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Steps for Rearing Microalgae (phytoplankton) in Your Classroom

 Microalgae (phytoplankton) are used as a source of nourishment for brine shrimp that you will feed your larval lobsters.  We will use a single species of phytoplankton that is routinely cultured at DEI.  If, at any time, you need help or assistance call the staff at DEI (497-5769), and if you need additional algal cultures, notify the DEI staff.

 The directions below are step-wise instructions that have been tested repeatedly at DEI and in classrooms throughout Washington County. 

 Two species of cultured phytoplankton (DEI - 2003)

 

1)    Obtain several plastic containers (that you have cleaned with warm soap and water and rinsed very well) to carry seawater from DEI back to your class.  One-gallon milk jugs (with screw caps) or larger plastic containers with tight-fitting lids will work well.

2)    Bring the containers with you to the Downeast Institute and ask the staff to fill each container with seawater that is passed through a one micron (1 µ) filter. (Although this seawater is filtered, you should not use it for rearing algae in this state).

3)    Obtain a 3L plastic soda bottle that you have cleaned with warm soap and water and rinsed very well.  You will fill the bottle approximately ¾’s full with the 1µ-filtered seawater.  Next, you will add 1.5 milliliters (mL) of Clorox bleach (sodium hypochlorite) to the seawater in the bottle. This is a sterilizing step, and is designed to eliminate any organisms remaining in the seawater after it has been filtered. (Even if your container is smaller than 3L, use the same volume of Clorox bleach to sterilize the seawater.)

4)    Stopper the top of the bottle with a 2-hole, #6.5 rubber stopper.  Cut a piece of flexible plastic tubing (3-inches), and push it halfway through one of the holes.  Next, take a plastic pipette and place a piece of flexible plastic tubing (18-inches) over the end opposite from the tip.  Then, insert the tip end through the other hole in the rubber stopper and push it through the hole until the tubing fetches up on the rubber stopper.

5)    Gently shake the contents of the bottle so that the bleach and seawater are thoroughly mixed.

6)    Let the bottle of bleached seawater sit untouched for a minimum of 18 hours (24 hours is best).

7)    After 18-24 hrs, add 1.5 mL of sodium thiosulfate solution.  Shake the contents of the bottle so that the sodium thiosulfate is thoroughly mixed.  Let the bottle of seawater sit for 1 hour.  The sodium thiosulfate will neutralize the bleach. (Again, if you use a smaller container, use the same volume of sodium thiosulfate.)

8)    After 1 hour, you are ready to add the nutrients and silicates that will help the microalgae grow.  First, add 1.5 mL of the f/2 nutrients.  These are liquid nutrients that should be in a container wrapped in duct tape.  The nutrients photodecay, so we use the duct tape wrapped around the container to ensure that no light enters the container.  Second, add 1.5 ml of the silicate solution.  You will see a white precipitate form almost immediately.  Do not be concerned about this precipitate.  Shake the contents of the bottle so that the nutrients and silicates are thoroughly mixed.  (The diatom species we will culture [Chaetoceros muelleri https://ncma.bigelow.org/node/1/strain/CCMP1316] is a unicellular species.  All diatoms have a cell wall that is called a frustule that is made of silica.  The addition of the silicate solution will help the diatoms form their frustules.)

9)    At DEI, you will receive approximately 400 mL of a sterile culture of a Chaetoceros muelleri.  This culture will come in a glass flask (500 mL) that you will take back with you to your classroom.  After you have added the nutrients and silicates to the seawater, you should pour approximately 150 mL of the algal culture into the 3L bottle.

10)           Repeat steps 3-9 so that you have two bottles of algae being reared at all times.

a) The algae added to each of the soda bottles is difficult to observe immediately, as it is very dilute.  Do not worry; with time, the diatoms in culture will begin to grow and the seawater will darken.

11)           For both bottles, place the end of the long section of tubing into an air-splitter and connect it to the BLUE air pump.  Plug in the air pump and adjust the flow of air into each soda bottle so that the seawater is well-bubbled.  This does not require a vigorous bubbling, and a small amount of air will do fine.

12)           If possible, place both soda bottles near a light or near a window.  Leaving them directly in a window that receives full sunlight will kill the algae, so do not place the bottles directly into the sunlight.  Your cultures will grow best if you could provide them a mix of sunlight and artificial light 24 hrs/day.

 Following these instructions carefully may not result in the best algal growth for a variety of reasons that include swings in temperature in your classroom over a 24-hr period, sunlight/artificial lighting conditions, or other uncontrollable variables that exist.  If your algae does not seem to be growing well (it should get a little darker each day, and should be the color of tea by day 8-10), get in touch with the DEI staff, and together we will work out a solution.

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