PLANT ANIMAL TISSUE CULTURE

Experiment 5

Principle

Animal cell culture and many of the cell culture applications that require use of cell suspensions, it is necessary to determine the concentration of cells. The device used for determining the number of cells per unit volume of a suspension is called a counting chamber. It is the most widely used type of chamber, since it was mainly designed for performing blood cell counts. It is now used to count other types of cells and other microscopic particles as well.

A number of stains have been employed to distinguish between viable and nonviable cells. This is based on the principle that live cells contain intact cell membranes that eliminate certain dyes, like trypan blue, Eosin, or propidium. In dead cells, the stain enters the cytoplasm and the cells take on the stain. If more than 25% of the cells are stained, the cell suspension is most likely not a viable one.

The trypan blue procedure is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is mixed with dye and then visually examined to determine whether cells take up or exclude dye.

Materials required

• Hemocytometer plus a supply of cover slips.

• Uniform cell suspension.

• 0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline.

• Tally Counter.

• Cell Suspension.

• Micropipettes.

• Inverted microscope (preferably phase contrast).

Procedure:

1. Obtain a uniform suspension of cells: Follow the typsinization/trypsin neutralization protocol for the specific cell type. Place the cell suspension in a suitably-sized conical centrifuge tube. For an accurate cell count to be obtained, a uniform suspension containing single cells is necessary. Pipette the cell suspension up and down in the tube 5-7 times using a pipette with a small bore (5 ml or 10 ml pipette). For cells thawed from cryopreservation (in 1ml cryopreservation medium), pipette up and down 7-10 times using a one ml pipette.

2. Prepare a 1:1 dilution of the cell suspension in trypan blue: Approximately 10 microliters of cell suspension will be required to charge one chamber of the hemocytometer. In a conical microfuge tube, add 10 microliters of 0.4% trypan blue solution. Gently swirl (finger vortex) the cell suspension and remove 10 microliters of it using sterile technique. Combine the 10 microliters of cell suspension with the 10 microliters 1 of trypan blue in the microfuge tube. Pipette up and down several times to ensure a uniform cell suspension using the same pipette tip and allow to stand for 5-15 minutes.

3. Load the hemocytometer: Moisten and affix cover slip to the hemocytometer. Ensure the cover slip and hemocytometer are clean and grease-free (use alcohol to clean). A small amount of trypan blue-cell suspension is transferred to one of the chambers of the hemocytometer by carefully touching the cover slip at its edge with the pipette tip and allowing each chamber to fill by capillary action. The chamber should not be overfilled or underfilled.

4. Determine the number of cells (total and viable): View the cells under a microscope at 100x magnification. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares. Count all the cells in the four 1 mm corner squares. If there are too many or too few cells to count, repeat the procedure, either concentrating or diluting the original suspension as appropriate.

5. For an accurate determination, the total number of cells overlying one 1 mm² should be between 15 and 50. If the number of cells per 1 mm² exceeds 50, dilute the sample and count again. If the number of cells per 1 mm² is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on both sides of the hemocytometer (8 x 1 mm² areas).

6. Keep a separate count of viable and non-viable cells. If more than 25% of cells are non-viable, the culture is not being maintained on the appropriate amount of media. Re-incubate the culture and adjust the volume of media according to the confluency of the cells and the appearance of the media. Include cells on top and left touching middle line. The cells touching middle line at bottom and right are not counted.

7. Note: i. Trypan Blue is the "vital stain"; excluded from live cells. ii. Live cells appear colourless and bright (refractile) under phase contrast. iii. Dead cells stain blue and are non-refractile.

Calculation

1. Count 4 corner squares and calculate the average. Each large square of the hemocytometer, with cover slip in place, represents a total volume of 0.1 mm³ (1.0mm X1.0mmX 0.1mm) or 10^-4 cm³. Since 1 cm³ is equivalent to approximately 1 ml, the total number of cells per ml will be determined using the following calculations:

2. %Cell Viability = [Total Viable cells (Unstained) / Total cells (Viable +Dead)] X 100.

3. Viable Cells/ml = Average viable cell count per square x Dilution Factor x 10⁴/

4. Average viable cell count per square = Total number of viable cells in 4 squares / 4.

5. Dilution Factor = Total Volume (Volume of sample + Volume of diluting liquid) / Volume of sample.

6. Total viable cells/Sample = Viable Cells/ml x The original volume of fluid from which the cell sample was removed.

7. Volume of media needed = (Number of cells needed/Total number of viable cells) x 1000.

Result:

%Cell Viability =

Question:

1. Applications of Cell counting.

2. Why typsinization is required for cell counting process?

Reference Material

I. Freshney- Animal Cell Culture.