We have Isolated tissue bath, we work on it, herbal, drug blocker....etc
If our krebs solution in organ is 10 ml, I read paper it said you can not adding more than 0.5 ml , is eqaul to 10.5 ml
If more than 0.5 all results become ERROR
Can any one help me ?
It really depends on the final concentration of your drug you need to test in the organ bath system. In general, to add less volume of any drug we tend to use a higher stock concentration of your drug. You can prepare 2, 4 an 10 fold more concentrated drugs depending on its solubility of course. Plus you need to make sure that your drug is prepared in the same krebs solution otherwise you need to add the appropriate diluent in the control baths at the same concentration to exclude a diluent effect.
Yes, the dilution can not be more than 1:20. Even if it is not wrong, it can effect the results. In our experiment, the dilution don't more than 1:50.
In a 10 ml organ bath we always added 100 microliters per evaluation, and if the evaluation is in a cummulative manner it was adjusted the concentration (taken in account if the dilutions were in log 10, medium in log 10 or quarter in log 10) in order to always added the final concentration calculated. At the end of the experiments you have to recalculated Final concentration to have real dilution to analyze results
Dear Samuel Estrada-Soto
If I have 7 doses from such drugs foe each dose we dose 100 microliters, the final volume become 10ml organ bath + 700 microliters is equal 10.7 ml , my question is this final volume 10.7 ml correct or not
My second question about:
what did you men by final concentration ?
If we prepare both 7 doses depending on our 10 ml organ bath, after we adding first dose final volume of organ bath become 10.1 ml, after adding 2nd dose become 10.2 , adding 3rd dose become 10.3 and etc
My second question I prepare all seven doses depending on 10 ml organ bath or depending on adding dose like 10.1, 10.2 . 10.3
so sorry may I cannot express carefully, becuase english language is not native.
Best regard
There is not a problem with the dilution, because, you have to adjust the quantity that you added to the organ bat, first you add 100 microliters, but, after that it is neccesary to add next adjusted dilution taken in account first dilution. I mean, if you add 100 microliters, and your dilution is in log 10, you have to add 90 microliters, and then and then.
It is advisable not to add to much fluid into the organ bath as this may significantly change the overall dilution or concentration of active compound in the organ bath. Having said this, the biological variation in response can be very high and if this is so, this consideration may not be as important but you may need to increase the number of observations to achieve a significant result.
Indeed it is best practice to minimize the dilution and deviation from the original starting volume. We target doses in the 10 uL range, with varying stock concentration to achieve the right physiological range, which is highly variable. There was one case, where the stock was not of sufficient concentration (solubility issues) and we had to lower the initial volume to achieve the correct final concentration. You need to be aware of final concentrations of your drugs while avoiding changing the concentrations/osmolarity of the buffer. Lastly, be aware that not all solvents are created equal in terms of physiology, for example ethanol can have profound physiological effects, but is often recommended as a solvent for certain drugs. Best of luck with your studies!
I'm agree with the last comment. Osmolarity is a crutial parameter ! Don't forget that ionic channels can be activated with osmolarity changes in particular with salt-based compounds ! In addition, very high concentrations ask us questions of the specificity of your molecule...
To keep added volumes as low as possible make a stock and serial dilution 1000 fold more concentrated that the start and endpoints of your concentration response curve. For a 10ml organ chamber you will add 10ul of -6 to get 1nM final bath concentration. Then 20ul of -6 to get 3x10(-9), then 7ul of -5 to get -8, then 20ul of -5 to get 3x10(-8), 7ul of -4..... and so on. That way there is minimal change in the volume of the organ chamber..... bearing in mind that you simply can't account for evaporation. As long as you're consistent.
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