• Microfabricated Enumeration Grid Chambers (MEGC)


Project Summary

Cryopreservation is an essential tool to bank valuable genetic resources. However, a lack of standardization and reproducibility impedes the development of germplasm repositories. The goal of this work was to evaluate the feasibility of microfabrication techniques to produce low-cost disposable counting chambers for standardized estimation of zebrafish sperm concentration. These microfabricated enumeration grid chambers (MEGC) (Fig. 1) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 × 100 μm) and a PDMS base platform (Fig. 2) to create a known volume with a 10-μm height to restrict sperm cells to a single layer. The results of cell counts (Fig. 3) estimated by two of three MEGC devices tested were not significantly different from a commercially available Makler® chamber. The material cost for a MEGC was < $0.1 compared to ~ $100 for a standard hemocytometer and $700 for a Makler® counting chamber. Although fabrication quality could be further improved, this study demonstrates the feasibility of soft lithography methods in creating low-cost, standardized counting chambers for the zebrafish research community to enhance development of comprehensive germplasm repositories.

Figure 1. Overview of the grid-patterned coverslip with 100 × 100 µm squares placed on top of posts that create a 10-µm tall chamber to create a space with known volume (1 × 104 µl for the space of 100 × 100 × 10 µm) for accurate counting of sperm concentration.
Figure 2. For use of the MEGC, sperm samples were pipetted onto the central platform of the base piece (left), followed by placing the coverslip on top (right).
Figure 3. To estimate sperm concentration, sperm samples were observed with a dark-field microscope (200-× magnification), and the number of cells (white dots indicated by the arrow) within each square was recorded.