Cell culture is a critical step in cellular biology research. To ensure healthy cell growth and facilitate downstream experiments, researchers must determine the cell growth curve and understand their growth status during cell culture, so cell concentration, viability and concentration need to be measured prior to downstream experiments. Typically, cell counting methods include manual counting and automated counting.

This article introduces the advantages and disadvantages of manual cell counting and automated cell counting, along with a detail introduction of these two methods.

Advantages and Disadvantages of Manual And Automated Cell Counting

Method	Counting Time	Single Counting Accuracy	Cluster Sample Analysis	Biological Hazard	Secondary Calculation	Visual Field Area	Data Management	Consumable Cost	Labor Cost
Manual Cell Counting	5min/sample	Low	Rough Calculation	Yes	Yes	1 sq. mm	Manual	Low	High
Automated Cell Counter	9sec/sample	High	Accurate Separation	No	No	3.5 sq. mm	Automated	Slightly Higher	Low

Mannual Cell Counting

Mannual cell counting, developed by the French anatomist and histologist Louis-Charles Malassez in the 18th century, has long been considered the gold standard for cell concentration measurement.

Procedure for manual cell counting by hemocytometer

1 Harvest and stain cells
Harvest the cell suspension and stain with trypan blue or AO/PI dyes.

2 Clean the hemocytometer
Clean the hemocytometer then air dry it or use a dryer. And you can also choose organic solvents such as 95% ethanol, ethanol absolute, or acetone to dehydrate it. Be sure not to scrub the hemocytometer with something hard.

3 Position the coverslip then load the sample
Place the appropriate coverslip over the haemocytometer to form a counting chamber. Pipette a drop of the cell suspension onto the edge of the coverslip and allow the cell suspension to flow into the chamber gently and evenly, avoiding bubbles.

4 Microscope observation and positioning
Find a suitable field of view under the microscope and observe the cell sample. Adjust the focus to obtain a clear view and find the target square. When using AO/PI dye, use a fluorescence microscope for observation.

5 Manual counting under the microscope
Look at the sample under the microscope and count the number of cells manually. This count is repeated three times, taking into account cells of different sizes, and the mean value is taken.

6 Calculate the concentration
Calculate the concentration of cells in the sample using the conversion formula and the recorded data. If trypan blue staining is used, separate live cells from total cells.

7 Clean the hemocytometer
Clean the hemocytometer as described above and store for future use.

Manual Cell Counting Application scenario

Low-throughput cell counting

Notes

● Casual cleaning of the hemocytometer may cause biological hazards.
● Due to the material of the hemocytometer, surface scratches may seriously affect experimental results.
● Impurities left behind may cause uneven cell distribution.
● Residues from cleaning agents may affect cell viability.
● Few clustered cells should be counted as one cell, while samples with more clusters need to be resuspended and measured.
● The counting principle for cells pressed on the gridlines should be determined in advance.

Automated Cell Counting

Although the blood cell counting board is considered the gold standard for cell counting, there are inherent problems with its design and use. Automated cell counters have been developed to overcome these problems.
There are three common types of automated cell counters: vision-based automated cell counters, Coulter-type cell counters, and flow cytometers.

Of these, the vision-based automated cell counter produces results similar to manual counting, which is now the gold standard, due to its high counting speed and similar principles. The automated counter can detect cells and particles and then apply algorithms to measure cell concentration, size and other parameters. This allows determination of cell seeding concentration and number, as well as cell viability and proliferation.

Procedure for Automated Cell Counting

(1) Harvest and stain cells.
(2) Load the sample onto a disposable counting slide.
(3) Insert the slide into the instrument.
(4) The instrument displays the count results immediately.

Automated Cell Counting Applications

High-throughput cell counting.

Automated cell counters can be used with various types of optical equipment and can therefore be upgraded to fluorescence automated cell counters for direct AO/PI dual fluorescence.

Notes

(1) Counting parameters should be set based on the sample.
(2) The predilution multiplier should be entered in advance.
(3) The accuracy of the autofocus should be checked and any deviation should be corrected immediately.

Summary

Manual cell counting with a hemocytometer is often error-prone. Besides, improper cleaning of the hemocytometer can pose a biological hazard. In comparison, automated cell counting is superior to manual cell counting in terms of data accuracy and speed efficiency. Furthermore, the United States Pharmacopeia-National Formulary (USP-NF) recommends the use of at least two or more methods to determine cell viability after recovery. In addition to brightfield trypan blue staining, the AO/PI staining method is commonly used for fluorescence-based live cell counting. Therefore, an automated cell counter with fluorescence capabilities is more convenient for both brightfield and fluorescence-based cell counting, making it more suitable for current cell biology research scenarios.