Protocol Database Basics

Preparing Overnight Cultures

Make sure you have PPE and your hair is back!

Start by spraying your entire bench and groves down with 70% ethanol. If creating parent overnight cultures, only use the LB media bottle (if working with knockout strains, use LB + kanamycin). Light your bunsen burner to create a sterile environment. Obtain plates with cultured E. coli bacteria strains and obtain sterile, round bottom culture tubes with caps and label them with the name of the E. coli strain you will grow, your initials, group #, and the date. 

• You will need four culture tubes, one for each bacteria and one for each control. Label them properly.

Flame the neck of the LB media bottles and transfer 5mL of LB media into each of your labeled culture tubes. Next pass an inoculation loop through the flame to sterilize it. Allow the loop to cool then use it to pick up a bacterial colony from a culture plate. Open the correctly labeled culture tube and submerge the inoculation loop into the media before removing it and replacing the culture tube cap. 

• For the control, place the sterilized inoculation loop in your media without bacterial cells.

Place your culture tubes in the large shaking incubator set at 37°C (200rpm) and incubate OVERNIGHT.

Preparing LB Agar Plates

Measure out the desired amount of the LB miller broth powder and bacteriological agar in a weigh boat and add it to the graduated beaker. Then transfer the desired amount of DI water to the beaker and loosely place the cap on. Autoclave on the liquid cycle for 25 minutes, then cool to a temperature that makes it easy for the user to hold on to the beaker. working quickly, pour out your plates in a sterile environment. Store the plates back in the plastic sleeve they came in and once solidified, store the plates upside down in the 4° fridge.

Making LB Liquid Media

Measure out the desired amount of the LB miller broth powder in a weigh boat and add it to the graduated beaker. Then transfer the desired amount of DI water to the beaker and loosely place the cap on. Autoclave on the liquid cycle for 25 minutes, then cool to room temperature before closing the cap and storing the beaker in the fridge.

Streak Plate Method

Plate Reader Phage Lysate Curve

Initial Bacteria Growth

1. Add desired amount of the media and bacteria to their wells.

2. Let incubate until the average value of those wells are at 0.500 absorbance OD.

3. Stop plate reader.
   
   

Long Term Phage Lysate Curve

1. Add desired amount of the appropriate phage to each well.

2. Let plate reader run the program for 16-18 hours:

   1. Shake at 37­°C for 5-20 minutes.

   2. Read Plate.

   3. Keep shaking until all readings are done.

3. Export results to Excel to interpret results.