Comparison of six different methods to calculate cell densities

Abstract

Background: For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).

Results: Our results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision.

Conclusions: Together, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.

Keywords: Automated cell counter; Cell counting; Flow cytometry; Fluorospheres; Hemacytometer; Image analysis; Microscopy; Microspore culture.

Fig. 1

a Three day-old eggplant microspore culture. Cultures at this stage are principally composed of regular eggplant microspores together with few slightly enlarged microspores (arrows). b Eighteen day-old eggplant microspore culture. Cultures at this stage are principally composed of regular eggplant microspores together with few enlarged microspores or microspore-derived embryos (arrows). c Fluorospheres. These particles are very regular in size and shape. Bars: 50 µm

Fig. 2

Box-and-whiskers plots for a mean densities of 17 different eggplant microspore cultures measured at days 3 and 18, b fluorospheres at three different concentrations: 1:10, 1:2 and 1:1 (undiluted, 1,030,000 microspores/ml). Dashed lines represent the expected microspore (in a) and fluorosphere densities (in b). Note that values in B are expressed as neperian logarithms of mean fluorosphere densities. See text for further details

Fig. 3

Bland–Altman comparisons of reproducibility of each method by comparing 3 and 18 day-old microspore culture data. Difference values of the Y axis are expressed in thousands

Fig. 4

Bland–Altman comparisons of agreement between methods. Difference values of the Y axis are expressed in thousands

Fig. 5

Comparison of microspore density measurements performed at days 3 (a), 18 (b) with four counting methods in cultures whose initial microspore density was adjusted to 400.000 microspores/ml (red dashed line) with each method. See text for further details