# HEMOCYTOMETER - (Oct/26/2006 )

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havent done a cell count in a while and needed some help since my basic math skills are horrible

I counted 230 cells within 1 of the four corner squares (consisting of 16 little squares). do i just multiply 230 x104 to get the density (cells/mL)?

If my cell density is 2.3 x 106 cells/mL, how do I dilute it down to 104 cells/mL..

Thanks so much

-BioDude1-

Suppose you have 5ml of your cell suspension containing 2.3 x 10^6 cells/ml
Then:

10^4 / 2.3 x 10^6 x Z ml = 5ml

Where Z is the amount of medium you have to add to obtain the desired 10^4 cells/ml.

Z = 5/(10^4/2.3 x 10^6) = 5 x 2.3 x 10^6 / 10^4 = 5 x 2.3 x 10^2 = 1150 ml.

So it is a 1:230 ratio (1150/5). Accordingly you can add 1µl of your initial suspension (2.3 x 10^6) to 229 µl of medium and obtain 230 µl which contain 10^4 cells.

-Jou-

Dear BioDude 1,

First of all 230 cells/ square (16 smaller squares) is to many cells to do an accurate cell count. You should really have between 25 and 100 cells/square. And you should count 4 squares and then average the count.
Are you doing a VIABLE count i.e. using Trypan blue as an EXCLUSION DYE. If you are you need to have the dilution factor within your calculation ......for example 100ul of cell suspension + 100ul of Trypan is a 1:2 dilution, thus the factor of 2 is put into the following calculation :

Mean Cells/square x 2 x 10,000 (10^4) = No of cells/ ml.

230 x 10,000 = 2.3 x 10^6

You need to dilute your cell suspension by a factor of 230 to get a cell suspension of 10^4

i.e 1ml of cell suspension to 229mls of media = 10^4 cells/ml

or 0.1ml of cell suspension to 22.9mls of media = 10^4 cells/ml

or 10ul of cell suspension to 2.29mls of media = 10^4 cells/ml

or as Jou has already stated eloquently 1ul of cell suspension to 229ul of media = 10^4 cells

Hope this is clear

Rhombus.

-Rhombus-

Ok Thanks for the help-

So 230 cells counted in 16 little squares total is too much?

-BioDude1-

its ideal to have between 50 and 100 cells, when u count the 16 little squares. Else the error rate increases.

-scolix-

QUOTE (BioDude1 @ Oct 26 2006, 09:16 AM)
Ok Thanks for the help-

So 230 cells counted in 16 little squares total is too much?

It is advisable to count 2 bigger squares atleast and then do the average. If we count more than 200 cells per 1 bigger square, that means sample need to be diluted more in order to get a good and accurate vcd = viable cell density.

we count all the cells in the 8 big squares ( 8 x 16 squares) and average them.

-scolix-

Check this attachment.

-exploresci-

Great pdf! thanks exploresci

-Pumuki-

QUOTE (Pumuki @ Dec 11 2006, 10:33 PM)
Great pdf! thanks exploresci

A hemacytometer (also spelled hemocytometer) is an etched glass chamber with raised sides that will hold a quartz coverslip exactly 0.1 mm above the chamber floor. The counting chamber is etched in a total surface area of 9 mm2 (see Figure 1).

Figure 1. Dimensions of a hemacytometer.

Calculation of concentration is based on the volume underneath the cover slip. One large square (see W in Figure 2) has a volume of 0.0001 ml (length x width x height; i.e., 0.1 cm x 0.1 cm x 0.01 cm).

In both methods, the hemacytometer is filled by capillary action - place the pipette filled with a well-suspended mix of cells at the notch at the edge of the hemacytometer and then slowly expel some contents so that the fluidis drawn into the chamber by capillary action.

Staining of cells often facilitates visualization and counting. Either mix cells with an equal volume of trypan blue [0.4% (w/v) tyrypan blue in PBS] to determine live/dead count (dead cells are blue) or kill cells with 10% formalin and then stain with trypan blue or other stain (to improve visualization of all cells.

Here are two simple methods for counting cells based on the surface area of the hemacytometer used to determine cell count. Other counting schemes are accetable also. The choice of methods depends upon the cell concentration - the accuracy of the procedure depends upon the number of cells counted. When cell concentration is low, one should count more grids.

Method A
Count the number of cells in the 4 outer squares (see the left panel of Figure 2).

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 4 squares x 2500 x dilution factor

Example: If one counted 450 cells after diluting an aliquot of the cell suspension 1:10, the original cell concentration = 450 x 2500 x 10 = 11,250,000/ml

Method B
Estimate cell concentration by counting 5 squares in the large middle square (see the right panel in Figure 2).

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 5 squares x 50,000 x dilution factor

Example: If one counted 45 cells after diluting an aliquot of the cell suspension 1:10, the original cell concentration = 45 x 50,000 x 10 = 22,500,000/ml

Figure 2. Counting procedure for Methods A (left panel) and B (right panel).

Source: Dr. Hansen

-ngtienhuy-

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