CELL STRUCTURE I LABORATORY

SIGN IN PROCEDURE:

At each of your desks you will find the following items:

  1. 2 slide boxes: on the outer surface the boxes will bear the same number followed by an a or b (e.g. 51a and 51b). These boxes have been assigned to you for the entire first semester. You are responsible for the slides in these boxes and will be charged for breakage or loss of slides (approximately $450.00 for the slide set). PLEASE TAKE GOOD CARE OF THEM.

  2. Sign-in sheet: a sheet of paper on which you record which slides in your slide boxes are present, absent or broken. At the end of the semester, the slides in your slide boxes will be compared against the information you record on this sheet to determine breakage/loss fees.

Please note that not all slides are present in every box. Some slides are ONLY in even-numbered boxes (EO), others in odd-numbered boxes ONLY (OO).

After you have checked every slide and their order, sign your sheet and place it on the table at the front of the room. Your sheet will be reviewed and if replacement slides are available they will be distributed to you.

The following suggestions are made to help prevent loss or breakage of slides:

  1. DO NOT leave your slide set in the microscope locker located in your assigned laboratory. Although this is very convenient, you share this locker with a second year medical student and they will not be as cautious with your slides as you will be.

  2. Be sure to remove the last slide of the day from your microscope stage and replace it in your slide box. Do not leave slides on the stage because your microscope will also be shared by the same second year medical student as noted above.

  3. Be sure your slide boxes are UPRIGHT before opening. All of your slides will fall out if the box is upside down and it is a BIG job to reinsert them correctly.

USE OF THE LIGHT MICROSCOPE

At half past the hour, your lab instructor will review the parts of the microscope and Koehler illumination with the entire lab. If you have not finished checking your slides at this time, you will need to finish after this review is completed.

During orientation, you received instruction sheets corresponding to your type of microscope. These will review the parts of the microscope and teach you how to set Koehler illumination. You may refer to these if you cannot follow everything covered by the instructor. If you need further help, review the section in the Appendix on use of the microscope.

At the completion of the review, take a few moments to look at any slide from your box. Try setting Koehler illumination at every magnification.

BUCCAL SMEARS (How to perform a buccal smear)

Supplies:
  1. Select two glass slides from the boxes provided. Using a pencil, write your initials on both slides, "stained" on the frosted end of one slide and "unstained" on the other slide.

  2. Using one end of a tongue depressor blade, scrape the inside of your cheek and smear on one slide. Flip the blade around and scrape the inside of the other cheek and smear on the remaining slide. Allow both slides to air dry for at least 2 minutes.

  3. Place the slide labelled "stained", smear side facing upward, on the staining rack. Flood this slide a drop at a time with Lui's A stain. BE CAREFUL NOT TO GET STAIN ON YOUR CLOTHES; IT WILL NOT COME OUT! Allow the stain to remain for 15 to 20 seconds.

  4. Add about 6-7 drops of Lui's B stain directly onto Lui's A. The stains will not mix well at first. Blow gently on the slide for a few seconds. Allow the stains to sit undisturbed for 2 minutes. You should see a green metallic scum form on the surface.

  5. Gently wash the surface of the slide with water from the water bottle to remove all the stain.

  6. Remove the slide from the stain rack, leaving water on the surface of the slide. If the slide looks dry, add one drop of water over the smear. Lay one edge of a cover slip against the water droplet and set it down gently over the smear.

  7. Place a drop of water on the unstained slide and coverslip in the same manner.

  8. Dry the undersurface of both slides.

  9. Discard tongue depressor blades in biohazard bags.

  10. Place stained slide under the 10x objective power of your microscope and set Koehler illumination. Scan the slide for your cheek cells. If done correctly, you will see clumps of and individual, flattened (squamous) cheek cells. Move to a higher magnification to see more detail. Note: some cheek cells may bear small, purple rods on their surfaces, these are bacteria (flora) which inhabit the oral cavity.

  11. Examine the unstained slide. Can you easily find the unstained cheek cells? Can you readily see nucleus, cytoplasm and location of cell membrane? You can turn your compound light microscope into a "phase" microscope by slightly lowering the condenser. Notice that this process increases the contrast in the unstained cells. What advantages does staining offer to the histologist? Discard glass materials only in Lilly buckets.

Any more than one X chromosome remains condensed in interphase nuclei. A useful way of determining sex clinically (no snickering please!) as well as several other X- linked syndromes, i.e. Klinefelter's, Turner's, by histological methods is to use one of several stains.

  1. Make another buccal smear as above and let the slide dry for 2 minutes.

  2. Place 3-4 drops of the 2% aceto-orcein stain on the slide. Let stand for 1-2 minutes then drain on paper towel

  3. Cover with a coverslip.

  4. Squeeze out excess stain by pressing (GENTLY) the slide between two pieces of paper towel.

  5. Examine on your microscope.

  6. Compare your slide to your neighbors' to assess presence or absence of Barr bodies. Even in females, not every nucleus will show Barr body in buccal smear so you'll need to look at a dozen or so cells before making your diagnosis.

At the front of the lab room, there is a demonstration slide set up so you can see mitotic figures in human tissue. During the pre-lab you were shown examples of where these dividing cells are observed in regions of the GI tract tissues. Try to find several examples of various stages of mitosis.

CELL STRUCTURE II LABORATORY

During this laboratory, you should examine the four slides listed below to find a number of cell organelles. Only some cell organelles may be identified by light microscopy; full resolution requires TEM. Later in the laboratory, your lab leader will present part of a review slide carousel.

Slide #42, liver:
This tissue section was embedded in paraffin and stained with hematoxylin (blue-purple dye) and eosin (pink-red dye) known simply as H & E. Hematoxylin binds to RNA and DNA staining these substances basophilic (blue); eosin binds to proteins staining them acidophilic (red). Please carefully examine the liver cells (hepatocytes) on this slide; they are the most prevalent cell type you see on the slide. Hepatocytes are large, round or polygonal cells which stain with red-pink cytoplasm and purple nuclei. Find the following hepatocyte structures: location of cell membrane, location of nuclear envelope, cytoplasm, nucleus, euchromatin, heterochromatin, nucleolus, and rough endoplasmic reticulum. Check the cytoplasm for globs of yellow colored material. The yellow substance is housed within lysosomes. What would you call this substance? Although lysosomes cannot be fully resolved by LM, you can see the pigment they contain.

Slide #86, liver:
Repeat the same process of examination for this slide that you did for the above slide. Can you see the cell organelles more or less clearly? This slide was embedded in plastic resin and stained with toluidine blue. Plastic embedment produces more clarity in cell structure but stain intensity is often reduced. Note the blue-purple patches in the cytoplasm. These are stacks of RER cisternae with bound ribosomes (remember the ribosomes stain basophilic?); "old-time" histologists called these patches ergastoplasm.

Slide #195, spleen:
This slide is paraffin-embedded and stained with prussian blue. The tissue is spleen removed from a patient suffering from sickle cell anemia. Scattered cells contain brilliant blue patches within their cytoplasm. This blue staining material is iron in lysosomes. The cells which have ingested the iron are macrophages.

Slide #21, epididymis:
You might require instructor assistance to find the cells lining cut tubes of the epididymis. Look for hollow, round structures. These are the tubes. Find the lumen (central open space) of a tubule. Identify the cells lining the luminae. These are the epithelial cells. Find the cell nuclei and the luminal cell membrane. Between these two structures is a region which did not pick up the blue stain. This non-staining region is the location of the Golgi complex which is very large in these cells.

CELL STRUCTURE III LABORATORY

Slide #31, Small intestine:
 Find the luminal surface of the small intestine. Move to high power and examine the apical surface of the luminal epithelial cells. Note a brilliant pink line at this apical surface. This is striated (brush) border formed of countless microvilli extending from cell surfaces.

Slide #21, Epididymis:
Find numerous cross-sections of tubular structures with large lumens. These are transverse views of one long, coiled tube, the epididymis. Note the very long extensions from the apical surfaces of the cells lining the tubes. These extensions are erroneously named stereocilia. Actually, the extensions are long microvilli. You may be able to see the small dark dots at the lumen where two cells meet. This is the terminal bar equivalent to the intermediate junction.

Slide #47, Trachea:
Examine the apical cell surface of the cells lining the trachea. Note the "eye lash-like" extensions from the apical cell surface which are motile projections, the cilia (singular: cilium or kinocilium).

Slide #113, Kidney:
This slide of kidney has been stained with PAS (periodic acid- Schiff). The basal laminae stain PAS positive (magenta or purple/pink). Look around the cross-sections of tubes for the PAS positive lines demarking the location of the basement membrane.