Peer Review #2 of "Noninvasive and safe cell viability assay for Euglena gracilis using natural food pigment (v0.1)"

Abstract

Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined 8 typical natural food dyes to find Monascus (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food dyes as cell viability assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA) . To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period. PeerJ reviewing PDF | (Manuscript to be reviewed 38 39 40 Abstract 41Noninvasive and safe cell viability assay is required in many fields such as regenerative 42 medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a 43 safe and inexpensive method which is a small load on cells and the environment is preferable 44 without requiring expensive and space-consuming equipment and a technician to operate. We 45 examined 8 typical natural food dyes to find Monascus (MP) or anthocyanin pigment (AP) works 46 as a good viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible 47 photosynthetic green microalga. This is the first report using natural food dyes as cell viability 48 assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field 49 microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral 50 imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To 51 confirm the ability of staining dead cells and examine the load on the cells, these two natural 52 pigments were compared with trypan blue (TB)...