Growth response and toxin concentration of cultured Pyrodinium bahamense var. compressum to varying salinity and temperature conditions.

The growth and toxin production of a Philippine Pyrodinium bahamense isolate in nutrient replete batch cultures were investigated under conditions affected by varying salinity, temperature and combined effects of salinity and temperature. Early exponential growth stage was reached after 7 days with a cell division rate of 0.26 div day(-1). The toxin content reached a peak of 298 fmol cell-1 at mid exponential phase and rapidly declined to 54 fmol cell-1 as it approached the death phase. Only three sets of toxins composed of STX, dcSTX and B1 were detected in which STX made up to 85-98 mol%toxincell-1. P. bahamense was able to grow in salinities and temperatures ranging from 26 per thousand to 36 per thousand and 23 to 36 degrees C, respectively. The optimum growth under varying salinity and temperature conditions was observed at 36 per thousand and 25 degrees C. Toxin content reached a peak of 376 fmol cell-1 at 25 degrees C and was lower (80-116 fmol cell-1) at higher temperatures (32-35 degrees C). Combined effects of salinity and temperature showed that P. bahamense was not able to grow at low salinity and temperature (i.e. below 26 per thousand-28 degrees C). Optimum growth was observed in higher salinities at all temperature conditions.

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture.

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.