Visualization of Cells

 

Objectives

·      Become confident using Nikon microscopes to visually assess cells.

·      Learn to assess confluency, degree of attachment, and morphology of several types of cultured cells.

 

Materials (cell preparation by Instructor)

Chinese Hamster Ovary cells (CHO)

Smooth Muscle cells (SMC)

Human Dermal Fibroblasts cells (HDF)

Bovine Aortic Endothelial cells (BAEC)

Cells at different confluencies

Flask of dead cells

Nikon microscope

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

 

Class Exercises During Lecture

1.      Look briefly at different cells types.

2.      Look briefly at flasks with different cell confluencies.

3.      Adjust filters to visualize different views.

 

Procedure

1.      Remove cover.  Turn microscope on.

2.      Set a flask of cells on a Nikon microscope.  The T-flasks should fit snuggly into the frame. 

3.      Turn up the light to mid-full power. 

4.      Focus using the gross and fine controls. 

5.      Cells can be viewed through a filter so they appear green/yellow.  Alternately, the filter can be removed.

6.      Complete the “Questions” below by answering the questions in your laboratory notebook.

EACH PERSON MUST LOOK UNDER THE MICROSCOPE FOR EVERY QUESTION. 

7.      Turn microscope off.  Replace cover.

 

Questions

Observations and answers to questions should be recorded in your laboratory notebook.

1.      Visualize a flask of cells using the 4x, 10x, and 20x lenses.  Estimate the number of cells in the visualized region for each magnification for one flask. 

2.      Cell attachment to the flask surface can be determined using the microscope.  To test for cell attachment, lightly move the flask and look for relative movement of cells compared to the flask.  Cells are DETACHED to the surface when the cells move even after the flask is still.  Cells are ATTACHED from the surface when the cells remain stationery after the flask is still.  Lightly move the flask around to see both attached and detached cells.  Make this observation on several flasks, including a flask with CHO cells.  Estimate the fraction of detached cells in one flask.

3.      Compare and contrast the shape of the four cell types.  Note such features as size, roundness, elongation, extension of pseudopodia, etc.

4.      Rounded up cells are dividing, dying, or just barely attached to the surface.  Estimate the fraction of rounded cells as compared to attached/extended cells in one flask.  Try to look at a flask of CHO cells. 

5.      Can you see the nuclear membrane or nucleus within the cells?  If so, describe.  Using high magnification (20x) is beneficial.  Make and record this observation on two flasks.

6.      Scan completely across one flask.  Do you notice any differences such as confluency, morphology, etc. across this flask?  If you see differences, postulate an explanation.

7.      Estimate the varying degrees of confluencies of the different flasks of CHO or HDF cells.  Sets of flasks with dilutions ranging from 1:2 to 1:32 will be provided.  Discuss with instructor or T.A. to help develop this skill.

8.      Compare the color and morphology of the dead cells to other living cells. 

9.      Do you see any locations where cells are overlapping?  What are the potential drawbacks to culturing cells at an overcrowded level?

 

Observations and answers to questions should be recorded in your laboratory notebook.  The answer to each question is worth 5 pts.