Sampling and Analytical Methodology:
\nIn the first trial, a Labyrinthulomycota culture held at 22 degrees C was divided into 18 sub-cultures that were incubated at 15 degrees C, 20 degrees C, 25 degrees C, 30 degrees C, and 32 degrees C in triplicate for three days. Culture temperatures and incubation period were based on previous visual observations of Labyrinthulomycota growth, where over-growth of culture flasks occurs after three days at temperatures of 25 degrees C and higher. In the second trial, nine sub-cultures were incubated at 20 degrees C, 25 degrees C, and 30 degrees C in triplicate for three days.
In the second trial, total protein was also assessed. After three days, the media was poured off, rinsed once with 3 mLs of 0.22 um-filtered artificial sea water, and replaced with 3 mLs of 0.22 um-filtered artificial sea water. With a sterile wooden dowel, the bottom of each culture was scraped until no Labyrinthulomycota growth was visible on the bottom of the culture. 700 uL of each culture was placed in a bead beater and mixed at 300 rpm for 30 sec; 400 uL was set aside for protein assays and 300 uL for cell counts and held on ice until use.
\nPrior to counting using a hemocytometer, cells were vortexed for about 20 sec. In each culture, cells were counted in triplicate.
\nTotal protein was extracted from each sample by adding 400 uL of extraction buffer (0.15 ug mL-1 DTT in Tris-HCl) to each tube. The contents of the tube were mixed and lysed for 2 minutes with the Fisherbrand disposable pestle grinder system, and incubated for 45 minutes on ice for extractions. Protein was measured using the DC protein kit (Bio-Rad), and read in triplicate using the Synergy HT multi-Detection microplate reader with KC4 software (Biotek Instruments, Vermont) at 750 nm.
Two types of assays were used in two trials to quantify Labyrinthulomycota cultures: cell counts using a hemocytometer and total protein concentration.