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One way valves attached to needles were inserted through the butyl stopper to release the gas produced via fermentation.
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After aerobic growth, cells from the late stationary phase were harvested, washed twice with sterile water, and later used to inoculate 100 ml YP medium supplemented with 40 g/l xylose or 80 g/l glucose and 40 g/l xylose in 300 ml bottles capped with rubber stoppers (syringe needles were inserted into rubber stoppers to release CO2 during fermentation) at an initial OD600 of 1.0 (0.63 g DCW/l).
The fermentations were carried out in cylindrical glass tubes, which were closed with a rubber stopper containing a glass pipe, sealed off with a cotton plug to release CO2. 100 ml fermentation medium was added to each tube.
Cysts were purified by sucrose flotation [ 142] and crushed against a 7.6-cm diameter 250-μm sieve with a rubber stopper partially submerged in water to release the eggs.
Syringe needles were placed through the stoppers of these 17 bottles to release produced biogas.
(Remove stopper from the funnel at intervals to release pressure).
(Note: In pre-heating the sulfoxide-acid mixture, remove the stopper of the separatory funnel at intervals to release the pressure).
Females were purified by sucrose flotation [ 40], and the females were crushed gently with a rubber stopper in a 7.5 cm diameter, 250 μm sieve to release the eggs.
To release the carbon dioxide produced at early stage, a syringe needle was pierced through the rubber stopper.
From stopper to co-stopper to setup man to, finally, co-setup man.
Attach the stopper to the lower rod.
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