Hello Professors,

I am trying to do an osmotic avoidance assay with NaCl / Trypan Blue solution…

Can you please tell me how to make the trypan blue solution?

P.S. - I have trypan blue in powder.

Thanks a lot.


in theory, trypan blue will go into solution in warm water up to 10mgmL-1 . But you only need it as a marker for the NaCl ring so you can use a much lower concentration than that. I suggest the following;

  1. Make up a 0.4% w/v solution of trypan blue (or buy it from SIGMA) and add 1mL of this solution when making 10mL of the 4M NaCl solution (e.g. 9mL water, 2.3376g NaCl and 1mL trypan blue solution). This produces a pale blue (but visible when pipetted as a ring) solution. N.B. store the trypan blue and the NaCl/trypan blue solutions at room temperature otherwise the trypan blue will precipitate out.

  2. Mark the inner and outer circumferences of the high osmality ring area on the assay plate by gently rotating tubes of a suitable dimension on the surface of the agar. You only need a small depression to act as a guide.

  3. Using a 100µL Gilson, add DROPWISE the NaCl/trypan blue solution in the area between the inner and outer circumferences so that you form a circle with a series of drops separated by gaps. This makes sure that the ring doesn’t overspill into the inner or outer zones of the plate.

  4. Now repeat 3. but adding drops to the gaps in the ring.

You should have a ring of the appropriate size that is fairly uniform.

Beware that some ready made solutions of trypan blue have azide added, not great for testing mnovement across your high osmolarity ring!



Thanks a lot Sir for your reply.

Just 2 questions -

How much volume of Nacl / Trypan Blue solution will I add per plate to make an Osmosis ring?

What will be the diameter of this ring?


so, amount of solution, dropwise is about 100µL…BUT this is dependent upon the ring size, could then be less. Ring size, in turn, is dependent upon the number of worms you are assaying.


for details on ring sizes and number of worms assayed.


Thanks :slight_smile: