Dye-Based Methods for Monitoring Cell Density and Viability

Trypan Blue Dye Exclusion

A very old, but still widely-used method for differentiation of viable and dead cells is the selective staining with trypan blue. This principle, investigated by Evans and Schulemann in 1914 (Evans and Schulemann 1914), relies on the cell membrane integrity dependent permeability for large molecules such as trypan blue. Viable cells (integrity of membrane) exclude the dye and appear unstained under the microscope, while the cytoplasm of non-viable cells with compromised cellular membranes is stained by the dye and appears (light-) purple-violet.


For manual determination of viable and dead cell density, a cell suspension sample is mixed with a trypan blue stock solution and analyzed in a hemocytometer. Commonly used is a Neubauer chamber. The squares engraved on its surface are counted out with bright field microscopy. The viable/dead cell density as well as the viability of the sample can subsequently be determined.


– Inexpensive Disadvantages

– Time consuming

– User bias (user-to-user variation)

– Low number of counts (inaccurate measure)

Automated Trypan Blue Based Cell Counting

To overcome the disadvantages of manual hemocytometer-based cell counting, automated cell counters based on trypan blue staining have been developed.

Those machines, such as e.g. Vi-CELL (Beckman Coulter, Brea, USA) or CedexHiRes (Roche, Basel, Switzerland) are image-based cell analyzers using a fully automated liquid handling systems with an integrated flow through (“pseudo hemocytometer”) chamber and a high-resolution image scanner. Image analysis algorithms (cell line dependent adaptation is required) enable the system to classify detected objects into debris, viable and dead cells, aggregates and other undefined objects. Besides the main parameters (viable-, total-cell density and viability), other parameters such as object-diameter can be examined. The cell diameter e.g. is a known indicator for deteriorating culture healthiness or ongoing apoptosis (Darzynkiewicz et al. 1997; Ishaque and Al-Rubeai 2002; Kroemer et al. 2009; Krysko et al. 2008).


– Cost effective trypan blue assay

– Avoids variability inherent in manual sample preparation and counting

– No user-bias Disadvantages

– High initial investment in equipment as compared to hemocytometer.

– Systematic errors can be caused by suboptimal settings of algorithm parameters.

Additional, general aspects of trypan blue based cell counting methods


– Visual inspection of cell population

– Long term “gold standard”; acceptance by regulatory agencies Disadvantages

– Serum and other FCS related substances can inhibit trypan blue (Bhuyan et al. 1976; Hu and el-Fakahany 1994).

– Staining artifacts can hamper interpretation of results.

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