A technique to detect cell damage due to chilling injury for fruits and vegetables using Trypan blue and Evans blue

Abstract

There are several methods to detect chilling injury symptom of produces such as the changes of electrolyte leakage (EL) or malondialdehyde (MDA) content. However, those methods detected whole injured tissue samples. They could not indicate the area of tissue or cell damage from chilling injury. Furthermore, there were several reports indicated that MDA content of some chilling injured tropical fruits were not concomitant with the EL and chilling injury index. In this study, a simple technique to detect cell damage caused by chilling injury was developed. Five tropical fruits and fruit vegetables which were susceptibility to chilling injury were kept at 4±1°C. Three samples from each fruit were collected daily and cross sectioned to 15 µm thick, then stained with either Trypan blue or Evans blue solutions. Stained tissues were examined under light microscope. The stained tissues were extracted and their optical densities (OD) were measured at 585 nm. The result showed that injured cells which lost cell membrane integrity were stained with Trypan blue and Evans blue, whereas live cells, of which their membrane is impermeable to the dyes, were excluded from staining. Thus these injured cells found mainly in the epidermal, sup-epidermal areas and were parenchyma cells. The ODs of crude extracts from Evans blue stained tissues were found to be related to the chilling injury index and MDA content of the chilling injured fruits. On the contrary, results from the control fruits kept at room temperature showed that the tissues were not stained with both Trypan blue and Evans blue. The OD of the solution from control tissues was not changed during study period. Moreover, Evans blue was more sensitive to detect cell damage compared to that of the Trypan blue. Therefore, this technique could be an alternative method to detect cell damage before chilling injury symptoms occur on the produce.