Overview:

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.

I.  Blood

Webslide 0003_D:  Human Blood Smear, Wright’s stain [DigitalScope]

First use low power to choose an area where erythrocytes are fairly close but not piled up or overlapping, and where white blood cells (they stain darkly) appear in reasonable numbers.  At high power use the Ruler Tool to measure the diameter of 10 well-formed red blood cells (RBCs) and enter those values into this online spreadsheet to calculate the average diameter.  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.

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 low power objectives to scan the slide for white blood cells. When you find a white blood cell, zoom in to the highest magnifcation to identify it as either a neutrophil, eosinophil, basophil, lymphocyte, or monocyte (if the cell is damaged or otherwise cannot be definitively identified, move on to another cell). Repeat this process for 50 white blood cells and enter the cell counts into this online spreadsheet to calculate the differential white blood cell count.  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! If you are having problems finding the specific cell types, there is an annotated version of slide 0003 available in which a single example of each of the 5 kinds of white cells is indicated with a different letter. Use this feature only after you've done some looking around on your own.

For your reference, the "normal" range of white blood cell counts is:

• Mature (3-5 lobes) neutrophils: 54-62%
• Immature neutrophils ("band" form with a non-segmented nucleus): 3-5%
• Basophils: 0-0.75%
• Eosinophils: 1-3%
• Lymphocytes: 25-33%
• Monocytes: 3-7%

ADDITIONAL BLOOD SMEAR SLIDES FOR REVIEW

Normal blood smear 63x (Wright stain) [DigitalScope]
Normal blood smear 86x (Wright stain) [DigitalScope]

Scan around the 63x and 86x slides at high magnification to see the various kinds of blood cells that were discussed in the lecture.  Most abundant, of course, are the red blood cells (RBCs) or erythrocytes, which are seen in large numbers everywhere you look.  In between the RBCs you should look for small, basophilic fragments which are platelets or thrombocytes that are important in blood clotting.  As you continue viewing, you will see occasional white blood cells (leukocytes).  Some of the white blood cells may defy identification, often because the cells were damaged during slide preparation, so look for characteristic examples, and ignore the equivocal cells. Refer to the images in your texts and from the lecture and try to find an example of each leukocyte type using the 63x and 86x slides (there's less area to cover in these high-mag slides and the cells present are excellent, although the 63x slide does NOT contain any basophils).

The most common white blood cell is the neutrophil, which has a distinct multi-lobed nucleus (often 3-5 lobes).  Also frequently seen arelymphocytes, which are small cells (often as small as RBCs) with a dark nucleus and very little cytoplasm.  Another cell type is the monocyte, the largest of the blood cells.  It has a large, relatively pale nucleus, and rather clear cytoplasm (granules are usually less apparent than those in the illustration in Wheater atlas).  You will also see an occasional eosinophil, with prominent reddish granules filling the cytoplasm, and a nucleus with 2 (or sometimes 3) lobes.  The exact color of the granules may vary from slide to slide, depending on how well the slide was prepared.  In your particular slides they may be anywhere from bright red to dull brown.  The remaining cell type you may see on your slides is the basophil, which is hard to find, since it constitutes less than 1% of the leukocytes (the 86x slide actually has THREE excellent examples).  The cytoplasm contains large, irregular granules in a "grape-cluster" appearance that usually stain dark blue or almost black. Basophil nuclei may often appear somewhat oval-shaped, so, at first glance, they may be confused with lymphocytes. However, the presence of the large, dark-staining granules should help you distinguish them; also, remember that basophils are rare.

After you've done some looking on your own, here some quick links showing examples of each type of leukocyte (in order of their normal frequency in a blood smear). USE THESE LINKS ONLY AFTER HAVING TRIED TO FIND THESE CELL TYPES ON YOUR OWN!

• Neutrophil (usually about 60% of WBCs)
• Lymphocyte (usually about 25% of WBCs)
• Monocyte (usually about 10% of WBCs)
• Eosinophil (usually about 3% of WBCs)
• Basophil (usually <1% of WBCs)

II.  Cellular (or "loose") Connective Tissue

Webslide 4000_D:  Colon, H&E [DigitalScope]

At the bottom of this slide, locate the surface epithelium and then study the cellular CT layer underneath 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 or "loose" connective tissue layer lying immediately beneath the moist epithelia that lines the gut, respiratory, and urogenital systems.  The lamina propria contains a higher proportion of cells to collagen than found in the dense irregular connective tissue in the skin (next slide).

Cellular (or "loose") 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 cells (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”).

III.  Dense, irregular connective tissue

Webslide 0058_D:  Thin Skin, human, H&E [DigitalScope]

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 (use the Ruler tool):

• nearest the epithelium, in the upper part of the connective tissue?
• deeper in the connective tissue, in the middle of the section?
  

IV.  Dense, regular connective tissue

Webslide 0306_D:  Plantar Skin, H&E [DigitalScope]

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 of the tendon to the dense, irregular connective tissue of the dermis found adjacent to the keratinized, stratified squamous epithelium at the bottom of the slide.

Between the dense, regular connective tissue of the tendon and the dense, irregular connective tissue of the dermis is a layer of adipose tissue, which is another specific type of cellular (or "loose") 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.

V.  Pathology Correlate

Webslide 026_40x:  Hypertrophic scar, H&E [DigitalScope]

This specimen is from a 24-year-old patient who had Mohs surgery (named for the technique developed by Dr. Frederick Mohs) to remove a basal cell carcinoma on his neck. Approximately 6 months post-surgery the scar had gotten larger than the original margins of the surgery and started to raise the skin. A biopsy was performed yielding the specimen shown.

First, take a look at the tissue covering the surface of the lesion and see if you can identify tissue as specifically as possible. Then, look at the tissue underneath and identify it as specifically as possible. You should notice that it is similar to the tissue seen under the skin in Webslide 0306_D above, except that the collagen fibers are much larger and much more abundant. Also, unlike the typical quiescent fibroblasts seen in the dermis [example], the fibroblasts in this lesion [example] have the hallmarks of being much more active (i.e. euchromatic nuclei with nucleoli and enlarged, basophilic cytoplasm due to abundant rough endoplasmic reticulum). Looking around within the lesion there are also numerous "mitotic figures" [example] in which you can see the chromosomes arranged in various phases of mitosis (the figure shown is a cell in metaphase).

The phenomenon observed here is a type of hypertrophic scar known as a keloid in which the fibroblasts of the dermis continue to deposit collagen in marked excess of what is typical in the wound healing process. The lesions themselves are benign and are not at increased risk of becoming malignant. Interestingly, keloids are found only in humans and occur in 5-15% of wounds. Although the process by which they arise is still not fully understood, there appears to be a genetic and age-related predisposition to keloid formation. The average age at onset is 10-30 years, whereas persons at the extremes of age rarely develop keloids.

Extra Slides

White Fat and Brown Fat
Webslide 0040_V:  Kidney, rat, H&E) [DigitalScope]

This slide features and adult rodent kidney, within which can be found abundant examples of simple cuboidal and coumnar epithelium lining kidney tubules. The organ itself is encapsulated in adipose tissue (fat), much of which is unilocular or white fat which is the predominant type of fat found within the adult and is characterized by a single (unilocular) fat droplet surrounded by a thin rim of cytoplasm. Unilocular fat is an important source of fat metabolism and storage. Another type of fat is multilocular or brown fat, which, as the name implies is brownish in appearance upon gross inspection. A pocket of brown fat can be seen in this slide surrounding the large renal artery and vein at the hilus of the kidney at the bottom of the slide. Brown fat is characterized by the presence of multiple lipid droplets within the adipocytes. The mitochondria in brown fat express an uncoupling protein within the mitochondrial inner membranes that uncouple the process of oxidative phosphorylation and thereby produce heat rather than ATP.