kinked, etc.) and how fibroblasts and other cells are positioned and dispersed distinctly in different tissues. A few general types of connective tissue patterns are readily learned. Don't be compulsive about the rest; do bear in mind that types may grade into one another.
Webslide 0003_D: Human Blood Smear, Wright’s stain
First use low power (10X) to choose an area where erythrocytes are fairly close but not piled up or overlapping, and where white cells (they stain darkly) appear in reasonable numbers. At 40X use the Tape Measure Mode to measure the diameter of 10 well-formed red blood cells (RBCs). The average diameter, estimated from this exercise, will serve you as a convenient benchmark for estimating other dimensions because red blood cells are so common in most tissue specimens prepared for microscopy.
RBC average diameter __________________.
Use this RBC benchmark to help identify blood platelets, which are typically 2-4 µm in diameter. They may be clumped together. Estimate how many erythrocytes are present for each platelet in your smear.
Use the 10X or 20X objectives to scan the slide. Find 50 white blood cells, and identify them with the 20X or 40X objective. Make a count of the different types: neutrophils, lymphocytes, monocytes, eosinophils, andbasophils. As described in lecture and the text, the key structural features are: cell size, shape and staining of the nucleus, presence/absence and color of cytoplasmic granules. Note that staining can vary from slide to slide. What looks orange-red on one slide many look pale purple on another. You may have to adjust your identifications to the staining spectrum presented on your slide, estimated from the appearance of RBCs, neutrophil granules, and nuclei. Basophils are rare, so share your find with your instructor and colleagues! The slide has an overlay marking a single example of each of the 5 kinds of white cells with a different letter. Use this if you are having problems finding a specific cell type (hit F2 to see overlay list).
WHITE CELL COUNT DATA
CELL TYPE NUMBER COUNTED PERCENT
Eosinophil Basophil Lymphocyte Monocyte
II. Cellular Connective Tissue
Webslide 0032_D: Ileum, monkey, H&E
Webslide 0098_D: Bladder
For each slide, first locate the surface epithelium and then study the cellular CT layer immediately below this epithelium. You will NOT be able to identify all of the cells in these CT regions, but you should be able to identify many of the cells described below.
Lamina propria is the term given to the cellular connective tissue layer lying immediately beneath the moist epithelia that lines the gut and urinary bladder. The lamina propria contains a higher proportion of cells to collagen than found in the dense irregular connective tissue in the skin (next page).
Cellular CT contains some of the white blood cells you examined above in circulating blood, namely lymphocytes (small round cell with little cytoplasm and a round darkly staining nucleus), neutrophils (each with lobulated nucleus and palely staining granules), and eosinophils (lobed or “C”-shaped nucleus with red granules in the cytoplasm). In addition, you should be able to recognize several resident CT cells including: (1) fibroblasts (each cell is elongated with elongated, darkly staining nucleus and scant cytoplasm), (2) plasma cell (relatively large ovoid cell with “clockface” or “cartwheel” nucleus at one side of cell and abundant pale staining cytoplasm in the rest of the cell), and (3) mast cells (round nucleus and cytoplasm full of granules; “fried egg with measles”).
The frequency of each cell type in a given section is variable, but as you search you should be able to find fibroblasts and examples of several of the other cells types in each slide. Webslide 32 contains many lymphocytes, along with plasmacells and mast cells, and Webslide 98 contains excellent examples of plasma and mast cells.
III. Dense, irregular connective tissue
Webslide 0058_D: Thin Skin, human, H & E
Look for the collagen fibers in the dense, irregular connective tissue region beneath the surface epithelium in this section of skin; they are stained a pink color. Note that the size of the collagen fibers tends to increase as one moves deeper into the connective tissue layer (away from the epithelium). In the lower part of the section (nearest the epithelium) there will be occasional smooth muscle fibers. Note that the muscle cells have elongated, oval nuclei inside each fiber. Collagen fibers are extracellular and have no nuclei, but you should see small, narrow, dense fibroblast nuclei among the collagen fibers. In the deepest part of the section, you'll see adipocytes in fatty connective tissue.
What is the diameter of a typical collagen fiber
...nearest the epithelium, in the upper part of the connective tissue? __________.
...deeper in the connective tissue, in the middle of the section?__________.
Can you see collagen fibrils?___________.
IV. Dense, regular connective tissue Webslide 0306_D: Plantar Skin, H&E
Go to the Duke Aperio website (aperio1.duhs.duke.edu) and click the link for the connective tissue lab to open this slide. Use the “Aperio WebScope” link for slide 0306.
At the top of the slide you will find part of the flexor tendon which is dense, regular connective tissue. Note the many parallel collagen fibers with fibroblast nuclei outside of the fibers. Compare the dense, regular connective tissue to the dense, irregular connective tissue next to the keratinized, stratified squamous epithelium at the bottom of the slide.
Between the dense, regular connective tissue and the dense, irregular connective tissue is a layer of adipose, which is another type of connective tissue. It is composed primarily of unilocular fat cells, each containing a single lipid droplet. The lipid is lost during sample preparation so the cells appear to be empty. In some cells, a single nucleus can be seen flattened against the periphery of the cell. Each unilocular fat cell has at most one nucleus. This is in contrast to a small blood vessel which might have a similar sized opening but would be surrounded by several endothelial cells.