Media Recipes

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Media Recipes

Use of agar

LB (Luria-Bertani) Broth, XXXX recipe

Be sure to amend the pH to 7.2±0.2.

Tryptic Soy Broth Agar (TSB Agar)

Important: Nicole comes to the lab to collect materials to autoclave around 10:30 a.m. Thus, make sure to fully complete this procedure and leave the media near the autoclave section before she arrives.


1. Turn on water bath to 50 degrees Celsius.

2. To prepare two 1.5 L solutions of medium, first obtain a 1L and 2L graduated cylinder.

3. Fill each graduated cylinder halfway with Milli-Q water (500mL; 1000mL respectively).

4. Place on separate scales and add a magnetic stirring pellet to each cylinder.

5. Weigh out Tryptic Soy Broth (30g/L = Final volume) on the scale.

6. Begin stirring the pellets. Make sure the pellet stir is controlled and not sporadic.

7. Using a spoon, slowly add 30g for the 1L graduated cylinder and 60g in the 2L cylinder.

       Important: Make sure to be gentle at this step or the powdered broth will form a “dust cloud” and be deposited throughout the lab 

8. Stir for 10-15 minutes. During this step you can slowly add Milli-Q water to the graduated cylinders to speed up the dissolving process.

9. While stirring, prep two 2L Erlenmeyer flasks. Add 1.5 % agar or 22.5g for a 1.5L (Final volume) solution.

10. To remove excess broth deposited on the sides of the graduated cylinders, use the magnetic stir bar retriever to dissolve any clumps into solution. Place the bar over the cylinder and clean with Milli-Q water from the squirt bottle. This ensures that any broth present on the stir bar retriever is placed back into solution.

11. After everything has dissolved, fill each graduated cylinder to 950mL and 1950mlL respectively.

12. Use the pH meter and the 5M NaOH solution (careful, a very strong base!) to adjust the pH of both graduated cylinders to 7.3 ± 0.2. Add the 5M NaOH in 50ul aliquots; this should take approximately 200ul per liter. Be sure to do this while the media is stirred.

13. Fill to the 1L or 2L line with water from the squirt bottle. Stir for an additional 20-30 seconds.

12. Remove each magnetic stirring pellet with the retrieving rods and clean with Milli-Q water.

13. Pour 1.5 L of dissolved tryptic soy broth into each 2L Erlenmeyer flask.

14. Add autoclave tape to the tin foil caps covering the erlenmyer flasks. Make sure to also add a piece of tape labeled with the lab and the type of media

15. Place Erlenmeyer flasks in the "to be autoclaved" basket by the door.

  • Nicole will return the autoclaved media to the water baths around 2. Blood plates need to be prepped shortly after the media warms to 50 degrees.

Todd-Hewitt Broth (THB)

Be sure to amend the pH to 7.8±0.2.

2. To prepare 1L of THB, first obtain a 1L graduated cylinder.

3. Fill the graduated cylinder halfway with Milli-Q water (500mL).

4. Place a hot plate and add a magnetic stirring pellet to the cylinder.

5. Weigh out Todd-Hewitt Broth (30g/L = Final volume) on the scale.

6. Begin stirring the pellets. Make sure the pellet stir is controlled and not sporadic.

7. Using a spoon, slowly add 30g for the 1L graduated cylinder.

      Important: Make sure to be gentle at this step or the powdered broth will form a “dust cloud” and be deposited throughout the lab 

8. Stir for 10-15 minutes. During this step you can slowly add Milli-Q water to the graduated cylinders to speed up the dissolving process.

9. To remove excess broth deposited on the sides of the graduated cylinders, use the magnetic stir bar retriever to dissolve any clumps into solution. Place the bar over the cylinder and clean with Milli-Q water from the squirt bottle. This ensures that any broth present on the stir bar retriever is placed back into solution.

10. While stirring, prep 5g of yeast extract to end up with a final concentration of 0.5% yeast extract.


11. After everything has dissolved, fill to the 900mL line with MilliQ water. Now, you may add the yeast extract and repeat steps 8 and 9 as needed. Fill only to the 950mL line though.

12. Use the pH meter and the 5M NaOH solution (careful, a very strong base!) to adjust the pH of the graduated cylinders to 7.8 ± 0.2. Add the 5M NaOH in 50ul aliquots; this should take approximately 200ul per liter. Be sure to do this while the media is stirred.

13. Fill to the 1L with MilliQ water.

12. Remove each magnetic stirring pellet with the retrieving rods and clean with Milli-Q water.

13. Pour 100mL of THB broth into separate beakers with caps.

14. Add autoclave tape to each covered beaker. Make sure to also add a piece of tape labeled with the lab and the type of media.

15. Place beakers in the "to be autoclaved" basket by the door. If running an overnight culture, also autoclave several test tubes and a rack.

  • THB needs to be stored away from direct sunlight. Therefore, once Nicole returns with the autoclaved media, make sure to place the beakers into the "media" cabinet.

Soft / Top agar

Blood plates

1. Create TSB agar as described above, but reduce the final volume of media by 5%. This translates into 1425ml total for 1.5L of media; 950ml for 1L; and 1900ml for 2L.

2. Be sure to amend the pH to 7.3±0.2.

3. After autoclaving, put in a 55°C water bath for several hours, so that the media is at 55°C.

4. Turn on the small water bath and set for 55°C. Add the bottle of defibrillated sheep blood to warm up; gently rotate/shake the blood to ensure there is not a pellet of cells at the bottom.

5. When everything is at 55°C, add 5% blood cells to the agar in the biological hood using sterile technique and a serological pipet. This is 75ml blood into 1425ml agar for a final volume of 1.5L. Or, 50ml blood into 950ml agar for a final volume of 1L.

6. Gently mix the agar+blood without causing bubbles.

7. Pour plates; feel free to return the flask to the water bath if you are anxious that the agar is becoming too cold. If the agar solidifies in the flask, there is nothing that can be done — we cannot re-melt it after blood is added.

8. To decontaminate the flasks, add 1 spoonful of Alconox; fill with water and let sit for at least 1 hour. Wash out the flask repeatedly in water to wash off the Alconox detergent.