A - Determination of active chlorine content in commercial
grade bleach (hypochlorite solution)
Active chlorine will release free iodine from potassium iodide
solution at pH 8 or less. The released iodine is titrated with a standard
solution of sodium thiosulphate, using starch as the indicator.
- acetic acid (glacial, concentrated),
- potassium iodide (KI) crystals,
- sodium thiosulphate (Na2S2O3 x 5 H2O,
i.e. 248.18 g/mol) 0.1 N standard solution:
- dissolve 25 g of sodium thiosulphate in 1 l of freshly boiled distilled
- improve its stability by adding 0.1 g of Na2CO3;
- store in a clean glass bottle in the dark,
- starch indicator solution:
- mix 5 g starch with a little cold water and grind in a mortar, pour into
1 l of boiling distilled water, stir and let settle overnight;
- use the clear supernatant preserved with 1.25 g salicylic acid and store
in a dark bottle.
- dissolve 0.5 to 1 g KI in 50 ml distilled water,
add 5 ml acetic acid,
- add 1 ml from sample to be checked,
- titrate away from direct sunlight: add 0.1 N thiosulphate
from a burette until the yellow colour of the liberated iodine is almost
disappearing, add 1 ml starch solution and titrate until the blue colour
- 1 ml of 0.1 N thiosulphate equals to 3.54 mg
B - Preparation of a disinfecting 500 ppm hypochlorite
solution from commercial grade bleach
- Check the content of free chlorine in the available bleach following the
- Say the content found is 6.5% free chlorine (typical range: 5 to 15% in
- A free chlorine 500 ppm solution means 500 ml of free chlorine in 1 000
000 millilitres, or 0.5 ml in 1 litre.
- 0.5 ml of free chlorine are found in 7.7 ml of 6.5% bleach (0.5/0.065);
- to prepare say 50 l of a 500 ppm hypochlorite solution add 385 ml (7.7
x 50) of 6.5% active chlorine bleach to 50 l of water.
- This is a practical method, not a stoichiometric preparation.
- When using a disinfecting hypochlorite solution for a prolonged time,
it becomes essential to check regularly its active chlorine content
C - Chemical methods to sterilize seawater (for algal,
rotifer and artemia cultures, both strain and mass level)
- The standard disinfecting concentration for culture seawater is 10 ppm
of free chlorine and the corresponding safe concentration of sodium thiosulphate
to deactivate residual chlorine is 12 ppm.
- Say the content of free chlorine in the available bleach is 6.5%.
- A free chlorine 10 ppm solution means 10 ml of free chlorine in 1 000 000
millilitres, or 0.01 ml in 1 litre.
- 0.01 ml of free chlorine are found in 0.15 ml of 6.5% bleach (0.01/0.065);
- to disinfect each litre of culture water add 0.15 ml of 6.5% active chlorine
bleach (final chlorine concentration: 0.01 ml/l),
- provide a gentle aeration for 30 minutes to mix well, then let stand overnight;
- add 12 mg of sodium thiosulphate to each litre of seawater (to have a 12
ppm solution, i.e. 12 mg in 1 000 000 mg) and aerate strongly for one hour,
- check for residual chlorine and use only if no blue stain develops.
- For practical purposes always prepare and keep at hand a relatively large
stock of disinfected seawater, say 100 l, to be used to fill small volumes
D - Identification of active chlorine residue
The same process indicated in the section A can be repeated in
a simplified way for a quick check of residual active chlorine in water. To one
ml sample add 1 ml of KI solution, 1 ml of 0.1 N thiosulphate solution and 1 ml
of starch indicator. If a blue colour develops it is a proof of presence of
active chlorine residues.
E - Preparation of disinfecting 10% (v/v) hydrochloric acid
- commercial grade hydrochloric acid (HCl or muriatic acid)
- tap water
- add 100 ml of HCl to 900 ml of tap
- fill the vessel first with water to drive out the acid fumes
- pour the acid without splashing to avoid acid splashes
- wear protective gloves, glasses and cloths when handling concentrated
- avoid breathing acid fumes
F - PREPARATION OF LUGOL SOLUTION
Lugol is a fixative staining solution for easier counting of
brine shrimp nauplii.
- solution A: dissolve 50 g potassium iodide (KI) and 25 g iodine (I2)
in 100 ml boiling water
- solution B: 25 g sodium acetate (NaOAc) dissolved in 250 ml water
- when solution A cools, mix the two solutions and store in a cool, dark
- Add a few drops to each 1-ml sample of rotifers or brine shrimps