Option two would indeed keep the osmolarity the same.  However most people prepare a thousand fold greater stock solution than the concentration required in the bath and therefor the volume of any drug diluent is kept to a minimum.  i.e if a bath concentration of 10 uM (micromolar) is required then an addition of 20 ul (microlitres) of a 10 mM (millimolar) stock would be added to the 20 mL bath.

Thank you so much Dr. Emma Moss

Jackapun, 

I have a couple of comments

1) I would not recommend the addition of ascorbic acid  as it may interfere with your measurements.  Just keep you concentrated agonist solutions (pure H2O) in ice- for the whole day. Dilute them about 1000 times directly in organ chamber ( eg add 20ul or less to the bath).

2) Sodium nitroprusside is light sensitive- protect the stock solution from light!

I agree with Emma, addition of small volume (10 or 20 ul) to a 20 mL is not going to change the osmolarity of  the solution that you are going to observe through the effect on isolated organ.

Thank you every body for comments

Of course, the whole volume 0.11 uL adding to organ bath is not change the osmolarity. Then I chose to dissolve NE or ACh in 0.01% ascorbic acid (ascorbic acid raise vascular relaxation 0.1 mM or more (K. de Saram et al. 2002. British Journal of Pharmacology)).

Usually we dissolve norepinephrine and acetylcholine on 0.001 N HCl, in 10-2 M which in organ bath is 10-4 (when using 100 microliter) also keep in aluminium foil and to norepinephrime 10 mg of ascorbic acid are prefered for 10 ml solution (10-2 M).

I wouldn't add ascorbate. Make your reagents fresh every day.

Like Emma said, make your stock solution a thousand fold more concentrated than the final concentration you want in your organ chamber. If you are doing a concentration response curve then do 1 in 10 serial dilation from your stock, so that you have -2, -3, -4, -5, -6 solutions. So then 20 ul of -6 will give you 1nM (-9) in your chamber, then add 40 ul of the same -6 solution to give you 3x10(-9) (approximately half log), then add 14ul of the next serial dilution (-5) to give you -8 in the bath, 40 ul of -5 to give you 3x10-8, 14 of -4 to give you -7, and so on. Working with small amounts as possible will not change the volume within the organ chamber too significantly (bearing in mind that you cannot really account for evaporation either.