If you do cell culture you will inevitably need to count your cells. Counting cells can be tedious, but it is important to do accurately. Your assessed quantity of living cells will affect all your downstream applications. In this article I will not only cover how to manually count your cells and how to do it right, but also tell you about automatic cell counting.

Manual Cell Counting

Traditionally, cell concentration is determined manually using a hemocytometer to count the cells. A hemocytometer is a thick microscope slide scored with a grid. To use a hemocytometer cells are counted within the squares of the grid and converted to cells/mL using the equation below:

# of cells counted / # of squares used to count
(I count the four large corner squares)
x dilution factor x 10,000
= cells/ mL

Easy in theory, but in practice sometimes we overlook important factors.

When I was an undergraduate intern, I spent almost an entire summer counting parasites. We were documenting the growth phenotypes of various parasite mutants. Depending on these parasite’s growth phase they were mobile. And as you can imagine, counting mobile cells that would wiggle from one end of the hemocytometer all the way to the other was incredibly difficult. Chasing these parasites made it nearly impossible not to miscount some of them. You may not have my challenge of mobile cells, but you still need to be careful for other reasons.

Tips for Manually Counting Cells Correctly

Dilute Your Cells Accurately

A good cell suspension will give you the most accurate and reproducible count. To do this, it is important to resuspend the cells carefully in a volume that equates to about 25-80 cells per large square on the hemocytometer. If the cell numbers are too low, they may not be representative of the concentration in solution. However, if there are too many cells, they can start to aggregate and lead to counting errors as well.

Make a Plan for Fence Riders

Count cells in the four large corner squares of the hemocytometer. As you begin to count, some cells will lie on the borders of the larger squares. Decide which of the two borders to include, and count any cells that lie on only two borders of these four borders. This way you are systematically including half of the fence riders, giving you an accurate and reproducible count.

Double Check Yourself

When I first began counting parasites, I also made sure that my values were reproducible and compared them to someone else’s values. This is especially good advice when dealing with swimming cells, but most anyone can benefit from an occasional audit.

After spending an entire summer counting parasites, I solidly vowed I would never take a job where counting cells was a big part of my responsibility. Boy was I wrong.

I am now a Postdoc working in a primarily cell culture lab where counting cells is extremely important for seeding, transfecting, as well as various assays requiring a specific amount of cells. For these experiments I also need to know the cell viability.

How to Count Only Viable Cells

To assess cell viability I use Trypan blue. Trypan blue is a dye used to stain dead cells. To stain with Trypan blue you need to:

  1. Wash your cell pellet with phosphate buffer saline (PBS) so that no dye from the medium remains.
  2. Resuspend your cells in a predetermined volume of PBS, again try to acquire a good cell suspension to ensure reproducibility and accuracy. The resuspension volume may change based on the type of cells you are using, whether large or small, and confluency of the flask.
  3. Dilute your cells from the resuspension stock in equal volumes of 0.4% Trypan blue.
  4. Load your sample onto the hemocytometer, add the cover slip, and count the live (opaque) from dead (dyed) cells under a microscope using the four large corner squares.
  5. Calculate cells/mL for both live and dead cells, and add them together to calculate the total number of cells.
  6. Finally, divide the live cell count by the total number of cells to determine the percent viability.

When using Trypan blue it is important not to incubate your cells in trypan blue for too long (more than 5 minutes) or even the viable ones will begin to take up the dye. And this would obviously defeat the purpose.

Automated Cell Counting

Cell counting is a necessary evil. Cell counting can easily take a few hours to complete, especially if you have to recheck some of the values manually. Therefore I know I am not the only one to think: “Is there a better way to count cells??”

I have tried to cut corners by counting only two squares instead of all four squares. However, this method probably doesn’t provide the most accurate and reproducible experimental results. I have also enlisted another person in the lab to help me count which I generally don’t suggest since there tends to be some variation between the way people count.

But there is a legitimate way to save time: automated cell counters.

Our lab recently got an automated cell counter and I love it. Automated cell counters provide reproducibility and accuracy between users at a fraction of the time.

There were two basic types of equipment we recently demoed. This was based on price of the equipment as well as the consumables. Surprisingly, you can purchase an automated cell counter starting at $1800 – a reasonable amount when you consider the cost of your time.

One of the instruments we demoed counted cells using the Coulter counting principle. This method uses electrical current that when disrupted by a particle creates a change in impedance. The other two instruments we demoed use typical imaging methods, such as looking under a microscope using bright-field imaging.

We chose to purchase an automated cell counter with the bright field imaging method for a few different reasons; the ability to program in a variety of different parameters for multiple cell lines; calculate cell viability; ease of use; speed and accuracy; price and cost of consumables.

Whichever method you use for cell counting still requires good preparation for reproducible and accurate results. However, I highly suggest trying to convince your boss into purchasing an automated cell counter. Especially if you constantly find yourself along side your co-workers staring into the microscope counting cells. It is fundamental part of cell culture, you can count on that!

Chelsey Kline, PhD