Planapo 100 oil objective

Between days 15 to 18, blood smears were prepared from gametocyte cultures, fixed with methanol and stained with Giemsa (Sigma, GS500), prepared as a 1:5 dilution in buffer (pH = 7.2) made using Gurr buffered tablets (VWR, 331942 F) and filtered. Slides were stained for 20 min, washed with buffer, and allowed to dry before observation using a Nikon E600 microscope with a PlanApo 100× oil objective. For calculation of gametocytemias and male: female ratios, at least 500 mature gametocytes were scored per slide. To count exflagellation centers, 500 μL of mature gametocyte culture was centrifuged at 500 × g for 4 min and the resulting pellet was resuspended in equal volume of prewarmed normal human serum. Temperature was dropped to room temperature to activate gametogenesis and after 15 min incubation 10 μl of culture was transferred to a glass slide and covered with a cover slip. Exflagellation centers were counted at 40x objective in at-least ten fields for each gametocyte culture. To avoid bias, microscopic examination was performed in a blinded fashion by a trained reader.

To measure cell size, samples from batch or continuous cultures were extracted, stained with BacLight^®LIVE/DEAD (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), placed on a cover slide, and imaged with phase‐contrast microscopy, using a Nikon Ti microscope with a Plan Apo 100× oil objective (numerical aperture of 1.45 and a refractive index of 1.515). The used camera was an Andor Zyla VSC‐02357, with a binning of 1 × 1, a readout rate of 200 MHz, and an exposure time of 200 ms. Conversion gain was set on 1/3 Dual gain, and the spurious noise filter was activated. The calibration from length units to pixel was defined as 0.07 μm px⁻¹. Measurements were performed with an activated perfect focus system, taking 10 × 10 image frames and moving the sample with a PriorScan III drive stage after each acquisition step. Cell areas were then manually determined using the Nikon software. For each growth rate, at least 300 cells were analyzed to determine averaged length and width. Cell volume was computed as V = π(w/2)² (l − w) + 4/3 π (w/2)³ where w and l are width and length of each cell, whose shape was considered as a cylinder [with base ray equal to w/2 and height equal to (l − w)] with two semi‐spheres at the ends (with ray equal to w/2).