Once the straws were thawed, the microalgae were inoculated in test tubes with 9 mL of sterile water nourished with Guillard and Ryther [16 (link)]’s F/2; they were then centrifuged at 3500 rpm for 10 min (Fisher, 225, Rochester, New York, NY, USA) to separate the microalgae from the cryoprotectant. Next, the concentrated microalgae biomass was inoculated into a new tube with enriched medium, from which five inocula of 1 mL were taken to perform five replications for each treatment, in which the viability of the cells and their population growth were recorded.
The viability of the cells was evaluated with the following criteria. The first was no cell damage (NCD), i.e., when the microalgae Scenedesmus sp. presented the central cells of the colonies with curved sides. At the same time, those located at the ends curved slightly to adopt a crescent shape. Usually, long appendages form a spine, a vibrant green color, an entire cytoplasm, a defined pyrenoid, a well-formed cell wall, chloroplasts, vacuoles, and visible starch granules. The next was cell damage (CD). This was considered to be when the microalgae Scenedesmus sp., despite preserving the number of cells and in some cases the mushrooms or spines, presented irregular shape; their cytoplasm was collected, they were without a defined pyrenoid. Their chloroplasts in some instances were not visible, and they had opaque coloration. The final criterion was marked lesions (ML). This was considered to be when Scenedesmus sp. Loses a number of cells in the row; isolated cells were observed, with an increase in size, deformity, and cytoplasm collected, and an accumulation of organelles in a sector of the cell, an invisible or undefined pyrenoid, rupture of the cell wall, and growth of the population of microalgae in culture. Cell counts were performed every three days in the Neubauer chamber to estimate the percentage of cells with different cell viability criteria. The rate of the viability of Scenedesmus sp. Cells was calculated immediately after thawing (day zero). On the fifth day, the recovery of the cells from damage caused by cryopreservation was evaluated. Subsequently, cell viability was assessed throughout culture (27 days).
The population growth curve throughout the culture was estimated by spectrophotometry after adjusting the absorbance with the help of the spectrophotometer, with a periodicity of three days in each of the 25 replicates until the microalgae reached its phase of decrease, in order to estimate the cell concentration for Scenedesmus sp. throughout 27 days of culture.
The viability of the cells was evaluated with the following criteria. The first was no cell damage (NCD), i.e., when the microalgae Scenedesmus sp. presented the central cells of the colonies with curved sides. At the same time, those located at the ends curved slightly to adopt a crescent shape. Usually, long appendages form a spine, a vibrant green color, an entire cytoplasm, a defined pyrenoid, a well-formed cell wall, chloroplasts, vacuoles, and visible starch granules. The next was cell damage (CD). This was considered to be when the microalgae Scenedesmus sp., despite preserving the number of cells and in some cases the mushrooms or spines, presented irregular shape; their cytoplasm was collected, they were without a defined pyrenoid. Their chloroplasts in some instances were not visible, and they had opaque coloration. The final criterion was marked lesions (ML). This was considered to be when Scenedesmus sp. Loses a number of cells in the row; isolated cells were observed, with an increase in size, deformity, and cytoplasm collected, and an accumulation of organelles in a sector of the cell, an invisible or undefined pyrenoid, rupture of the cell wall, and growth of the population of microalgae in culture. Cell counts were performed every three days in the Neubauer chamber to estimate the percentage of cells with different cell viability criteria. The rate of the viability of Scenedesmus sp. Cells was calculated immediately after thawing (day zero). On the fifth day, the recovery of the cells from damage caused by cryopreservation was evaluated. Subsequently, cell viability was assessed throughout culture (27 days).
The population growth curve throughout the culture was estimated by spectrophotometry after adjusting the absorbance with the help of the spectrophotometer, with a periodicity of three days in each of the 25 replicates until the microalgae reached its phase of decrease, in order to estimate the cell concentration for Scenedesmus sp. throughout 27 days of culture.
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