Investigating photosynthesis using algal balls
Technical Notes
In this protocol, the algae Scenedesmus quadricauda are suspended in alginate beads. Hydrogen
carbonate indicator solution (also called bicarbonate indicator) can then be used to investigate the
rate of photosynthesis under different environmental conditions.
Overview
This document will explain how to:
1. Prepare an algal culture and sodium alginate ready for students to use in lessons
2. Immobilise the algal culture in alginate beads
3. Make up standard indicator solutions for students to compare their results against
4. Select appropriate lighting for the investigations
5. Prepare equipment for a range of investigations using algal balls
6. Source equipment
1a. Preparing the algal culture
Method for bulking up the algae (3-4 weeks before the lesson)
You will need a small starter culture of algae (see supplier information) and a large round bottomed
flask (5dm3) or volumetric flask (2dm3).
Add the starter culture to a large round bottomed flask (5dm3) or a volumetric flask (2dm3)
containing a two litre solution of enrichment medium (see supplier information).
This culture will need to be aerated gently with a pump (a simple aquarium pump, tube and airstone is suitable for this) and maintained at approximately 18-22°C. Growth will be fastest when
24h illumination is provided (see notes on lighting). After 3-4 weeks, when this bulked algal culture
is dark green, like pea soup, it is ready for use.
At least one hour prior to the lesson this bulked algal culture will need to be measured out into
50cm3 batches (in beakers or measuring cylinders) and allowed to settle. You are aiming for about
5cm3 to settle out of 50cm3 of the bulked algal suspension. If the bulked algal solution is not dark
green, you will need to pour out a greater quantity of bulked algal solution to get a quantity of 5cm 3
settling out.
Buying the culture ready concentrated
If you need the algae at short notice, concentrated cultures can be purchased from the supplier
(see supplier notes) in 50cm3 quantities. Each student needs 5cm3 of this concentrated culture.
1b. Preparing the sodium alginate
This should be made up the day before the lesson. A 2-3%* w/v solution should be made by
adding the sodium alginate powder to water. A magnetic stirrer should be used to mix the solution.
The powder does not mix in immediately and will need to be left overnight to fully dissolve. The
alginate should not be heated as this compromises the gel strength. Once made, it can be kept in
bottles/beakers covered with cling film for up to two weeks at 4°C.
*As sodium alginate is a natural product, its viscosity can vary from batch to batch. The alginate
required for this investigation should be fluid enough to pour slowly, so it is advisable to test your
alginate for viscosity in advance of the lesson.
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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2. Immobilising the algae in alginate balls
Apparatus and chemicals for class practical
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2 litres of bulked algal culture (divided up into 50cm3 batches in advance)
2-3% sodium alginate solution (100cm3 should be enough for a class of 30
students)
2% calcium chloride solution (50-100 cm3 per student)
7 ml bijou bottles with lids (1-2 per student for the concentrated algae)
fine nosed syringes (20ml)
100 or 250 ml beakers (1 per student)
retort stands, bosses and clamps (1 set per student)
tea strainers/small sieves
plastic tea spoons or plastic tweezers
Note
See our video guide to this protocol at https://www.youtube.com/watch?v=fI3x68CkKW0
Making the algal balls is an activity that engages students and takes about 45-60 minutes. Once
made and rinsed in distilled water, the algal balls call be stored in distilled water at 4°C for several
months. If made in advance and kept refrigerated the algal balls should be removed and brought to
room temperature for at least 30mins in advance of the lesson. Students will use approximately 20
algal balls per bijou bottle for most experiments except where they are altering the number of algal
balls.
The method described on the next page gives the quantities needed for students to make their own
algal balls. It will provide each student/group with 100-200 algal balls.
If lesson time is limited, the algal balls can be made in bulk in advance using larger quantities of
the algal suspension and sodium alginate solutions mixed together in a beaker before using the
same technique. However, enjoyment and ownership of students creating their ‘own’ balls
contributes to the engagement in undertaking this activity.
Preparation
At least one hour prior to the students making their own algal balls, the bulked algal culture should
be poured into beakers in 50-100cm3 batches. This should be allowed to settle so that the algae
sink to the bottom of the beaker (this seems to work best if the beakers are in the dark). If the algal
culture was not a dark green colour, more than 50cm3 of the algal culture should be poured into the
beakers so that enough algae are incorporated into the alginate balls later on – you are aiming for
pea green coloured (or darker) algal balls. See earlier notes on preparing the algal culture.
Sodium alginate can be made several days in advance and stored at 4°C until the lesson.
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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2. Immobilising the algae in alginate balls
Student Method (makes up to 200 algal balls)
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Take the beaker with the settled green algae in the bottom. Pipette off and dispose of the
supernatant above the algae and retain the concentrated green algal solution.
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Pipette approximately 5 cm3 the concentrated green algal solution into a bijou bottle.
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Pipette 2.5 cm3* of sodium alginate solution into the same bijou bottle.
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Tighten the lid on the bottle and mix gently for a few minutes until a homogenous solution is
obtained.
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Place a beaker of calcium chloride underneath a syringe (with the plunger removed) held
by a clamp on a retort stand.
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Transfer the algae/sodium alginate mixture to the syringe.
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The mixture will slowly start to drip out of the syringe into the
calcium chloride. The alginate reacts with the calcium chloride
causing it to solidify and form uniform sized balls. The algal balls
need to be left in the calcium chloride for 5-10 minutes to allow this
to take place.
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After the algal balls have been in the calcium chloride solution for
5-10 minutes, they should be tipped into a plastic tea strainer and
then rinsed well in distilled water. If they are to be stored, they
should be transferred to a clean beaker of distilled water and kept
covered with cling film (or in a lidded wide-necked jar) at 4°C.
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Plastic spoons or plastic tweezers can be used to transfer the algal
balls into bijou bottles when required.
*the exact volume of sodium alginate required may vary according to its
viscosity. If it is very viscous, more may be required. We suggest that you
trial making the algal balls in advance of the lesson to ensure the ratio of
the algal culture and sodium alginate works.
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
The algae/sodium
alginate mixture
drips out of the
syringe into a beaker
of calcium chloride
solution
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3. Standard indicator solutions
Colour change / pH value as an indicator of photosynthetic rate
In these investigations an indicator solution is used to measure the change in rate of
photosynthesis in different conditions. (Note that Universal indicator cannot be used.)
The colour change/pH of the hydrogen carbonate indicator solution can be determined using a
colorimeter or by eye using a colour chart for comparison. Charts can be laminated and reused. Or
by using standard solutions as below
Indicator colour chart and corresponding pH values
Download from http://www.saps.org.uk/attachments/article/235/ colour%20chart%20showing%20hydrogencarbonate%20indicator.pdf
← Increasing CO2 in indicator(more yellow) pH 8.4=Atmospheric level of CO2 (0.04%)
pH 7.6
pH 7.8
pH 8.0
pH 8.2
pH 8.4
Decreasing CO2 in (more purple) indicator →
pH 8.6
pH 8.8
pH 9.0
pH 9.2
Making standard indicator solutions for comparison to test results
Hydrogen carbonate indicator can vary from supplier to supplier and the colour of your solution
may vary from that shown in the colour chart . You may want to set up standard solutions (of
known pH) so that students can compare the colour of the solution in their bijou bottles to the
prepared standards, as described below.
Apparatus for standard indicator solutions
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bijou bottles labelled with each of the 9 pH values
beakers / volumetric flasks
distilled water
boric acid (12.4g)
borax (sodium tetraborate decahydrate, 9.5g)
hydrogen carbonate indicator
Method
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As hydrogen carbonate is a sensitive indicator, it is important to rinse all glassware
thoroughly in distilled water beforehand
Dissolve the boric acid and make up to 1 litre in distilled water
Dissolve the borax and make up to 1 litre in distilled water
Add 25cm3 of the boric acid solution to each of 9 clean 100cm3 volumetric flasks
Use the table below to add a set amount of the borax solution to each of these flasks
Top up each flask to the 100 cm3 mark using distilled water and swirl to mix
Add 4.5 cm3 of each solution to the correctly labelled bijou bottle
Just before the lesson, add 0.5 cm3 of the hydrogen carbonate indicator that the students
will be using to each bottle
pH value
Vol. borax
7.6
1.00
7.8
1.55
8.0
2.45
8.2
3.60
8.4
5.70
8.6
8.70
8.8
15.00
9.0
29.50
9.2
57.50
25.00
25.00
25.00
25.00
25.00
25.00
25.00
25.00
25.00
(cm3)
Vol. boric
acid (cm3)
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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Lighting and Safety
4. Choosing appropriate lighting
For all of the investigations using algal balls, a portable floodlamp, preferably a compact
fluorescent lamp (CFL) type of approximately 42W - see suppliers notes - or 150-200W Halogen
lamp is required to bulk up the algae culture. When students are investigating distance from the
lamp, several students can share a flood lamp easily. When investigations are being conducted in
front of flood lamps upturned trays or lab jacks can be placed in front of the lamp so that bijou
bottles can be placed in the light.
If ordinary desk lamps are used, fitted with 40-60W filament bulbs or 8-11W CFL (often referred to
as energy saving bulbs), the indicator solution will change colour, however this process may take
over 60 minutes. CFL/energy saving bulbs in excess of 60W equivalent (15-22W) will provide
sufficient intensity of light to obtain results in a 60 minute lesson.
We suggest portable energy saving compact fluorescent flood lamps or “site lights” (such as the
42W model available from Screwfix – see suppliers notes). These lamps are large and can be
shared by several students, they give off a bright light equivalent to ~250W (halogen) with little
heat compared with halogen lamps.
Cheap portable 150W halogen lamps are available that are also suitable although these will cause
a significant heating effect. If using these, a heat sink such as a chromatography tank filled with
water should be placed between the lamp and the bottles of algae to minimise heating effects.
Safety
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Care is needed if using lamps near water sources. Halogen lamps operate at high
temperatures and can warm ‘heat sinks’ very quickly, care should be taken when emptying.
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Calcium chloride granules are irritant, so avoid contact with skin/eyes and do not inhale the
dust when making this into a solution. A 1.5% solution required for this investigation is nonhazardous. See CLEAPPS recipe 19 and Hazcard 19A or other appropriate health and
safety guidelines followed at your organisation.
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Sodium hydrogen carbonate indicator – avoid inhaling dust when dissolving the dyes – see
CLEAPPS recipe 48 and Hazcard 32 or other appropriate health and safety guidelines
followed at your organisation.
When making up the standard indicator solutions
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Boric acid – weigh out the solid in a switched off fume cupboard to avoid stirring up any
dust. At low temperatures, the aqueous solutions are low hazard - see CLEAPPS Hazcard
14 Boron Compounds and Bulletin 139 or other appropriate health and safety guidelines
followed at your organisation.
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Borax (sodium tetraborate decahydrate) – weigh out the solid in a switched off fume
cupboard to avoid stirring up any dust. At low temperatures, the aqueous solutions are low
hazard - see CLEAPPS Hazcard 14 Boron Compounds and Bulletin 139 or other
appropriate health and safety guidelines followed at your organisation.
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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5. Equipment required for algal ball investigations
For these investigations students may need stop clocks and pens to label their bottles. They may
also need sticky tape to fix paper/filters to the bottles.
The hydrogen carbonate indicator will need to be equilibrated to atmospheric CO2 concentration by
bubbling air through the solution until it is bright red (pH 8.4).
For investigation 1: Light and dark
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Hydrogen carbonate indicator solution (enough for each student / group to fill 2 bijou
bottles, plus spare)
Bijou bottles and lids (2 per student / group)
1 bright lamp (e.g. 42W portable CFL or 150W halogen floodlamp with heat sink) or several
smaller desk lamps fitted with 100W filament or 100W equivalent energy saving (CFL)
bulbs (see notes on lighting)
Black sugar paper cut into strips to wrap around the bijou bottles
For investigation 2a: Light intensity (distance from lamp)
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Hydrogen carbonate indicator solution (enough for each student / group to fill 2 bijou
bottles, plus spare)
Bijou bottles and lids (2 per student / group)
1 bright lamp (e.g. 42W portable CFL or 150W halogen floodlamp with heat sink) or several
smaller desk lamps fitted with 100W filament or 100W equivalent energy saving (CFL)
bulbs (see notes on lighting)
Black sugar paper cut into strips to wrap around the bijou bottles
50cm rulers
For investigations 2b and 3: Light intensity / compensation point (neutral density filters)
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Hydrogen carbonate indicator solution (enough for each student / group to fill 5 bijou
bottles)
Bijou bottles and lids (5 per student / group)
0.15, 0.3 and 0.6 neutral density filters, cut to wrap around bijou bottles (1 of each per
student / group)
Colorimeter, cuvettes and pipettes if accurate compensation point investigations are
required
Neutral density (ND) filters block out light of all wavelengths and can be used as an alternative to
moving the bottles further from the lamps. As the bottles can be placed at the same distance from
the lamp, this removes the varying heat effect over distance when using halogen lamps. The ND
filters suggested above have given good results (see supplier information for more details) and can
be used to find the compensation point between photosynthesis and respiration.
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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For investigation 4: Light colour/wavelength
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Hydrogen carbonate indicator solution (enough for each student / group to fill 5 bijou
bottles)
Bijou bottles and lids (5 per student / group)
1 bright lamp (e.g. 42W portable CFL or 150W halogen floodlamp with heat sink) between
2-6 students or several smaller desk lamps fitted with 100W filament or 100W equivalent
energy saving (CFL) bulbs (see notes on lighting)
green, red, blue and clear acetate films, cut to wrap around bijou bottles (one of each per
student / group)
Black sugar paper cut into strips to wrap around the bijou bottles (one per student / group)
For accurate filters, we recommend Primary Red, Primary Green and Bray Blue. These can be
obtained from Lee Filters (see suppliers section).
For investigation 5: Number of algal balls or concentration of algae in each ball
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Hydrogen carbonate indicator solution (enough for students to fill 5 bijou bottles each)
Bijou bottles and lids (5 per student)
1 bright lamp (e.g. 42W portable CFL or 150W halogen floodlamp with heat sink) between
2-6 students or several smaller desk lamps fitted with 100W filament or 100W equivalent
energy saving (CFL) bulbs (see notes on lighting)
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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6. Supplier information
Most of the chemicals and equipment you will need for these investigations are easily sourced from
standard school science suppliers.
The following information is given to help you source less common apparatus and materials.
Algae
Scenedesmus quadricauda algae can be obtained from Sciento, 61 Bury Old Road, Whitefield,
Manchester M45 6TB, Tel: 0161 773 6338
http://www.sciento.co.uk/catalog/category/49/
You can order 30 ml starter culture or a 50 ml culture (both under £10) and the powdered
enrichment media (£6).
Photosynthesis kit
NCBE based at Reading University have put together a kit for this investigation. It contains a
starter culture of algae, powdered enrichment media, a bottle of hydrogen carbonate indicator
solution, bijou bottles (75), fine-nosed syringes (15) and light filters. This is enough equipment for a
class of 30 students to conduct several experiments and the equipment can be reused. Details for
ordering can be found at http://www.ncbe.reading.ac.uk/. The cost is £65 + VAT
Colourimeter
Many suppliers provide suitable colourimeters for use in schools and colleges.
A suitable model that provides reliable results is the WPA CO7500 from Biochrom Ltd.,
22 Cambridge Science Park, Milton Road, Cambridge, CB4 OFJ. Tel: 01223 423723
http://www.biochrom.co.uk/
This unit works best when using the green (550nm) filter that is supplied with the instrument. It is
simple to use and robust.
Filters
Reliable filters that have been successfully used in these investigations came from Lee Filters. The
products used and their codes are: Colour filters: Primary Red (106), Primary Green (139), Bray
Blue (722); Neutral density light control technical filters: 0.15 ND (298), 0.3 ND (209) and 0.6 ND
(210). These can be obtained from: Lee Filters: www.leefilters.com
Filters can also be obtained from the NCBE, http://www.ncbe.reading.ac.uk/menu.html
Lamps
To bulk up the algal culture portable energy saving CFL flood lamps /site lights are recommended
(such as the 42W model available from www.screwfix.com ; product code 23370; approximately
£26). These lamps are large, can be shared by students in subsequent investigations and give of
very little heat compared with halogen lamps (as of November 2012 these lamps may be
incorrectly listed as halogen on the screwfix website although they are actually CFLs)
Cheap portable 150W halogen lamps are available that are also suitable (e.g. via Rapid
Electronics; ~£8) although these have a significant heating effect. If using these, a heat screen
such as a chromatography tank filled with water should be used.
When illuminating student investigations into algal balls if many lamps are required across the lab
standard desk lamps fitted with energy efficient (CFL) lamps of an equivalence brightness of at
least 75 -100W (tungsten filament) are recommended. These are often rated 15-22W, are
available from most hardware stores, again give off little heat and give satisfactory results in
photosynthesis experiments (please note local guidance on disposal of energy efficient bulbs, as
these often contain mercury compounds).
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Photosynthesis with Algal Balls: Technical notes (Revised 2012)
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