Week 5 - October 14, 2022

 Thursday, October 13, 2022

Making LB Broth for E. coli 

On Thursday, we came in and learned how to make LB broth for E. coli. We were given the recipe to make 1 liter of broth and asked to make the conversions for the amounts necessary in order to make 200 ml and 100 ml of broth. The difference between them is that for the 100 ml concentration, we added Agar to the mix. The amounts necessary to make 1 liter of broth are included in the first table, Figure 1.1. Figure 1.2 contains the amounts necessary to make 200 ml of broth. In Figure 1.3, I have included the amounts necessary to make the LB broth with agar for 100 ml concentration.  

Recipe for 1 Liter of Broth

Ingredients

Amount 

Tryptone 

10 g 

Yeast Extract 

5 g

Sodium Chloride 

10 g

D.I. Water 

Up to 1 liter 

Figure 1.1

Recipe for 200 ml 

Material

Amount 

Tryptone 

2 g 

Yeast Extract 

1 g

Sodium Chloride 

2 g

D.I. Water 

Up to 200 ml 

Figure 1.2



Recipe for 100 ml with Agar 

Material 

Amount 

Tryptone 

1 g 

Yeast Extract 

0.5 g 

Sodium Chloride 

1 g

D.I. Water 

Up to 100 ml 

Agar 

1.5 g

Figure 1.3

Making the Broth: 

  1. Rinse out the necessary equipment with distilled water

    1. Graduated cylinder, scoop, beaker 

  2.  Cut squares of aluminum foil 

    1. This is where we will measure out the material (ingredients)

  3. Place one square of aluminum foil onto the scale and zero it out 

  4. Fill the graduated cylinder with about half of the distilled water you will need

  5. Begin to measure out each material on its own individual piece of foil 

  6. After the first material has been measured out, carefully remove the piece of aluminum foil from the scale

    1. Gently fold the foil (with the material on it) in half   

  7.  Put the material into the graduated cylinder with the distilled water 

  8. Repeat this process for the rest of the materials 

  9. Using another graduated cylinder, fill the first graduated cylinder with water until it reaches where it needs to be

    1. In this case, we filled it up tp 200 ml and 100 ml 

    2. When adding in the rest of the water, make sure to use the water to clean off the sides of the graduated cylinder so all of the materials incorporate 

  10.  Cover the top of the graduated cylinder with a piece of parafilm

  11.  Gently shake the cylinder until all of the materials have dissolved

  12. Once they are all incorporated, transfer the broth into a beaker that holds double the amount of liquid that you need

    1. For the 200 ml concentration, we used a 500 ml beaker

    2. For the 100 ml concentration, we used a 200 ml beaker

  13. Cover the top of the beaker with aluminum foil

  14. Put a small piece of autoclave tape on the beaker

    1. We put it on top of the aluminum foil

  15. Put it into the autoclave

For the LB Broth with Agar

  1. Steps 1-9 will be the same

  2. After the water has been added, measure out the agar

  3. Put the agar into the graduated cylinder 

  4. Continue with steps 10-15

    1. The agar will not dissolve completely while shaking it 

      1. It will dissolve in the autoclave  


Friday October 14, 2022 

Plate Pouring 

With the broth that we made on Thursday, we were taught to pour plates. We used the LB Broth with Agar 100 ml concentration. It had solidified so we boiled it on the hot plate in order to get it to its liquid state again. It boiled for about five minutes before we turned the plate off and removed the beaker from the plate. We let it cool and used the baby bottle test to determine if it was the correct temperature. To do this, we placed the bottom of the beaker onto the fleshier part of our forearm (right under the crook of our elbow). We wanted it to be warm, not too hot or too cold. If it were too hot, it could affect the antibiotic and if we let it cool down too much, it would begin to solidify. During this whole time, we had our antibiotic in ice until it was time to use it. We then began to pour the plates. 

  1. Label the bottom of the plates with

    1. Our initials 

    2. The date

    3. What the liquid is 

      1. We labeled LB Broth

    4. The antibiotic 

      1. We labeled Ampicillin 

    5. The concentration amount

      1. 50 μg/ml 

  1. Boil the broth to dissolve anything that has solidified 

  2. Do the baby bottle test to ensure it is not too hot or too cold

  3. Add antibiotic into the broth

    1. We added 100 μl/ml of ampicillin

    2. After getting the desired amount of antibiotic, we put it back into the ice 

  4. Move the plates towards the edge of the table to make it easier and faster to pour

  5. Gently lift the top of the plate up and pour the broth and antibiotic mixture into the plate 

    1. You can shake the plate around to make sure it is evenly distributed

  6. Quickly put the lid back on the plate and move onto the next

  7. Let them sit out to solidify and then store in the fridge

    1. Took about an hour to solidify



 

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