Description

Lysogeny broth (LB), more commonly called Luria Broth,  agar plates are typically used as a growth substrate for the culture of bacteria (e.g., E. Coli). Selective growth compounds may also be added to the media, such as antibiotics. Individual microorganisms placed on the plate will grow into individual colonies, each a clone genetically identical to the individual ancestor organism (except for the low, unavoidable rate of mutation). Thus, the plate can be used either to estimate the concentration of organisms in a liquid culture or a suitable dilution of that culture, using a colony counter, or to generate genetically pure cultures from a mixed culture of genetically different organisms, using a technique known as streaking. In this technique, a drop of the culture on the end of a thin, sterile loop of wire is “streaked” across the surface of the agar leaving organisms behind, a higher number at the beginning of the streak and a lower number at the end. At some point during a successful “streak”, the number of organisms deposited will be such that distinct individual colonies will grow in that area which may be removed for further culturing, using another sterile loop.

Procedure

This recipie makes about 1 L of media, sufficient for 30 plates. Preparation time is approximately 2 hours.

To a flask of volume at least 2 L, add:

• 10 g Tryptone
• 5 g Yeast Extract
• 5 g NaCl
• 800 mL of distilled water

1. Stir the solution until everything is completely dissolved.
2. Add 400ul of 5N NaOH with stirring to adjust the pH.
3. Bring the liquid level up to 1000 ml with distilled water.
4. Add 15g of granulated agar to the liquid and stir until the agar is dissolved (about 1 minute).
5. Remove the stir bar, cover the flask with aluminum foil and autoclave for 20 min using the liquid cycle.
6. Cool down the medium until it is cool enough to be held in the hands (about 40^oC).
7. While the media is cooling, spray and wipe the bench with 95% ethanol.
8. Open a bag of sterile 3″ empty plates and place them in stacks of 10 plates with the lids up. Save the bag for later storage of the plates.
9. When the media has cooled, add the appropriate amount (reference sheet) of antibiotic to the medium and gently swirl to mix
10. At this point, you can pour the LB agar from the flask into a sterile 500-mL beaker for easier transfer onto the plates.
11. Sterilize the flask mouth by flame. If any bubbles are present in the agar, you can burst them passing the flame quickly over the LB agar solution.
12. Open the lid of the top plate and flame the beaker mouth, then pour the LB agar onto the plate until about half-way full.
13. The plates should stand at room temperature for a day before being bagged and stored. They may be used for experiments later the same day if required.
14. Label the plates for proper identification:

• LB only – single vertical black band
• LB + Ampicillin – (optional black band) single vertical red band
• LB + Chloramphenicol – single vertical blue band
• LB + Kanamycin – (optional black band) single vertical green band

1. Store the plates upside down inside the bag, to prevent them from drying out, and store at 4^oC.