Cell counting: basics in sample preparation

…89 click …90 click …91 click and 92 CLICK. Wait did I count that cell or not? And the one over there? Yes, no, yes… well let’s start counting again just to be sure ...1 click …2 click ….3 click. Sounds familiar, doesn’t it? We all had to count cells manually. I still remember messing up my first sample and the disgruntled look from my instructor whilst doing so. It is a tedious job that needs to be done properly, be it automated or manual cell counting.

However you wish to determine your cell concentration, you need a proper sample for an accurate value. An incorrect sample is one of the main causes for invalid counts. What can be done to prevent incorrect samples from being analyzed?

Maintain a homogenous suspension

When analyzing your sample, it is assumed that the cell distribution within the sample is representative of the whole suspension. Yet it is well known, that when a cell suspension is allowed to rest for a short period, cells will gravitate towards the bottom of the tube. This results in a concentration gradient within the suspension and thus any sample taken will be unrepresentative.

By using a vortex before sampling or by using the classic “finger-flick”, you regain a homogeneous distribution. Some operators prefer to re-suspend the cell suspension when taking a sample with the pipette. Whatever procedure is performed, be sure to make it the standard protocol.

Eliminate debris inclusion

With most counting protocols, chances are there will be some level of debris present. This can be a significant cause for misclassification when not properly adjusted for. Both for when debris is included as a cell (false-positive) or a cell is excluded as debris (false-negative).

Accuracy of the results will improve by minimizing the amount of debris included in the sample. Otherwise, decreasing misclassification by training for manual counting or improved detection parameters for automated systems will also give more accurate results.

Standardize your protocols

Each operator has their own way to handle cell counting, starting with trypsinizing cells to the way pipettes are handled. By standardizing protocols across your whole facility and making sure these are properly followed (via routine assessments), you should increase the accuracy of the counts from sample to sample.

It may take some time and effort to get everybody in line to follow the protocol. But in the long run, this will result in more accurate counts, leading to better results from experiments and saving you money.


To improve your sample and thus your results, focus upon standardizing protocols and follow up on these as best as you can. This will minimize errors caused by sample preparation but also inter-operator variation. Also, by keeping the sample as homogenous as possible and by excluding debris, the accuracy of the count can increase greatly.