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WHAT'S OUTSIDE THE CELL MEMBRANE ?


INTRODUCTION:

If individual cells such as cheek cells are examined under the light microscope the outer boundary or cell membrane is visible. However, if that same cell is examined with an electron microscope the boundary is revealed as a double membrane. The lipids and proteins which make up this double membrane have carbohydrates attached to them that form a "coat" around the cell. The "coat" or pericellular matrix, which can be viewed under certain conditions, is involved in intracellular interactions which enable cells to bind together to form tissues.

Cells which are grown in culture will produce a coat which can be seen under the light microscope. Two types of cells; fibroblasts which make up loose connective tissue, and chondrocytes which are found in cartilage, can be grown under sterile conditions in small petri dishes for 48 hours. These cells are actively producing a matrix which will exclude particles that are released on or near the cells.


OBJECTIVES:

To examine the cell coats (matrix) of chondrocytes and fibroblasts by observing these cells actively exclude particles which are released around them. To observe the function of the cell membrane as a gatekeeper as it prevents large particles from approaching the cell boundary.


MATERIALS:

  • Formalin fixed horse red blood cells (1 x 108 cells/ml in .1% BSA/PBS)
  • Methylene Blue
  • 35mm dish with growing chondrocytes or fibroblasts ( 1 X 10 5 cells)
  • Pipettes
  • Small beakers
  • Microscope
  • Dish of confluent cells to demonstrate tissue


PROCEDURE:

  1. Obtain a microscope, a sterile pipette and a dish of living cells. HANDLE THE DISH OF CELLS CAREFULLY. The liquid in the dish is called cell culture media and contains all the nutrients necessary for the cells to live. The dish is specially coated so that the cells will attach to the bottom and continue to grow and divide.

  2. Remove the cover and tip the dish to one side. Place the tip of the pipette at the edge of the dish and withdraw most of the culture media from the dish. You must leave a small amount of media in the dish so the the cells do not dry out. DO NOT DISCARD THE MEDIA put the media in the beaker or other container provided by your teacher.

  3. Place the dish in the center of the microscope stage and turn the diaphragm so it allows the minimum amount of light to pass through the specimen. Turn the low power (10X) objective into position and roll the body tube down as far as possible. While looking through the eyepiece, slowly turn the coarse adjustment knob until you notice the small transparent cells. If your teacher directs, add a small drop of methylene blue at the edge of the petri dish.

  4. Carefully examine the measuring pipette and compare it with the following diagram. Locate the line which indicates 1 milliliter, also locate the 0.5 line. Find the line between 1 ml and 0.5 ml, this is equal to 0.75 ml. Add 0.75 ml of fixed horse red blood cells to your dish of cells. Leave the dish on the microscope stage and DO NOT MOVE OR JAR the dish for 10 minutes.

  5. Examine the contents of the dish carefully. The small reddish brown particles are horse red blood cells. The chondrocytes or fibroblasts should no longer seem to be so plentiful because some are completely covered by the horse red blood cells. Some cells should have an area surrounding them that is clear of horse blood cells. This clear area outside of the cell membrane is the pericellular matrix or "coat" .


QUESTIONS

  1. Describe the structure of the cell membrane.
  2. What is the function of the cell membrane.
  3. What tissue in the human body are chondrocytes part of ? What is the function of this tissue ?
  4. What type of molecules form the cell matrix or "coat" ? What is the function of the matrix?
  5. Explain the relationship between cells and tissues.



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