Premium
Assessment of the Cell Viability of Cultured Perkinsus marinus (Perkinsea), a Parasitic Protozoan of the Eastern Oyster, Crassostrea virginica , Using SYBRgreen–Propidium Iodide Double Staining and Flow Cytometry
Author(s) -
SOUDANT PHILIPPE,
CHU FULIN E.,
LUND ERIC D.
Publication year - 2005
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2005.00058.x
Subject(s) - propidium iodide , biology , flow cytometry , staining , viability assay , microbiology and biotechnology , andrology , cell , biochemistry , programmed cell death , apoptosis , genetics , medicine
. A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold‐ and heat‐shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28°C, were incubated in triplicate for 30 min at −80°C, −20°C, 5°C, and 20°C for cold‐shock treatments and at 32°C, 36°C, 40°C, 44°C, 48°C, 52°C, and 60°C for heat‐shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at −20°C and the lowest PVC was obtained at −80°C (54.0%). After 30 min of heat shocks at 40°C and 44°C, PVC decreased slightly but significantly compared to cells maintained at 28°C. When cells were heat shocked at 48°C, 52°C, and 60°C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at≥44°C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28‐day‐old cultures and ranged 11–25% in 38 to 47‐day‐old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.