Media Recipes

General Guidelines

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. Media needs to be autoclaved the day that it is made; otherwise, it will be colonized by microbes in the air. This translates to only making media before 11am from Monday-Friday. Outside of these times, please talk to Eric.

Generally, use this method to make media.

1. Add MilliQ water to a graduated cylinder up to approximately 3/4 of the volume of media that you are making.
2. Weigh out and add in any dissolvable components of media (so, NOT agar yet).
3. Use a stir-bar and stir-plate to completely dissolve the media.
4. Fill up the graduated cylinder to ~25ml short of the final volume. Return to the stir-plate to mix in the water.
5. Adjust the pH of the media, while it is being stirred.
6. Fill to final volume; return to the stir-plate to mix in the water.
7. Put agar in bottles or flasks, if needed.
8. Measure out media into containers to be autoclaved.

Use of agar

Set the water bath to 55 degrees for keeping agar warm, before pouring it. Turn on the water baths after you are finished making media for the day, so that they are warm for when Nicole returns the media.

pH of Media

Our water pH varies greatly week to week! Be sure to calibrate and use the pH meter to adjust the pH of any media that you make. There is 5M NaOH and 5M HCl next to the pH meter to adjust the pH of media.

Media Recipes

LB (Luria-Bertani) Broth, Miller recipe

• Add 25g LB powder per 1000ml of broth.
• Amend the pH to 7.2±0.2.
• Optional: For plates, add 1.5g agar per 100ml of LB broth into the flask/bottle before autoclaving.

TSS Buffer

• 5g PEG 8000
• 1.5 mL 1M MgCl2
• 2.5 ml DMSO
• Add liquid LB to 50mL

Amend the pH to 6.5.

LeMaster-Richards Minimal Media (LeMaster and Richards, 1982)

This minimal media is used when working with our E. coli carbon mutants. This media has multiple components that need to be combined after being autoclaved separately

For 100ml / 250ml media

• 70ml / 175ml Sterile water (autoclave in the bottle that you plan to combine in these solutions)
• 25ml / 62.5ml 4x Minimal Salts:
• 5ml / 12.5ml carbon/sugar source (0.2% final sugar concentration)
• 100ul / 250ul 2M MgSO4
• 25ul / 62.5ul trace mineral solution (on chemical shelf)

Your favorite carbon/sugar source

10% (w/v) glucose is often used.

100g glucose (also called D-glucose, or dextrose) into 1000ml total volume; autoclave.

2M MgSO4

• 24.1g MgSO4 into 100ml total volume; autoclave.

4000x Trace Mineral Solution

• 811mg FeCl3 (0.1M solution)
• 81mg MnCl3 (0.01M solution)
• 68mg ZnCl2 (0.01M solution)
• 134mg [Co(NH3)6]Cl3 (0.01M solution); find chemical in Bio Superlab
• 62mg CuSO4 5H2O (0.005M solution); in Bio Superlab
• XXXX Boron (0.01M solution) [if possible to find]

Tryptic Soy Broth Agar (TSB Agar)

1. To prepare two 1.5 L solutions of medium, first obtain a 1L and 2L graduated cylinder.
2. Fill each graduated cylinder halfway with Milli-Q water (500mL; 1000mL respectively).
3. Place on separate scales and add a magnetic stirring pellet to each cylinder.
4. Weigh out Tryptic Soy Broth (30g/L = Final volume) on the scale.
5. Begin stirring the pellets. Make sure the pellet stir is controlled and not sporadic.
6. 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 

1. 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.
2. While stirring, prep two 2L Erlenmeyer flasks. Add 1.5 % agar or 22.5g for a 1.5L (Final volume) solution.
3. 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.
4. After everything has dissolved, fill each graduated cylinder to 950mL and 1950mlL respectively.
5. 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.
6. Fill to the 1L or 2L line with water from the squirt bottle. Stir for an additional 20-30 seconds.
7. Remove each magnetic stirring pellet with the retrieving rods and clean with Milli-Q water.
8. Pour 1.5 L of dissolved tryptic soy broth into each 2L Erlenmeyer flask.
9. 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
10. Place Erlenmeyer flasks in the "to be autoclaved" basket by the door.
11. Turn on water bath to 55 degrees Celsius.

• Nicole will return the autoclaved media to the water baths around 2. Blood plates need to be prepped shortly after the media cools down to 55 degrees.

Be sure to amend the pH to 7.3±0.2.

Todd-Hewitt Broth (THB)

1. To prepare 1L of THB, first obtain a 1L graduated cylinder.
2. Fill the graduated cylinder halfway with Milli-Q water (500mL).
3. Place a stir-plate and add a magnetic stirring pellet to the cylinder.
4. Weigh out Todd-Hewitt Broth (30g/L = Final volume) on the scale.
5. Begin stirring the pellets. Make sure the pellet stir is controlled and not sporadic.
6. 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 

1. 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.

1. 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.
2. While stirring, prep 5g of yeast extract to end up with a final concentration of 0.5% yeast extract.
3. 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.
4. 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.
5. Fill to the 1L with MilliQ water.
6. Remove each magnetic stirring pellet with the retrieving rods and clean with Milli-Q water.
7. Pour 100mL of THB broth into separate bottles with caps.
8. 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.
9. Place bottles 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.

Be sure to amend the pH to 7.8±0.2.

Soft agar / Top agar

This is agar that keeps melted at a lower temperature, but it is not as 'firm' as normal agar. Use this to add to bacteria to create a lawn, but without killing the bacteria.

1. Make or use Trypic Soy Broth (TSB).
2. Plan which bottles to use; be sure that they fit into the laboratory microwave.
3. Add 0.8% agar (0.8g per 100ml)
4. Put to be autoclaved.

To use, melt agar in the laboratory microwave. Then, aliquot into the necessary volume in the hood, using pre-warmed 13mm test tubes still in a heat block (at 45 degrees).

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 50°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 / 50°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.