Cells were cultured over night in YES containing glucose at 0.5% weight per volume until they reached mid-log phase, concentrated by centrifugation, and visualized using phase microscopy using a Zeiss Axioscop microscope with a Plan NeoFluor 40× objective.
Cell movement was followed by phase contrast microscopy using a 10× objective.
The diversity of protozoa in 10-fold concentrated wastewater samples was also determined by phase contrast microscopy using a Leica DMR microscope after staining the samples for 1 h with 1 µL each of neutral red (100 µg/mL; Sigma-Aldrich) and acridine orange (1 µg/mL; Invitrogen), and diluting samples with an equal portion of glycerol to decrease protozoan motility.
In some experiments, cells were viewed by phase light microscopy using an Olympus IX51 inverted microscope at a × 100 magnification and cells were visualised using the NIS-Elements BR (Nikon, Melville, NY, USA) imaging software.
In order to test whether or not reduction of Indy/CeNAC2 in C.elegans also shares characteristics of caloric restriction, whole body fat stores were analyzed by phase contrast microscopy using the azo compound Oil Red O to stain triglycerides.
Cell numbers were enumerated by phase-contrast microscopy using a Bürker-Türk counting chamber at ×1,000 magnification.
After treatment, the morphological features of the cells were evaluated by phase-contrast microscopy using an inverted microscope Axio Observer Z1, equipped with a camera, AxiocamHRc Ver.3 (Carl Zeiss Inc., Germany).
Egress assays were performed as previously described [36]. 2 µM A23187 was used for 10 mins and results were observed by phase microscopy.
The microspore cultures were monitored by bright field and phase contrast microscopy using an in vivo live cell imaging microscope (Leica AF6000 LX with Electron multiplier CCD camera Hamamatsu C9100-02).
Cell morphologies of treated and untreated cells were analyzed by phase contrast light microscopy using an inverted Axiovert 200 microscope.
