Hemacytometer Workbook

This workbook was developed for use with Module 2 of the InVitro Insights Cell Culture Training Program, developed by Becton Dickinson. The problem that follow are presented in increasing degress of complexity. They were designed to assist in the development of skills necessary to determine the number of viable cells available for subculturing and the amount of cells necessary for different types of experiments.

In order to better understand the concepts presented in the video, it is recommended that viewers use the Hemacytometer Workbook in conjunction with the Hemacytometer Reference Guide.

Below is a diagram of the two hemacytometer counting grids, with corresponding labels and terminology used throughout this workbook.



Typically, 5 counting squares from each grid are counted. The total count from the ten squares is then divided by 10 to determine the average count per square.

Hemacytometer Calculations
Viability Assessment & Cell Density Determination Total volume
(cell suspension sample + trypan blue solution)
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Volume of cell suspension sample = Dilution Factor Total number of cells counted — Number of non-viable, blue-stained cells = Number of Viable Cells Number of viable cells counted
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Total number of cells counted = % Viability The number of cells counted in 10 squares
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Nunber of squares counted (10) = Average Number of Cells Per Square Average number of cells per square x 10⁴ = Number of Cells Per ml Number of cells per ml x Total mls of original cell suspension = Total # of Cells in Original Cell Suspension Total number of cells in original cell suspension x % Viability = Total # of Viable Cells Available in Original Cell Suspension

Sample Problem #1:

Two T-75 flasks containing anchorage-dependent cultures wre trypsinized. The cells from both flasks were pooled, and the resulting cell suspension was transferred to a conical tube. The total volume of liquid (cells suspended in media) was 20.5 mls. Although these cells are routinely split 1:2, anyone handling these cultures is requested to assess the viability of the population.

0.5 ml of the cell suspension was added to a tube containing an equal volume of trypan blue, the tube was aqitated, and a hemacytometer was loaded with the resulting suspension. Both grids were covered with appropriate ammount of cell suspension, and the following counts were obtained: Grid #1: 5 squares counted for a total of 176 cells.
(5 out of 176 cells were stained blue) Grid #2: 5 squares counted for a total of 195 cells.
(5 out of 195 cells were stained blue)

What is the average number of viable cells per square?

What is the % viability?

What is the number of viable cells per ml in the tube containing the cells suspension availble for subculturing?

What is the total number of cells available for subculturing?

Since these cells are routinely split 1:2, approx

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