Protocols for the Sterilization of water using Sodium Hypochlorite (Bleach) or Acid

The Easy Bleach Method
For sterilizing seawater of average cleanliness, this protocol will yield water of acceptable sterility and quality for most microalgae (greenwater) and larval culture.

**Be sure to familiarize yourself with proper chemical handling techniques before attempting these procedures. Always wear protective goggles and gloves when handling any chemicals**

1.  Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add 0.5ml unscented laundry bleach per liter of water.

3.  Seal the vessel and swirl the media so that all internal surfaces are wet.

4.  Allow the vessel to sit at room temperature for at least 6 hours, preferably 12-24.  The vessel may be stored for long period of time with the bleach inside, provided the vessel remains uncontaminated.

5) Prepare a 1M stock solution of Sodium Thiosulfate.  The most commonly available crystalline form is the pentahydrate, and should be added at 248 grams per liter.  This solution may be filter sterilized or autoclaved to ensure sterility.

6.  To de-chlorinate the water, add 0.1ml of 1M sodium thiosulfate per liter of water.

7.  Aerate at least 2 hours.

8.  Confirm that no residual chlorine exists before using the medium.  Dip tests for pools work well, as do the DPD reagent available in convenient liquid or powder pillow-packets.

Acid Sterilization

**If you are not familiar with the safety hazards associated with handling concentrated acids, DO NOT attempt this method**

1. Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add sufficient hydrochloric acid or muriatic acid to reduce the pH of the solution to 3.6.  The amount needed will vary depending on the concentration of the acid stock, the initial pH of the water, and its buffering capacity, which may also be affected by residual calcium deposits in the vessel.  A good starting point for muriatic acid (about 32% concentration) is 2ml/L.

3.  Confirm pH with a pH meter or narrow range test strips.

4.  Swirl the sterilized solution so that all internal surfaces are moistened.

5.  Allowing a sufficient sterilization time (2-4 hours).

6.  Return the pH to normal with sodium bicarbonate (baking soda).  A good starting point is 1/8 tsp/L.

6.  Confirm pH is appropriate for culture with a pH meter or narrow range test strips.