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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)
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
Total number of cells counted
= % Viability
The number of cells counted in 10 squares
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, approximately how many millions of cells will be used to seed each of four flasks?

If these cells needed to be split 1:4, approximately how many millions of cells would be use to seed each of the eight flasks?

Sample Problem #2:

Use the data from sample problem #1 to determine what volume of the pooled cell suspension in the tube needs to be transformed to a tissue culture dish requiring one million cells.

Sample Problem #3:

A sample of cell suspension is ready for counting and viability assessment; however, after loading the hemacytometer and microscopically observing the cells on the counting grids, it is determined that the counting ranges are too high (i.e. there are too many cells to count.)
What must be done to obtain good counting ranges?

What volume of medium must be added to 0.1 ml of the original cell suspension to obtain a 1:10 dilution?

What is now the Dilution Factor?

If a sample of this 1:10 dilution is added to an equal volume of trypan blue solution, what is the final Dilution Factor?

If the new counts are now in the range of 3 to 5 cells per counting square, are they still valid? Explain.

What must be done to obtain valid counting ranges?

What are optimal counting ranges?

Sample Problem #4:

A confluent T-75 flask is trypsinized, and the cells are resuspended in 15 ml of complete medium. The number of viable cells/ml have been determined to be 1.3x106 cells/ml.

What is the total number of viable cells in the flask?

What was the number of viable cells per unit area (cm2) in original 75 cm2 flask?

How many T-25 flasks can be seeded with this cell suspension, if the target seeding density for each of the T-25 flasks is 2.5x104 cells per cm2 of area?

Sample Problem #5:

You have just been told that someone in the lab needs eight T-25 flasks seeded this afternoon. They have told you that you can use one T-175 flask of their stock culture to seed the eight flasks. After discussing the typical seeding densities for this particular cell type, you determine that you will need 4.5x105 cells for each of the eight flasks that you will be seeding.

What is the minimum number of cells that you will need to meet the seeding density requirement for these eight flasks?

Sample Problem #6:

After counting the cells from the T-175 flask in the sample problem #5, you have determined the average cell count per counting square to be 31.

If your dilution factor was two, what is the number of cells per ml?

If you have 20 mls of the cell suspension from the original flask, will you have enough cells to seed eight T-2 flasks?

Sample Problem #7:

A static suspension culture of non-adherent cells (e.g., leukimia cells) growing in a single T-7 flask is to be subcultured into 5, 60mm dishes (e.g., BD Falcon™ 353002). An aliquot of the original 15 ml cell suspension in the original flask is counted with the hemacytometer, and the cell concentration is found to be 2.5x106 cells/ml. You wish to seed the 5 dishes at a density of 7x105 cells/dish, and in order to promote good gas exchange in the dishes you have decided to limit the depth of the medium in the dishes to 6mm (0.6cm). The area of the dish bottom is 28 cm2. (Ref. InVitro Insights Program Supplement #103).

How do you dilute the original cell suspension to achieve this goal?


Sample Problem #1

  • Average # of viable cells per square = 36.1
    Grid # 1: 176-5 = 171
    Grid # 2: 195-5 = 190
    171+190 = # viable cells per 10 squares
    360/10 = # viable cells per square
  • % Viability
    361 viable cells / 371 total cells = 0.97 = 97%
  • # Viable cells per ml = 72.2x104 cells/ml
    36.1x104x2 (Dilution Factor) = 72.2x104 cells/ml
  • Total # cells available for subculturing = Approx. 14 million cells
    72.2x104 cells/ml x 20 ml = 72.2x104x20=14.44x106 cells
    (# viable cells/ml)
  • # Cells per flask (for each of 4 flasks) = Approx. 3.61 million cells
    14.44x106 cells / 4 = 3.61x106 cells
  • # Cells per flask (for each of 8 flasks) = Approx. 1.8 million cells
    14.44x106 cells / 8 = 1.8x106 cells
Sample Problem #2
  • # mls needed = 1.4 mls
    We have 72.2x104 cells/ml or 0.722x106 cells/ml
    We need 1.0x106 cells
    Therefore; we divide (1.0x106 cells)/(0.722x106) = 1.385 or 1.4 mls
Sample Problem #3
  • Sample must be diluted to obtain good counting range
  • 0.9 ml medium must be added
  • Total Dilution Factor = 10
  • Final Dilution Factor = 20
  • The 3-5 range is not valid. There are too few cells to determine a statistically significant count.
  • To obtain valid counting ranges, try an intermediate dilution.
  • Optimal counting ranges are 20-40 cells per square (200-400 cells per 10 squares)
Sample Problem #4
  • Total # of cells available = 1.95x107
    1.3x106 cells/ml x 15 ml = 1.3x106x15 = 1.95x107 cells
  • # of viable cells/cm2 = 1.9x107 cells / 75 cm2 = 2.6x105 cells per cm2
  • 31 T-25 flasks can be seeded with this cell suspension.
    2.5x104 cells/cm2 x 25 cm2/T-25 flask = 6.25x105 cells per T-25 flask
    1.95x107 cells / 0.0625x107 cells/flask = 31.2 = 31 flasks
Sample Problem #5
  • 3.6x106 cells are needed to seed eight flasks.
    4.5x105 cells/flask x 8 flasks = 36x105 or 3.6x106 cells total
Sample Problem #6
  • 6.2x105 cells per ml
    31x104 cells/ml x 2 (dilution factor) = 62x104 = 6.2x105 cells/ml
  • Yes - we need 3.6x106 cells (Ref. Sample Problem #5) and have 12.4x106 cells
    We have 62x104 cells/ml x 20 ml = 12.4x106 cells total
Sample Problem #7
  • First, determine the volume of medium per dish:
    0.6cm x 28.0 cm2 = 16.8 cm3 = 16.8 ml
  • Next, since you need 7x105 cells per dish in 16.8 mls - you will need a cell suspension concentration of 4.17x104 cells/ml (7x105cell/16.8 mls)
  • Since there are 5 dishes, you will need 5x16.8 or 84 mls of the cell suspension at the above concentration.
  • Next, you need to add "X" mls of the original cell suspension
    (2.5x106 cells/ml)X = 84 ml (4.17x104 cells/ml)
    (2.5x106 cells/ml)X = 3.5028x106 cells
    X = (3.5028x106 / 2.5x106) mls
    X = 1.401 mls
    Therefore; 1.4 mls of original cell suspension must be added to 82.6 mls of medium to make 84 mls.
    Note: Usually for practical reasons, the volume of final cell suspension can be adjusted to a rounded-off number (i.e., 90 or 100 mls)

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