Imt 2 inverted light microscope

Methanol extractions of the toxin contents from V. rugosum and S. acuminata strains were performed. An aliquot of 5 mL was taken from one of the culture flasks and fixed using Lugol’s iodine solution. The exact cell concentrations for each strain were then determined using a Nageotte counting chamber via an Olympus IMT2 inverted light microscope.
Secondly, toxin extractions were performed according to Abadie’s protocol [59 (link)]. Exactly 20 mL of the culture suspensions was sampled from the flasks and centrifuged (2000× g, 15 min, 20 °C). The supernatants were then carefully removed and discarded. 1 ml of 100% methanol (VWR chemicals, Radnor, PA, USA) was then added to the remaining pellets. The extraction was performed by two consecutive sonications of 1 min each, followed by filtration over a 0.2 µm membrane (Whatman Mini-UniPrepTM). The filtered extracts were then stored at −21 °C until quantification.

After optical examination, feces were dislocated and put in culture plates (96 wells) with 200 µL of ENSW. Potential germination was checked every two hours under optical Olympus IMT2 inverted light microscope. The germination rate (in hours) was estimated by the time of first observation of pelagic cells. The germination percentage (percentage of temporary cysts that germinated after gut passage) could not be determined as V. rugosum cells encyst naturally in laboratory conditions, making it impossible to distinguish the cells that went through guts as cysts from those that encysted during the incubation.