LAB PROTOCOL:HOW TO MAKE ALGAL BALLSPREPARATION - BY LAB TECHNICIAN•1To make a 3% solution of sodium alginate, add 3g sodium alginate to
100mL of distilled water in an Erlenmyer flask. Stir continuously for at least
4 hours, or overnight using a magnetic stirrer. Do not apply any heat.

•2To make 500mL of 2% calcium chloride, add 10g of calcium chloride to
500mL of distilled water and stir to dissolve.

•3You will need approximately 400mL of relatively dense chlorella culture
to make 40mL of the chlorella-alginate mixture. Using this solution, youshould be able to make at least 500 algal balls; enough for a class. To learn
how to culture chlorella, visit our Living Organism Care - Algae resource on
our website. Once your chlorella culture has grown enough to be a brightto darkish-green, siphon out as much of the denser areas into a measuringcylinder. Leave the culture to rest overnight to allow the algae to settle tothe bottom of the cylinder. After this time, there should be a dark “plug” ofdense algae at the bottom.

MATERIALS•••

Figure 1: Supernatant separated from “plug” of dense algae•4To prepare the chlorella-alginate solution, take note of the volume of theplug, then carefully remove the supernatant until it accounts for 2/3 ofthe total volume in the measuring cylinder, with 1/3 dense alginate. For
example, if you have 10mL of chlorella down the bottom, remove all but
20mL of supernatant so that you are left with 30mL in total. Keep thesupernatant aside. If the mixture is too thick later to drop easily throughthe syringe, then you can use this to thin it out a little. Mix the remaining
chlorella and supernatant in the measuring cylinder and add to an equalvolume of your sodium alginate, e.g., 30mL of chlorella and 30mL ofalginate to make 60mL altogether.

•5To set up retort stand apparatus, attach a 30mL syringe barrel to the retort
stand clamp so that the outlet is pointing downwards. Position the syringe
barrel over a beaker of calcium chloride solution.

•6To make algal balls, transfer your algae/alginate mix to the syringe, andallow it to drop through into the solution. Keep at least 1.5mL of mixture insyringe so that gravity will help push it through. Check the rate of the drops
and the shape of the balls as they fall to the bottom.
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If the rate is slower than a drop or two per second, add a small
amount of the supernatant.
If the balls flatten on entering the liquid, the syringe is too high andshould be lowered.
If the mixture comes through the syringe is too low, you may end up
with “sausages” of algae rather than balls.

Leave the algal balls in solution for 5-10 minutes then rinse with distilledwater. Store the algal balls in distilled water. Loosen the lid on the container
to allow airflow. The chlorella within the balls is alive and therefore needs
light to stay active. Place the balls in a well-lit area until required. Do
not place them in direct sunlight, or expose them to too much heat. We
recommend using the algal balls as early as you can; otherwise, they are
best used within 2 weeks.

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3g sodium alginate
10g calcium chloride
400mL of (bright to darkish-green)chlorella culture
Distilled water
Retort stand apparatus
30mL syringe barrel
Plastic pipette
Measuring cylinder
Large beaker

SAFETY PRECAUTIONS•

Wear appropriate personal
protective equipment (PPE).

•Know and follow all regulatory
guidelines for the disposal of••laboratory wastes.
Avoid direct contact with any culture.
Wash your hands thoroughly after