LB media preperation (500mL;~25 plates)
Introduction
In order for bacteria to be successfully cultured, they must be grown in the appropriate media. LB, also known as Lysogeny broth, is a nutrient rich broth that is a standard for culturing Escherichia coli, as it allows for quick growth and high yields. Therefore, the proper preparation of LB will be crucial to maintaining our bacterial stock throughout the summer. Furthermore, addition of agar to LB broth creates a gel for bacteria to grow upon, and is therefore used for plating bacterial cultures on petri dishes.
** WATCH THESE VIDEOS BEFORE ATTEMPTING THIS PROTOCOL***
Making LB agar: https://www.youtube.com/watch?v=74Ndr9AVowM
Pouring plates: https://www.youtube.com/watch?v=X4L16Vu5MSk
BASIC TERMINOLOGY AND CONCEPTS
Agar vs. Agarose: Agar is used for making petri plates to culture organisms, while agarose is used for making gels, in the likes of SDS-PAGE and gel electrophoresis
SAFETY PRECAUTIONS
SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets.
PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.
Autoclave: The autoclave should only be handled by leads and managers. ***Note that any autoclaved materials may still be hot and should therefore be handled with caution. Be careful not to burn yourself.
Materials
- Reagents
- 5g Bacto-tryptone
- 2.5g yeast extract
- 5g NaCl
- 7.5g agar (Only necessary if making LB agar plates)
- 500mL of dH2O (distilled water)
- Materials
- 1L pyrex bottle
- 1L graduated cylinder
- filter paper and scupula
- Stack of sterile plates (this protocol makes approximately 25)
- Bunsen Burner/Ethanol burner
- 70% EtOH wash bottle
- Paper towels/wipes
Procedure
- Part 1: Making the LB broth
- This step can be carried out at a regular lab bench.
- Obtain a clean 1L pyrex bottle
- Obtain a graduated cylinder with 500mL of dH2O and add to the bottle. Record the amount added
- Using filter paper, separately measure out 5g of NaCl, 5 g of Tryptone, and 2.5g of yeast extract on a scale and add them to the bottle. Swirl the bottle in a circular motion to mix. Remember to recalibrate your scales in between measurements.
- If you are making LB agar plates, weigh and add 7.5g of agar and swirl to mix. Record the amount added.
- Note the contents do not necessarily need to be completely in solution before autoclaving
- Part 2: Autoclaving ***ONLY DONE BY MANAGERS AND LEADS***
- Lightly seal the top of the beaker with aluminium foil, and label the beaker with autoclave tape. Include LB (agar)- your name- date- media number- IGEM.
- ** Unless you have been trained to use the autoclave, you will not be conducting the following steps 6-9.
- Use appropriate transportation protocols to bring the LB bottle into the autoclave room. Remember to store the beaker in an autoclavable basin, in case of spills.
- Check the water level on the autoclave, if necessary. Autoclave on the liquid setting for approximately 20 min.
- ** The contents of the beaker will be hot after autoclaving, therefore take the necessary measures to prevent burns. **
- After auoclaving, allow the LB media to cool to 55oC before handling
- Use lazer thermometer to check the temperature of the glass.
- The LB broth can be stored in sterile conditions at room temperature, and should be good for 3-4 months. Flame the lip of the bottle each time the LB is used. If the LB contains antibiotics: store in a -4oC freezer.
- However it is not recommended to store LB with antibiotics as the antibiotics will degrade over time
- Part 3 Pouring the plates (if you are making LB agar)
- *** While pouring the plates it is crucial to maintain a sterile environment. This should be done in room WB 303, with a sterile environment provided by a lit bunsen burner.
- Note: While you are waiting for the autoclave, steps 1-3 can be done in the meantime, in addition to the clean up from Part 1.
- Sterilize the workspace with 70% EtOH before depositing your materials. Light the bunsen burner.
- Obtain a stack/roll of empty plates. The plates should still be in their plastic sleeve/wrapping, as they should be sterile. Dont throw out the wrapping as it can be used to store the plates. It is essential that you minimize any chance of contaminating the plates. Make sure that you open the package at the top and expose the plates as minimally as possible.
- Note that this protocol makes approximately 25 plates.
- Once you take the plates out, store them upside down on your lab bench. Label the plates with "your name-IGEM 2016-date prepared- designated stripe* (if using antibiotic) - media number. Once labelled, you may stack the plates to free up workspace.
- Allow the LB media to cool before pouring. The LB will start to settle at about 30oC.
- If you are preparing selective media, add antibiotic to the mixture. Use the recommended antibiotic concentrations (see table below). Swirl the flask in a circular motion to mix. If you dont know whether or not you are preparing selective media, ASK.
A | B | |
1 | Antibiotic | Recomended concentration |
2 | Chloramphenicol (CAM) | 25 μg/mL |
3 | Ampicillin (AMP) | 100 μg/mL |
Table1
- One stripe along the sides corresponds to CAM, two stripes corresponds to AMP
- Take an empty plate and open it slightly. You do not need to open it all the way to pour the agar. When pouring the agar, pour until 2/3 of the plate has been covered, or approximately half of the plate has been filled when viewed from the side. Pour the agar slowly, to prevent the formation of bubbles. Swirl the plate in a circular motion to distribume the media evenly on the plate.
- If you pour too much LB, you will only be able to produce <25 plates. If you don't pour enough media, it may minimize bacterial growth.
- After pouring, set the plates to cool in stacks of 4-5, as this takes up less space. Dont stack plates too high - we want to minimize the risk of spills.
- Rinse the pyrex bottle with water before the remnants solidify and become hard to remove.
- Allow the plates to cool for at least 20 minutes until the agar has solidified. Flip the plates to prevent condensation forming on the agar. The plates can then be stacked and stored in plastic bags (ideally re-use the plastic bags that the plates came in.)
- The LB agar plates should be stored in a 4oC freezer, should be good for 1-2 months.
- Leaving the lab
- Prior to leaving the lab, you should:
- - Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual. - Wipe down your workspace. - Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.
- References
- Addgene: Protocol – Making LB Agar Plates for Bacteria. From: https://www.addgene.org/plasmid-protocols/bacterial-plates Department of Ecology and Evolutionary Biology, UCLA. Making LB Agar Plates. From: https://www.eeb.ucla.edu/Faculty/Barber/Web%20Protocols/LB%20Agar%20Plates.pdf U of T iGEM Luria Broth (LB) protocol Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli Physiology in Luria-Bertani Broth . Journal of Bacteriology. 2007;189(23):8746-8749. doi:10.1128/JB.01368-07.
- Changelog
- Created 5/16/2016
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