# About Trypan Blue Cell Counting

Majority of experimental procedure in cell cultures, such as transfections, cryopreservation, cell fusion techniques and subculture routines, it is necessary to count the cell number prior to use.

Using a consistent number of cells will help to maintain optimum growth of cells and also help researchers to optimize experimental procedures using cell cultures.

Accurate cell number in various experiments in turn gives results with better reproducibility.

The most common method of cell quantitation is by using hemocytometer.

The hemocytometer, one of the most important device was invented by Louis-Charles Malassez. Hemocytometer is a thick glass microscope slide with a rectangular indentation that creates a chamber.

Hemocytometer chamber is engraved with a laser etched grid of perpendicular lines.

The ruled area of the hemocytometer consists of several, large, 1 x 1 mm (1 mm2) squares.

These are subdivided in 3 areas; 0.25 x 0.25 mm (0.0625 mm2), 0.25 x 0.20 mm (0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2).

The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares.

This is used in RBC counting while 1 x 1 quadrant is used in WBC counting.

The raised edges of the hemocytometer glass slide hold the coverslip 0.1 mm off the marked grid.

This gives each square a defined volume.

This gives each square a defined volume.

The hemocytometer is a counting-chamber device originally designed and usually used for counting blood cells. Image adopted from BioRender

## Principle of Trypan Blue Cell Counting

When a liquid sample containing cells is placed on the chamber covered with a coverslip, capillary action completely fills the chamber with the sample.

Looking at the chamber through a microscope, the number of cells in the hemacytometer chamber can be determined by counting.

The cells to be counted are those which lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square.

The cell number in the hemacytometer chamber is used to calculate the concentration or density of the cells in the mixture from which the sample was taken.

In an improved Neubauer hemocytometer (common medium), the total number of cells per ml can be calculated by simply multiplying the total number of cells found in the hemocytometer grid (area equal to the red square) by 10^4 (10,000).

Concentration of cells in original mixture

= (no. of cells counted) x dilution factor/volume

= number x dilution factor /10-4ml

= number x 10^4 x dilution factor/ml

**An example: **

Total cell suspension after trypsinisation = 1ml

Cell suspension for counting =100μl cells+100μl trypan blue = 200μl

The cell number in individual quadrants = 90, 78, 65, 85

Average = 318/4 = 79.5 or whole number 80

Total cell count = Avg. cell no. x dilution factor x10^4

= 80 x 2 x 10^4

= 1.6 x 10^6 cells/ml

Adopted from BioRender

### Requirements of Trypan Blue Cell Counting

**Sterile:**

**1.** Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).

**2.** Trypsin, 0.25%

**3.** Growth medium

**4.** Pipette tips

**5.** Pipette

**6.** Microfuge tubes

**Non Sterile: **

**7.** 0.4% Trypan blue solution

**8.** Hemocytometer (Improved Neubauer’s chamber)

**9.** Inverted Microscope, centrifuge, CO2 incubator, Biosafety cabinet

##### Procedure of Trypan Blue Cell Counting

**1.** Remove the media from flask containing cells.

**2.** Add 2ml of 0.25% Trypsin-EDTA.

**3.** Incubate for 2 minutes at 37oC in CO2 incubator. Tap occasionally to verify that the cells are releasing. Check in microscope to visualize detachment of cells.

**4.** Remove trypsin-EDTA. Add fresh 2 ml of medium and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells.

**5.** Mix the suspension thoroughly to disperse the cells, and transfer a small sample (~0.1 ml) to a vial.

**6.** Clean the surface of the slide with 70% alcohol or IPA, taking care not to scratch the semi silvered surface.

**7.** Clean the coverslip, wet the edges very slightly, and press it down over the grooves and semi silvered counting area.

**8.** Mix the cell sample thoroughly, pipette vigorously to disperse any clumps and collect 20 μl into the tip of a pipette.

**9.** Transfer the cell suspension immediately to the edge of the hemocytometer chamber, and expel the suspension and let it be drawn under the coverslip by capillarity action. Do not overfill or underfill the chamber, or else its dimensions may change due to alterations in the surface tension; the fluid should run only to the edges of the grooves.

**10.** Blot off any surplus fluid (without drawing from under the coverslip) and transfer the slide to the microscope stage.

** 11.** Select a 10X objective and focus on the grid lines in the chamber. Move the slide so that the field you see is the central area of the grid and is the largest area that can be seen bounded by three parallel lines. This area is 1 mm2 With a standard 10X objective, this area will almost fill the field or the corners will be slightly outside the field, depending on the field of view.

**12.** Count the cells lying within this 1mm2 area using the subdivisions (also bounded by three parallel lines) and single grid lines as an aid for counting. Count cells that lie on the top and left hand lines of each square, but not those on the bottom or right-hand lines, to avoid counting the same cell twice.

**13.** If there are very few cells (<100/mm2), count one or more additional squares (each 1 mm2) surrounding the central square.

**14.** If there are too many cells (>1000/mm2), count only five small squares (each bounded by three parallel lines) across the diagonal of the larger (1 mm2) square.

**15.** Determine number of viable cells by mixing 100 μl of cell suspension and 100 μl of 0.5% trypan blue (trypan blue is excluded by live cells).

**16.** Load on hemocytometer and count the viable and non-viable cells. Calculate percentage of viable cells by: % viable cells = number of viable cells/total number of cells × 100

#### Trypan Blue Cell Counting Precautions

There are several sources of inaccuracy:

The presence of debris or air bubbles in the hemacytometer chamber.

Overfilling the hemacytometer chamber such that sample runs into the channels or the other hemacytometer chamber.

Incomplete filling of the hemacytometer chamber.

Cells not evenly distributed throughout the hemacytometer chamber.

Too few cells to count. This can be overcome by centrifuging the cells, re-suspending in a smaller volume and recounting.

The haemocytometer use can be time consuming, susceptible to subjective judgements by the operator and thus greatly influence the variability.

In some cell types, such as those that form clusters, are particularly difficult to count using this method.

Trypan Blue is toxic and is a potential carcinogen. Protective clothing, gloves and face/eye protection should be worn.

Do not breathe the vapour.

###### Trypan Blue Cell Counting Citations

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