Algae Culture Manual Species for use:

1. Chaetoceros gracilis Stock from USA and Thailand (good for all stages of culture)
2. Thalassiosira weissflogii Stock from USA (especially for early zoea to help prevent Z2
syndrome)

Nutrients:

1. AGP-C (Algae Growth Promoter Complete)

Plates and tubes : 250ppm (= 0.25 ml/litre)
200 ml and 500 ml bottles : 250ppm (= 0.25 ml/litre)
2 litre and 20 litre bottles: 100ppm (= 0.2 ml/2 litres & 2 ml/20 litres)
2 mt tanks: 25ppm (= 50ml/2 mt tank)
6.5 mt tanks: 15ppm (= 97.5 ml/6.5 mt tank)

2. Sodium metasilicate solution

60 g of sodium metasilicate and make up to 2 l with distilled water in a 2 l flask for lab up to 20
litres. Filter through 0.5 micron filter paper and use 1ml/litre of water in lab;
180g of sodium metasilicate and make up to 1 litre with distilled water
Then use 200ml/2 mt tank or 650ml/6.5mt tank.

3. EDTA

10ppm of EDTA (either powder or preferably Verses 100) for all cultures = 65 g(ml)/6.5 mt
tank, 20g(ml)/2 mt tank, 0.2g(ml)/20 litre bottle.



250

Cleaning and sterilizing equipment:

Cleaning: All equipment (Petri dish, tubes, flasks, pipettes, funnels etc) must be washed with
freshwater and neutral detergent, left in a solution of 100ml HCL in 10 litres of water for 12-24
hours, and then rinsed with distilled water.

All the glassware and metal loops, once washed must be wrapped in aluminum foil and sent for
autoclaving.

Sterilizing in autoclave: All this equipment must be autoclaved for 15 minutes at 121
o
C at a
pressure of 15 bar. The autoclave is located next door to the algae lab.

1. Put water in bottom of autoclave
2. Put the tray with the equipment for autoclaving inside
3. Open the valve and turn on the gas full
4. Once steam starts escaping, close the valve and wait until pressure is 15 bar
5. Set the timer for 15 minutes and turn down heat to maintain 15 bar
6. After 15 minutes, turn off the heat and wait for pressure to go down to <5 bar
7. Open the valve to allow steam to escape
8. Once steam all escaped, lever up lid with screwdriver, unlock and remove
9. Take tray and equipment into algae lab, remove from tray, close lids or wrap with foil
and allow to cool

Checking/counting algae cells:


3
= 0.0001cm
3
=10-
3ml. If a total of α cells are counted in the space over this area-(1), the estimated number of cells
per ml in the sample is: α×10
4
. In order to obtain the algal density in the culture, get average
counts from at least 4 replicates of grids (samples): (α1+α2+α3+ α4) cells per ml.

i.e. if α = 100 then there are 100*104 = 1 million cells/ml
or if α = 10, then there are 10*104 = 100,000 cells/ml.

It is best to count in a pattern going up and down or across and back. If the algae cells are
touching the line of the box, it is to be included in a count. If a size of algae is very small and it is
only touching the outside of the borderline, do not include in a count (see Fig. 3). 3

Figure 3. Determining micro-algae cell counts Algal culture rooms

The algae culture system is divided into 4 separate rooms:

1. Algae lab: for fridge containing bactoagar plates, 10ml tubes, 20 ml tubes, 200ml flasks
and 500 ml flasks
2. Bottle room: for 2 litre flasks and 20 litres bottles


4
(If the pH drops too much, increase with drops of Tris buffer until pH is 7.2-7.5)
Then distribute from here to fill the lab glassware with:

10 ml for the 10ml test tubes,
18 ml for the 20ml test tubes,
160ml for the 200ml flasks, and
400ml for the 500 ml flasks

Then take to autoclave and sterilize for 15 minutes, remove, allow to cool and innoculate with
algal culture as follows:

Into each10 ml agar tube: put one colony of algae from a plate and leave 4-7 days
Into each10 ml dilution tube: 2 ml of algae from an agar tube and leave 4-5 days
Into each 20ml tube: put 2 ml of the best quality 10 ml test tubes and leave 4-5 days
Into each 200ml flask: put 2 * 20 ml of the best quality 20ml tubes and leave 3 days
Into each 500ml flask: put 0.5 (100ml) of a 200ml flask and leave 3 days

At full capacity we require:

105 * 10 ml test tubes, with 15 new prepared every day
250 * 20 ml test tubes, with 50 new prepared every day
60 * 200 ml flasks, with 20 new prepared every day
90 * 500 ml flasks with 30 new prepared every day
167 * 2 litre flasks with 56 new prepared every day
167 * 20 litre bottles, with 56 new prepared every day
21 * 2 mt tanks, with 7 new prepared every day
21 * 6.5 mt tanks, with 7 new prepared every day.


• Take a 1 ml glass pipette (already sterilized and wrapped in foil) and take 0.1 ml of algae
from the 10ml tube
• Again, hold the mouth of the tube briefly over the alcohol burner to sterilize it and
replace the cap of the tube
• Open the Petri dish and slowly drip the 0.1ml algal sample over the top of the agar in a
zig-zag pattern
• Close the Petri dish and seal with paraffin and place upside down on a lightly illuminated
shelf
• Mark on the Petri dish the date and species of algae with permanent market
• Leave to grow for 4-20 days, then use this plate to re-inoculate new agar 10 ml tubes Inoculating Petri dishes to 10 ml agar tubes (done once a week):

Preparing the agar test tubes:

• Retrieve test tubes from the bucket with 10% HCl solution
• Wash the tubes well with water and detergent then dry upside down in test tube rack
• Use cleaned (autoclaved) 10 ml glass pipette to transfer 10 ml of culture medium (as
above) into the test tube.
• Screw cap lightly (still loose) to tube
• Take tubes to autoclave and steam for 15 minutes as described above
• Take back to lab, tighten screw caps and leave to cool in test tube rack

Inoculating agar tubes from Plates

• Select a plate with clean, isolated colonies without bacterial infections
• Clean bench, hands and wire loop with alcohol
• Take paraffin of Petri dishes
• Place wire loop in flame until red, then take out of fire and cool in air

• Leave in test tube rack for 4-5 days until ready for inoculating 20ml tubes
• Shake test tubes once per day

Inoculating 20 ml production tubes from 10 ml dilution tubes

Preparing the production test tubes:

• Retrieve test tubes from the bucket with 10% HCl solution
• Wash the tubes well with water and detergent then dry upside down in test tube rack
• Use cleaned (autoclaved) 10ml glass pipette to transfer 20 ml of culture medium (as
above) into the test tube.
• Screw cap lightly (still loose) to tube
• Take tubes to autoclave and steam for 15 minutes as described above
• Take back to lab, tighten screw caps and leave to cool in test tube rack

Inoculating dilution tubes from agar tubes

• Select a 10ml dilution test tube with dense, high quality algae without bacterial infections
• Clean bench, hands and wire loop with alcohol
• Take cap off 10ml dilution test tube and pass mouth over flame quickly
• Using autoclaved 5ml pipette, remove 2 ml from tube
• Take top off new 10 ml test tube and add 2 ml of algae solution
• Pass top of test tube through flame quickly and reseal with cap
• Write species and date on test tube with permanent marker
• Leave in test tube rack for 4-5 days until ready for inoculating 200ml flasks
•
Shake test tubes once per day Inoculating 200 ml flasks from 20 ml production tubes

Preparing the 500 ml flasks:

• Retrieve 500 ml flasks from the bucket with 10% HCl solution
• Wash the flasks well with water and detergent then dry upside down
• Use cleaned (autoclaved) 500ml glass measuring cylinder to transfer 400 ml of culture
medium (as above) into the flask
• Close top of flask with aluminum foil
• Take flasks to autoclave and steam for 15 minutes as described above
• Take back to lab, tighten foil cap and leave to cool on bench

Inoculating 500 ml flasks from 200 ml flasks

• Select a 200 ml flask with dense, high quality algae without bacterial infections
• Clean bench, hands and wire loop with alcohol
• Take cap off a 200 ml production test tubes and pass mouth over flame quickly
• Take top off new 500 ml flask and add half of one 200 ml flask (i.e. 100ml) to each of
two 500 ml flasks
• Pass top of flasks through flame quickly and reseal with foil
• Write species and date on flasks with permanent marker
•
Place a rigid air pipe connected to airline into the bottom of the flasks and open air valve
• Leave on shelf in front of double 40 watt bulbs for 2-3 days until ready for inoculating 2
litre flasks
• Shake flasks once per day

Room 2: Bottle room: 8
This room should be kept strictly clean with constant air conditioning so that the temperature is

• 1.8 litres seawater 18 litres seawater
• 0.2 ml AGP-C 2 ml AGP-C
• 2 ml silicate solution 20 ml silicate solution
• 0.02 g (ml) EDTA 0.2 g (ml) EDTA
• 0.2 ml Vitamin B solution 2 ml Vitamin B solution
+ +
• 0.25 litres algae (0.5*500ml flask) 2 litres algae (1*2l flask) 1000 liter outside tank area

This area should be kept strictly clean with transparent plastic roof tiles to allow light to pass
inside the room. The unit should also have fluorescent tubes left on 24 hours per day for this high
volume algae culture. Temperature is ambient, but should not be allowed to rise too high. Try to
maintain at 24-28
o
C by opening windows or installing fans in the hot season.

After each harvest of the tanks and before refilling, the tanks are scrubbed with scrubbing
brushes, soap and freshwater. The a 15-20 litre bucket containing 10 litres of water and 100ml of
HCL is used to wash the entire inner surface of the tanks with the use of a long handled brush.
The workers must wear rubber boots and gloves during this procedure. After this, the tanks are
flushed again with clean seawater and are then ready for refilling.

9

Water for this room is seawater from the algae culture reservoirs, via the cartridge filtration and
35gpm UV system.

The 2 mt tanks are then filled up (to 1.84 mt) with seawater. Then liquid chlorine is added at 184

brushes, soap and freshwater. The a 15-20 litre bucket containing 10 litres of water and 100ml of
HCL is used to wash the entire inner surface of the tanks with the use of a long handled brush.
The workers must wear rubber boots and gloves during this procedure. After this, the tanks are
flushed again with clean seawater and are then ready for refilling.

Water for this room is seawater from the algae culture reservoirs via the cartridge filtration and
35gpm UV system.

The 6.5-mt tanks are then filled up (to 4.5 mt) with seawater. Then liquid chlorine is added at 450
ml/tank

In the morning, the chlorine is neutralized with 180ml of sodium thiosulphate solution/6.5 mt
tank.

After checking with the chlorine test kit to ensure that the chlorine is all gone, the tanks are
inoculated with nutrients and then the starter culture of algae added. This is 1 *2 mt tank/6.5 mt
tank, giving a total of 6.5 mt.

10

11

Additions for these inoculations are as follows:

Per 1 mt of tank 6.5 mt tank

• 900 litres seawater 4.5 mt seawater
• 15 ml AGP-C 100ml AGP-C
• 100 ml silicate solution 650 ml silicate solution
• 10 g (ml) EDTA 65g (ml) EDTA


All the air-stones and airlines used should be washed in soap and water after each use and then
wiped with a cloth with 10% HCL solution (100 ml Acid in 10 litres water) before reinstalling
into the tanks for the next stocking.

All the floors and walls of each room should be washed down daily with chlorinated water (20g
chlorine in 10 litres of water, using a brush. All surfaces in the room 1 & 2 should be wiped down
with alcohol on a cloth daily.

Once per month, all water and air tubes should be filled with 10% solution of HCL (100ml acid in
10 litres water), left for 12 hours and then washed out with clean freshwater.