Tissue Culture Laboratory (BIOE342) - Protocol
Index

Determination of Cell Concentration

Objective

·        Determine the cell concentration of a cell suspension.

·        Become familiar with the hemocytometer and Coulter Counter.

·        Apply statistical framework to measured values.

Materials

Cell suspension at ~106 cells/mL (prepared by instructor)

Hemocytometer and coverslip

Micropipette

Tally counter (optional)

Isoton

Coulter Counter vials

Calculator

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

Procedure

1.      Determine the cell concentration using the Hemocytometer and a Nikon microscope following the “Hemocytometer Instructions”.

2.      Prepare three samples, each with 500 mL of cells and 9.5 mL Isoton.  First, aliquot Isoton into Coulter Counter vials.  Place vials in laminar hood;  do not sterilize.  Add cells to vials.

3.      Determine the cell concentration using the Coulter Counter following the “Coulter Counter Instructions.” Remember to gently mix the solution two or three times by inversion before placing it on the stand.  Run each sample on the Coulter Counter  in triplicate.

4.      Dispose of biohazardous material properly.

5.      Calculate the cell concentrations using the notes below.

6.      On the computer, record your cell concentrations and the stock suspension from which you got your cells for the hemocytometer and Coulter Counter measurements.

7.      Calculate the mean and standard deviation for your measured cell concentrations.  Record these calculated values on the computer.

8.      Instructor to email data to class.

Calculations

Hemocytometer

The area of the center square is 1 mm2.  The depth of the chamber is 0.1 mm.  Determine the cell concentration (#/mL) measured in each chamber.  NOTE:  You will not need to accurately know the cell volume that you added into the chamber.

Coulter Counter

The dilution of the cells into Isoton (typically, 10X or 20X) and the volume sampled by the Coulter Counter (500 mL) are both required to calculate the cell concentration based on the cell count number determined by the Coulter Counter.

Questions

1.      How is a constant volume of liquid assured in the hemocytometer chamber?  In other words, why don’t you have to worry about getting the entire sample into the chamber?

2.      The hemocytometer required two measurements.  How meaningful is a standard deviation with only 2 data points?

3.      Compare the concentrations calculated using the hemocytometer to the concentrations determined using the Coulter Counter for your data.  If there are discrepancies or differences, postulate an explanation.

4.      Record the mean and standard deviation for the all the cell concentrations measured by members of the class in your lab notebook.  Indicate whether the data is from the hemocytometer or Coulter Counter.  Calculate a mean and sample standard deviation for all the experimental values using each counting technique.

5.      Are there any outliers in the measured cell concentrations based on the data from the whole class?  If so, on what statistical criteria could you remove outlier data from the data set?

6.      What are the sources of error that may account for differences in measured cell concentrations within one instrument?  What differences could be present between different operators?

7.      Do you think that the hemocytometer or the Coulter Counter is more accurate?  More precise?

8.      Would you recommend using a hemocytometer or Coulter Counter for routine cell counting?  Why?  [Practical reasons such as time, cost, etc should also be included.]

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