Fluorescent dyes that stain DNA can be used to identify nucleated cells in a mixed cell population. Acridine orange (AO) is a nuclear staining (nucleic acid binding) dye permeable to both live and dead cells. It stains all nucleated cells to generate green fluorescence when imaged with the Cellometer X2.

Because mature mammalian red blood cells don’t contain nuclei, only mononuclear cells produce a fluorescent signal. Fluorescent-positive nucleated cells are easily counted. There is no need to lyse red blood cells, saving time and eliminating an extra sample preparation step.

Use of a fluorescent dye, such as propidium iodide (PI), is highly recommended for accurate viability analysis of cell samples containing debris.

Propidium iodide (PI) is a nuclear staining (nucleic acid binding) dye that enters dead cells with compromised membranes. It stains all dead nucleated cells to generate red fluorescence.

Because the Cellometer X2 can count dead cells in the red fluorescence channel, there is no interference from debris and live cells.

Brightfield and fluorescent imaging

The Cellometer X2 Image Cytometer performs simple, 1-step determination of yeast concentration and viability. The Cellometer X2 is useful for research laboratories looking to automate fermentation monitoring.

Brightfield images can be viewed to confirm cell morphology. Counted brightfield images show counted cells within cell clumps. Fluorescent images show dead cells for confirmation of viability results. Analysis of both brightfield and fluorescent images from each sample makes 1-step concentration & viability determination possible.

Brightfield and fluorescent images of yeast cells. Cells appearing in both the brightfield and fluorescent image are dead.

The Cellometer X2 Image Cytometer automatically generates a cell size histogram based on cell diameter.

The minimum and maximum cell diameter settings can be optimized to count specific cells in a sample.

Concentration dynamic range figure 1 depicts the dynamic range for cell concentration measured by Cellometer X2. This data set was taken on a concentration series of cultured yeasts.

Samples from 2.5 x 10⁵ - 5 x 10⁷ cells/ml can be counted without further dilution.

The %CV at each concentration was below 10%.

Figure 1: Table of results for cell concentration dynamic range.