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(Source: M. Westerfield from Zebrafish Book 5th Edition)

Several different types of water are used for procedures described in the following sections.  These solutions have been adapted to balance ease and cost of production with the changing needs of zebrafish during their life cycle.

In general, adults can be maintained in tap water, conditioned by letting it set.  This depends critically on the quality of the local water source and on the demands for embryo production.  Poor water quality will adversely affect the health of the fish, their susceptibility to disease, and their breeding potential.  If there is any question about water quality, deionized or distilled water should be used to which a small amount of salts and minerals are added.  If an adequate supply of deionized water is unavailable, a closed‑ system that recirculates the water after purification can be used.

Embryos and young larvae have stricter requirements and should be raised in egg water.  Embryos removed from their chorions require additional calcium and should be maintained in embryo medium.

System (or tank) water:  Water out of the facility's water system.  This water is dripped into clean tanks and is used for setting up pairwise crosses.
Egg water:  Used for in vitro fertilization and raising young embryos.

Stock salts:  40 g "Instant Ocean®" Sea Salts added to 1 L distilled water

Egg water: 1.5 ml stock salts added to 1 L distilled water = 60μg/ml final concentration.

Embryo medium:  Do not confuse with "egg water" above.  Use this medium for handling dechorionated embryos and storing young embryos in dishes.  This is basically 10% Hank's with full strength calcium and magnesium.

Embryo medium:  1.0 ml Hank's Stock #1, 0.1 ml Hank's Stock #2, 1.0 ml Hank's Stock #4, 95.9 ml ddH2O, 1.0 ml Hank's Stock #5, 1.0 ml fresh Hank's Stock #6.  Use about 10 drops 1 M NaOH to pH 7.2     

Hank's solutions: Hank's solutions can be made from stock solutions (kept refrigerated, they will last for several months).  A premix of the salts can be stored in the refrigerator for several weeks.  Sodium bicarbonate does not store well, so it is made up fresh each time Hank's solution is made.

Full Strength Hank's: 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4  , 1.3 mM CaCl2  , 1.0 mM MgSO4  , 4.2 mM NaHCO3

Hank's Stock Solutions:
Stock #1  8.0 g NaCl 0.4 g KCl in 100 ml ddH2O
Stock #2  0.358 g Na2HPO4 Anhydrous,  0.600 g KH2PO4 ,   in 100 ml ddH2O
Stock #4  0.72 g CaCl2  in 50 ml H2O
Stock #5  1.23 g MgSO4×7H2O in 50 ml ddH2O
Stock #6  0.35 g NaHCO3 10.0 ml ddH2O

Hank's Premix:
Combine the following in order:
10.0 ml Solution #1, 1.0 ml Solution #2, 1.0 ml Solution #4, 86.0 ml ddH2O, 1.0 ml Solution #5
Store Hank's Premix in the refrigerator along with the Hank's solutions.

Final Hank's:
9.9 ml Hank's Premix, 0.1 ml fresh Stock #6


  1. Anonymous

    Am I reading this incorrectly or are the proportions stated differently for Embryo Media and Embryo Media Premix?  It seems to say 1 : 0.1 : 1 : 1 of #1, #2, #4, #5 for the Embryo Media, but then says 10:1:1:1 for these same components when describing the premix.  The amount of Solution #2 seems to be off in one of them.

    1. "Embryo medium pre-mix"?  I don't see this anywhere in the protocol.  Please explain - maybe I'm missing something.  

    2. Ah - maybe the issue is that embryo medium is almost, but not exactly the same as 10% Hanks.  With respect to calcium and magnesium (from stock solutions #4 and #5 respectively), embryo medium is 100% Hank's.  

  2. Anonymous

    Stock #2  0.358 g Na2HPO4 Anhydrous,  0.600 g KH2PO4 ,   in 100 ml ddH2O

    It is a wrong recipe???0.358 g Na2HPO4 Anhydrous should be 0.358 g Na2HPO4-12H2O???Because embro medium is basically 10% Hank's with full strength calcium and magnesium, the concentration of Na2HPO4 should be 10%*0.25 mM.

    So where is the wrong???