Dear Hamed, I dont think you need to sterilize everything separately, except urea solution which is not suitable for autoclaving and better needs be filter sterilized. Before you decide how to make the required solution, may I please ask what exactly the pH you are intending to have is and, secondly, how important is it for you have a more stable pH? The pH 9.0 is quite high and in the upper most limit of the buffering range of Tris (pKa of 8.1). Are you sure you need pH 9.0? If yes then go ahead. Using Tris base will give you much higher pH >10 so you will have to add a lot of conc HCl to bring it down to 9.0.

If you need a very roburst buffering strength then the best option of using a combination of Tris base and Tris HCl. You can have further information of this link….

http://www.sigmaaldrich.com/Graphics/Supelco/objects/4800/4709.pdf

Also the working temperature is important. Please keep in mind that as Tris solutions decrease in temperature from 25°C to 5°C, the pH value increases an average of 0.03 units per °C. As the solution increases in temperature from 25°C to 37°C, the pH decreases an average of 0.025 units per °C.

Tris solutions can be autoclaved (121°C, 15 psi, 15 minutes).

So here is how you can go ahead with your preparation:

1. Add weighed amount of Tris in a 1 L glass bottle.

2. Pour in 900 ml water.

3. Mix and adjust pH to 9.0 with conc HCl.

4. Add weighed amount of nutrient agar in the above solution of Tris (this would save time and effort of autoclaving both the Tris solution and agar separately).

5. Bring volume up to 980 ml with more water (remaining 20 ml will be for urea solution as urea is highly soluble in water viz 1.08 g/ml. Moreover, it is easy to handle 20 ml while filter sterilizing than 50 ml).

6. Autoclave the mixture.

7. Prepare separately the urea solution (20 ml) and warm by keeping in 37°C water bath or , if in hurry, by microwaving briefly to warm a bit. You may skip this step but this will prevent any lumps formation by adding cold urea solution to warm autoclaved agar and Tris mixture.

8. After autoclave, bring the temperature of the bottle down to near warm or by leaving the bottle at 50-55°C until it comes down to that temperature or, if in hurry, by placing in running water under a tap.

9. When just warm (feel by the back of your hand and it should appear just ok not too hot), wipe exterior of the bottle down and bring it near the flame.

10. Carefully add filter sterilized urea solution and mix well.

The change in pH should not be a problem as with pH 9.0 you are still in buffering zone of Tris (7.1 to 9.1).

Hope that helps. Cheers.

pH will change a bit after autoclave. If your work requires accurate pH, you have to adjust after autoclave. Urea solution will not change pH if it is pure.

pH of Tris-HCl buffer doesn't change much after autoclave either

Its ok if you are preparing 1% of agar solution only. You could add more agar if you want a solid medium.

My concern for asking the question is pH adjustment , should it be adjusted after mixing the tris buffer solution with other ingredients or before that? Because i prepared according to what i explained above (i adjusted the pH before mixing with ingredients) but no growth occured. I measured the pH and found that it is 7.95 instead of 9. What can be the problem?

Dear Hamed, I dont think you need to sterilize everything separately, except urea solution which is not suitable for autoclaving and better needs be filter sterilized. Before you decide how to make the required solution, may I please ask what exactly the pH you are intending to have is and, secondly, how important is it for you have a more stable pH? The pH 9.0 is quite high and in the upper most limit of the buffering range of Tris (pKa of 8.1). Are you sure you need pH 9.0? If yes then go ahead. Using Tris base will give you much higher pH >10 so you will have to add a lot of conc HCl to bring it down to 9.0.

If you need a very roburst buffering strength then the best option of using a combination of Tris base and Tris HCl. You can have further information of this link….

http://www.sigmaaldrich.com/Graphics/Supelco/objects/4800/4709.pdf

Also the working temperature is important. Please keep in mind that as Tris solutions decrease in temperature from 25°C to 5°C, the pH value increases an average of 0.03 units per °C. As the solution increases in temperature from 25°C to 37°C, the pH decreases an average of 0.025 units per °C.

Tris solutions can be autoclaved (121°C, 15 psi, 15 minutes).

So here is how you can go ahead with your preparation:

1. Add weighed amount of Tris in a 1 L glass bottle.

2. Pour in 900 ml water.

3. Mix and adjust pH to 9.0 with conc HCl.

4. Add weighed amount of nutrient agar in the above solution of Tris (this would save time and effort of autoclaving both the Tris solution and agar separately).

5. Bring volume up to 980 ml with more water (remaining 20 ml will be for urea solution as urea is highly soluble in water viz 1.08 g/ml. Moreover, it is easy to handle 20 ml while filter sterilizing than 50 ml).

6. Autoclave the mixture.

7. Prepare separately the urea solution (20 ml) and warm by keeping in 37°C water bath or , if in hurry, by microwaving briefly to warm a bit. You may skip this step but this will prevent any lumps formation by adding cold urea solution to warm autoclaved agar and Tris mixture.

8. After autoclave, bring the temperature of the bottle down to near warm or by leaving the bottle at 50-55°C until it comes down to that temperature or, if in hurry, by placing in running water under a tap.

9. When just warm (feel by the back of your hand and it should appear just ok not too hot), wipe exterior of the bottle down and bring it near the flame.

10. Carefully add filter sterilized urea solution and mix well.

The change in pH should not be a problem as with pH 9.0 you are still in buffering zone of Tris (7.1 to 9.1).

Hope that helps. Cheers.

Urea warming leads to generation of ammonia https://www.google.de/url?sa=t&source=web&cd=2&ved=0CCgQFjAB&url=http%3A%2F%2Fwww.inchem.org%2Fdocuments%2Fsids%2Fsids%2F57136.pdf&ei=cgFgUvq4NsPNtAae5oCoDg&usg=AFQjCNEV2SdbFnr8aMcfWd5ZmGH9qf3g8Q&bvm=bv.54176721,d.Yms

Ok this media to grow what

Casadei Nicolas, yes I agree with you but the problem is that Hamed wants to prepare nutrient agar that has urea as one of the components. Since agar solidifies at around 45C so we are left with no option except that we may skip pre-warming of urea solution before adding to molten nutrient agar. Cheers.