Urgent - ATC

For those who have read earlier, please read again. It has been edited.

Here is an important concept for ATC:

The calculation of cell counting and step-by-step reasoning:

A x B x C x 10^4

"Divide total viable cell by 8 to get C" This is what the TA say. But Dr Lim say NO! In the test, whenever you are given a viable cell number, divide it with 4, not 8, to get average viable cell. Why 4? Because normally, we use only one chamber instead of both of them. In one chamber (see figure 1) there are 4 corners with 4x4 boxes. Each of them is 1mm (width) x 1mm (length) x 0.1mm (depth) = 0.1mm^3.

Figure 1: One chamber of the haemacytometer

1. We need to calculate how many cells are present in only ONE of the corners (the corners are marked RED in Figure 1). Since there are 4 of them, you calculate all the viable cells in all 4 corners (like what we did in the lab), then divide by 4. Last time, we calculate for 2 chambers. So, 2 chambers x 4 corners = 8 corners. That's why we divide 8 for our last experiment. But in the test, please divide by 4. Because normally we use ONE chamber. Let's say we calculated 20 cells for all 4 corners. So, 20 cells /4 = 5 cells.

Then, 5 cells x 2 (dilution factor) = 10 cells.

2. Now, we get 10 cells. 10 cells refers to the number of cells, on the average, in ONE CORNER. But, how much is the volume of the cells is in ONE CORNER? According to the manual, the volume of 1 corner is 0.1 mm^3 (as calculated as above). But we don't want mm^3, we want ml! So, to change 0.1 mm^3 to ml, you need to divide 0.1 mm^3 by 10^3 . So,

(0.1 mm^3/10^3) x 1 ml = 1 x 10^-4 ml

The ml value is now 1 x 10^-4 ml. But how to get a conversion factor?

3. Still remember your microbiology? If you do a dilution of 10^-5 (the diluted value), your dilution factor is 10^5. Right? You just have to do this to get the factor: 1/(diluted value). So, 1/(10^-5) = 10^5. See? That's the dilution factor. In our case, to get the conversion factor, just do the same thing:

1/(converted value) = 1/(1 x 10^-4 ml) = 1 x 10^4 per ml or 10^4/ml.

TADAA!! Look carefully, your conversion factor's unit is PER ML, or /ml, because you divide 1 by millilitre (1/10^-4 ml).

4. When you get the 10 cells in step 1 & 2, you are only calculating for one corner, still remember? And 1 corner is 0.1 mm^3 or 1 x 10^-4 ml. So, what we want to know now is, how many cells are there in ONE MILLILITRE? To get that, you multiply 10 cells with the conversion factor (10^4/ml):

10 cells x 10^4/ml = 1 x 10^5 cells/ml

Yay. Now we are almost done. Now, the LAST STEP.

5. This step is VERY IMPORTANT. We didn't do this in the report. Now, what we want to know is how many VIABLE CELLS ARE IN THE WHOLE CULTURE. Just now, we only calculated how many viable cells in ONE CORNER per ml. To get the number of cells for the whole cell culture (in the T flask, or petri dish), just multiply with the total volume of the culture. Let's say the culture in the T flask is 20 ml. So, the total of viable cells in the whole cell culture is:

1 x 10^5 cells/ml x 20 ml = 2 x 10^6 cells (see that the unit ml is cancelled off)

So now, your answer is the unit of CELLS.

In short, the formula is:

A x B x C x 10^4/ml

where A is the TOTAL CELL CULTURE VOLUME
B is dilution factor
C is the average viable cell => total viable cells in all FOUR CORNERS divided by 4
and 10^4 is the conversion factor from mm^3 to 1 ml.

Just a slight modification from what you have learnt in the lab manual.

PS. Do the same thing for dead cell count. Good luck in your lab test!

PPS. For the unit of conversion factor, if you want to delete the "/ml", it is okay. Dr Lim allows this. But if you do that, you also have to delete the "ml" in the total cell culture volume (A). Otherwise, you will find that you can't get "cells" as the unit.

PPPs. Some more, the example above assumes that we use only 1 chamber. If the question specifies that we use TWO chambers (so got 8 corners), then you have to do one by one. DON'T SEKALI GUS count all and divide by 8 like what we did in our report. After you do both, plus the both answers to get the total cells. Although the answer is the same as dividing 8 , Dr Lim insists on doing separately. Go ask her why yourself.



Source: Dr Lim and http://www.abcam.com/index.html?pageconfig=resource&rid=11454 .