Manual vs. Automated Cell Counting
Overcoming Four Major Sources of Error in Manual Cell Counting
Manual cell counting is the standard method of cell counting in many labs. But there are several issues when results are obtained by manually counting the number of cells using trypan blue and a hemocytometer.
Automated cell counting using image cytometry provides a solution to these sources of error.
This article presents a review of manual vs. automated cell counting methods.
The NucleoCounter® image cytometers count suspension, adherent and aggregated cells, enabling fast and precise cell measurement in situations where concentrations are challenging to estimate.
This improved methodology provides an immense advantage in precision and reproducibility of mammalian cell culturing.
Challenges in Manual Cell Counting of Mammalian Cells
The time spent at the microscope counting cells is both laborious and time-consuming. As mammalian cell cultures are delicate systems, they require high reproducibility of experimental parameters during setup and culture.
It is vital to know the specific cell concentration and viability of a cell sample to obtain reproducibility in sub-culturing, to monitor growth rates or for cryopreservation1,2.
However, manual cell counting is often associated with large variations in calculating cell concentration and viability. The four biggest sources of error in manual cell counting are:
1. Human Perception of What Defines a Cell
Manual definition and recognition of a cell versus cell debris or other particles can be challenging, even for the trained eye.
Each person performing the manual cell count adheres to a certain set of criteria that defines a cell along with the stain intensity threshold to count it as viable or dead.
Such variations in human perception when counting manually can be extremely detrimental to experimental setup and analysis when counting cells manually.
If multiple users count the same sample, it is not uncommon to see a variance significantly higher than the mean of a Poisson distribution3.
Cell samples with cell debris are often very challenging to count correctly whilst performing a manual count. In comparison, fluorescent events are clearly visible.
2. Volume, Dilution and Pipetting Errors
The preparation and loading of the cell sample in the hemocytometer can give rise to errors.
Though the hemocytometer contains a given volume, the space between the counting chamber and the cover glass might be slightly increased when the chamber is filled with liquid.
This can result in an underestimation of the sample volume causing overestimation of cell concentration, leading to errors based on estimating the volume incorrectly.
Volumetric inaccuracies can also arise from pipetting or from serial dilutions.
3. Viability Determination: Trypan Blue or DAPI?
Trypan blue stains dead cells with a permeable cell membrane whereas viable cells are not stained.
When estimating cell viability manually, trypan blue is used as a marker for dead cells. Since the intensity of the stain can vary in any given sample, it can be difficult to determine whether a cell stains positive with trypan blue.
Given that trypan blue is toxic to cells, viable cells are eventually stained if not analyzed in a certain timeframe, usually within 5 to 30 minutes, depending on sample conditions.
For these reasons, trypan blue is known to underestimate the viability of cell populations and caution must be taken when interpreting trypan blue-based vitality4.
Therefore, selecting a membrane-impermeable DNA-binding dye as 4′,6-diamidino-2-phenylindole (DAPI) for definition of dead cells will increase the precision of viability determinations.
4. Counting Enough Events for Statistically Significant Calculations
Manual cell counting in the Neubauer hemocytometer is standardized to ten chambers corresponding to 1 µl total volume counted1.
However, the standard practice of manual cell counting is usually to count ~100 cells, or a specific volume such as 0.4 µl, regardless of the concentration of cells. Using such a low volume and cell count increases the effect of stochastic variables.
If only 100 cells are counted, the standard variation will be at minimum 10% due to the inherent statistical limitations, assuming the variation follows the Poisson distribution.
According to the Poisson distribution, the expected standard deviation is equivalent to the square root of the number of events recorded, even without human-introduced variations.
Test Your Manual Cell Counting Procedure
Do you know how well you and your colleagues count cells? To validate your cell counting procedure, you need to examine coefficient of variation between the people performing manual cell counting in your lab. Here is a short protocol for doing so:
- Take a minimum of five aliquots of the same cell sample (e.g. five tubes of 200 µl test sample in each). Thoroughly mix the sample before aliquoting
- Ask five colleagues to count one aliquot each (without talking to each other or sharing data!)
- Count using your favorite hemocytometer and your normal standards of counting. Do not count more squares or more cells than you would normally
- Calculate the cell count for each aliquot (see formula)
- Calculate the arithmetic mean, standard deviation, and coefficient of variation in percent (see formulas)
- CV = 0 – 5%: Did you cheat? Your cell counting skills are advanced!
- CV = 5 – 15%: Excellent! You are a very well-trained cell counter
- CV = ≥ 15%: Your score is about average. Manual cell counting often carries large variations, which give data inconsistency between experimental setups and have important impact on the reproducibility of your research
Avoid Human Interference Using the Via2-Cassette™
The Via2-Cassette™ is designed to overcome human interference in cell counting:
- No human bias to influence results:
The software algorithm defines a cell based on acridine orange fluorescence consistently for every analysis
- Pipetting errors eliminated from sample staining and handling:
Cassettes are pre-loaded with immobilized acridine orange and DAPI to define total cell and dead cell populations
- No human errors in calculations:
Cassettes have a pre-calibrated volume of the measurement chamber for precise and reproducible results
- Statistically significant data generation:
The instrument will alert you if you have too few or too many cells in the sample
You can easily load a cell sample into the cassette by submerging the built-in pipette into the cell suspension and pressing the piston.
The Via2-Cassette™ is designed for fast and efficient one-step viability and cell concentration count.
Acridine orange stains the total population of cells and dead cells are stained with DAPI. Learn more.
Automated Cell Counting for Higher Precision and Reproducibility
The NucleoCounter® developed by ChemoMetec is the most precise and easy-to-use automated cell counter 5.
These image cytometers use fluorescent microscopy of fluorophores for detection and analysis of cell cultures with stable, long-lived LED light sources and fixed emission filters for minimal variation.
The sample is excited with LEDs and then light passes through emission filters that match the dyes. The user loads the sample, which automatically stains the sample within the cassette, before inserting it into the instrument.
Focused and filtered light from the LEDs illuminates the sample window of the Via2-Cassette™ and the built-in camera takes a picture of the fluorescent event in the sample. Next, the instrument software algorithm analyzes the images and calculates results.
The NucleoCounter® instruments not only provide a platform for obtaining high-quality data, but also allow for visual inspection thereof, as images can be viewed with the accompanying instrument software.
The NucleoCounter® instrument’s LED lights pass through an excitation filter before passing through the Via2-Cassette™, which contains the sample.
Here, fluorophores bound to cells will emit light, which is focused and passed through an emission filter to enhance the signal. The focused emitted light is captured by a digital camera.
Fluorophores for Cell Counting
Automated cell counting with the Via2-Cassette™ (for NucleoCounter® NC-202™ and NucleoCounter® NC-200™) or Via1-Cassette™ (NucleoCounter® NC-200™ and NucleoCounter® NC-3000™) is based upon two spectrally and biologically different dyes defining total cell numbers and unviable cells: Acridine orange and DAPI.
Acridine orange is cell-permeable, and binds primarily nucleic acids6, i.e. DNA in the cell, which makes it an efficient dye for counting total cell numbers. Upon excitation at 505 nm, acridine orange emits green fluorescence with maximum emission at 525 nm.
DAPI is an efficient stain for dead cells, as living cells are impermeable to low concentrations of DAPI (a few µg per ml). DAPI fluoresces blue upon binding to AT-rich clusters in the minor groove of double stranded DNA7.
Upon excitation at 365 nm, DAPI emits blue fluorescence with maximal emission at 461 nm.
The NucleoCounter® instruments detect the interaction between cells and DAPI or acridine orange by two excitation LED light sources with peak wavelengths at 365 nm and 505 nm.
To detect emission, a single dual-band emission filter of 410-460 nm and 540-650 nm is used.
- MC Phelan: Basic techniques in mammalian cell tissue culture. Curr Protoc Cell Biol. 2007; 36: 1.1.1-1.1.18.
- RI Freshney: Basic Principles of Cell Culture. 2006; p. 1–22.
- L Nielson, G Smyth and P Greenfield: Hemacytometer Cell Count Distributions: Implications of Non-Poisson Behavior. Biotechnology Progress. 1991; 7(6): p. 560-563.
- D Shah, M Naciri, P Clee et al.: NucleoCounter – An efficient technique for the determination of cell number and viability in animal cell culture processes. Cytotechnology: 2006; 51(1): p. 39-44.
- JR Tennant: Evaluation of the Trypan Blue Technique for Determination of Cell Viability.Transplantation. 1964; 2: p. 685-94.
- E Robbins and PI Marcus: Dynamics of Acridine Orange-Cell Interaction. I. Interrelationships of acridine orange particles and cytoplasmic reddening. J Cell Biol. 1963; 18: p. 237-50. 10.1021/bi00388a057
- M Kubista, B Akerman and B Nordén, Characterization of interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry. 1987; 26(14): p. 4545-53.
- Webinar: How Precise Are Your Cell Counts?
- Webinar: Why is Trypan Blue Toxic and Unsafe to Use?
- Blog post: Spilling the Secrets: How to Count Cells with a Hemocytometer
- Blog post: Revealing the Precision of Your Manual Cell Counts
- The NucleoCounter® range: Automated Cell Counters & Analyzers