WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2024 41 FIGURE 3. Isochrysis growth data including the amount of cells/mL in each measurement. Results Isochrysis sp. samples were cultured using the standard methods used by the CFK Aquaculture Laboratory, using three different saltwater treatments. Raw seawater, water sourced from the ocean containing all-natural nutrients but no living organisms, was used for our first sample. This batch was created on March 3. Evaluation was done on March 7 using a hemacytometer to average cells, which found an average of 12.8 cells per square unit. This equated to 3,200,000 cells/mL for the first measurement (Figure 3). On March 15, a measurement of cells was completed with an average of 17.4 cells per square unit and 4,350,000 cells/mL. On March 15 two samples were also used to create 2 different cultures, one using instant ocean and one using reef salts. On March 17, there was no change in the color of the beaker, so two more samples were used to make our fourth and fifth cultures. They followed the same order of two different salt mixtures. By March 23, 2 samples had succeeded in proliferating, one from the March 15 batch and one from March 17. On March 27, the second sample from March 15 had successfully been cultured, indicating both salt mixtures worked. On April 1, measurements were taken from all sample beakers. The first sample, created on March 7th, had 29.4 cells per square and 7,350,000 cells/mL. The second sample created on March 15 revealed 12.2 cells per square and 3,100,000 cells/mL. The other sample created on March 15 had 18.4 cells per square and 4,600,000 cells/mL. The successful third sample created on March 17 showed 28 cells per square and 7,000,000 cells/mL. The final sample that was created on March 17 never completed its culture; it stayed white (Figure 4). This culture was measured nonetheless and showed 0.6 cells per square unit and 150,000 cells/mL. The sample data is presented in Figure 3, including the amount of cells/mL in each measurement. Discussion/Conclusion On March 3rd, the first Isochrysis sp. culture was grown with raw seawater. It was split on March 27. The split was successful and resulted in two new cultures identified as Raw A and Raw B. Raw A had more algae cells when compared to Raw B. This may be due to many issues with splitting algae. Dilutions used to properly separate the algae into two separate samples can be incorrect due to human error and other variables. On March 15th, the second set of Isophrysis sp. cultures were created and had a steady rate of growth over time. One of the cultures had a higher number of cells when compared to the other. On March 17th, the third set of Isochrysis sp. cultures were created. One of the two cultures was observed to be successfully growing by March 23. The other culture never acquired pigmentation and stayed a white hue, with less than 4 cells identified within the entire hemacytometer. This culture was discarded on April 1st. There are many potential variables influencing growth rate differences between cultures. Due to space restrictions all the cultures were grouped closely together, causing half of the cultures to be directly in the light and the others to be in front of those cultures. When removing samples from the beakers, they may have been exposed to ventilation and contaminants. A pipette was placed in the beaker, suctioned by a human finger, then placed on a microscope slide. The samples were also cultivated in the main laboratory with other lab projects where different solutions could have been introduced into the dilution buckets. Algae are used throughout the CFK lab as feed for many different species, including copepods and clownfish. The brown algae will help the lab test new nutrients and improve mixed feed solutions for these organisms and create new opportunities for the lab to be more of a controlled oceanic ecosystem. Notes Tiffany Clavijo, The College of the Florida Keyes. Corresponding Author: tiffany.clavijo@cfk.edu CFK laboratory assistant Jessie Appelhans helped tremendously to cultivate these samples safely and effectively, as they would hopefully be used in the lab. 1 Instant Ocean Spectrum Brands 3001 Commerce St. Blacksburg, VA 24060-6671 References Alkhamis, Yousef and Jian G Qin. 2013. Cultivation of Isochrysis galbanain in phototrophic, heterotrophic, and mixotrophic conditions.” BioMed Research International 2013(983465):1–9 https://doi.org/10.1155/2013/983465. Cock, J. Mark, Akira F Peters and Susana M Coelho. 2011. Brown algae. Current Biology 21(15):R573-5. doi:10.1016/j. cub.2011.05.006 FIGURE 4. Four successful cultures of Isochrysis galbana and one that was a failure. It can be noticed by the clear/white hue.
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