Lysogeny broth (LB) Preparation

LB is the most widely used bacterial culture medium but its roots lie in the area of genetics of bacteriophage. Guiseppi Bertani developed the LB recipe when he was trying to increase the amount of plaque that formed on an indicator strain of Shigella (Bertani 1952). As per Bertani, LB has been often misinterpreted to mean “Luria Broth”, “Luria-Bertani” medium, or “Lennox Broth”; however the original acronym stood as “Lysogeny Broth” (Bertani, 2004). The agar version of the medium is identified as LA however, it is frequently called LB. While initially developed to study bacteriophage in addition to Shigella growth, LB subsequently became the most preferred medium to grow Escherichia bacteria and other species of the enteric.

Introduction

To allow bacteria to be successfully cultivated the bacteria must be grown in the correct medium. LB is also known as Lysogeny broth is a nutritious broth that is the common method for cultivating Escherichia coli because it permits rapid growth and yields. So, the correct preparation of LB is essential for maintaining our bacterial stock all through the summer. In addition, adding agar into LB broth forms the perfect environment for bacteria to thrive on which is why it is used to place bacterial strains on petri dishes.

Materials Required

Reagents

Equipment

• 1L Pyrex bottle
• 1L graduated cylinder
• Filter paper and scoopula
• Stack of sterile plates (this protocol makes approximately 25)
• Bunsen burner/ethanol burner
• 70% EtOH wash bottle
• Paper towels/wipes

Preparation of Lysogeny broth (LB) 

It is the Preparation of Lysogeny broth (LB) is broken down into distinct steps like;

Note: This procedure will yield 500mL of broth or about 25 plates.

Step 1: Preparation of LB broth

1. Make sure you have one clean 1L bottle of pyrex
2. Get a graduated cylinder that contains 500mL of dH2O. Add it in the bottle. Keep track of the amount you that was added.
3. Filter paper to take 5g of NaCl and 5g of Tryptone as well as 2.5g in yeast extract using the scale. Add them into the bottle. Make the circular motion in order to mix. Be sure to calibrate your scales every time you take measurements.
4. If you’re creating LB plates for agar weight and then add 7.5g of agar. Then swirl to mix. Note the amount of agar of agar added.

Note: This procedure can be performed at any regular laboratory bench

Note that the contents do not necessarily require to be fully in solution prior to autoclaving.

Step 2: Autoclaving

1. Seal on the sides of the bottle using aluminum foil. Label the beaker using autoclave tape with the words the LB.
2. Utilize the appropriate transport protocols to transport the LB bottle to the autoclave area. Make sure you store the beaker in an autoclavable container in the event of spills.
3. Verify the level of water on the autoclave. If needed. Autoclave using the liquid setting for around 20 minutes.
4. It is likely that the contents in the beaker are likely to be hot following autoclaving, so take the appropriate precautions to avoid burns.
5. After autoclaving, let to allow the LB medium to cool down to 55 degrees Celsius prior to handling. Utilize a laser thermometer to determine the temperature of the glass.
6. The LB broth is stored in sterile condition at room temperature. It will last 3 to 4 months. Flaming the lid of the bottle every whenever the LB is utilized. If the LB has antibiotics, keep it in a freezer that is rated at -4°C. It is however not recommended to keep LB that contain antibiotics since the antibiotics are degraded as time passes.

Step 3: Pouring the plates (for LB agar)

As you pour the plates it is essential to ensure the sterility of the environment. This can be accomplished in the room WB303 in a clean and sterile atmosphere created by the burning of a Bunsen burner.

1. Clean the work area with 70 70% EtOH prior to putting your materials in. Then, light your Bunsen burner.
2. Take a roll of plates that are empty. The plates should wrapped in the plastic wrapper or sleeve and should be clean. Be sure to keep the wrapper since it can be used to protect the plates. It is vital to eliminate any possibility of contamination of the plates. It is important to remove the packaging from high and then expose your plates as little as you can.
3. When you’ve taken the plates off, put the plates upside-down on the lab bench. Label the plates. After labeling, you can stack the plates in order to free up space for work.
4. Let to allow the LB medium to cool prior to pouring. The LB will begin to settle around 30°C.
5. If you’re making specific media, add antibiotics to the mix. The flask should be twisted in an circular motion to mix. If you’re not sure whether or not you’re creating targeted media ASK. Make concentrated liquid stocks to help with the antibiotics.
6. Concentrations of recommended antibiotics Chloramphenicol (CAM) 25 mg/mL. Ampicillin (AMP) 100mg/mL
7. Use a plate that is empty and let it open a little. It is not necessary to fully open it to pour the Agar.
8. Pour the agar in a steady stream until two-thirds of the dish is coveredor about one-half of the plates is full when seen by the sides. Pour the agar slowly in order to stop any bubbles from forming. Then, swirl the plate in circular motions to distribute all the media evenly over the plate.
9. If you pour in too many LB it will not be able to make 25 plates. If you don’t use enough of the media can reduce the growth of bacterial.
10. After pouring, allow the plates aside to cool in stacks of 4-5 to conserve space. Then flip the plates to avoid condensation from forming over the agar. Avoid stacking plates overly high – we’re looking to minimize the possibility of spills. Let the plates cool for at minimum 20 minutes until the agar has sunk.
11. Wash the Pyrex bottle with water prior to the remaining solidifies and becomes difficult to get rid of.
12. The plates are then put in a stack and then stored inside plastic containers (ideally recycle the plastic bags the plates were packaged in.)
13. Place LB Agar plates in a freezer that is 4 degrees. They should last for up to a year.

Note: the steps 3 through 4, along with the cleaning up of Part 1 are possible while waiting for an autoclave.

Application of LB

• The LB medium is a nutrient-rich medium which is employed to grow individuals of Enterobacteriaceae and also for coliphage plaque testing.
• LB and the related media (SOC Terrific Broth 2xYT, SOC, etc.) are widely used in recombinant DNA research as well as other molecular biology processes.
• In most cases, an antibiotic is often added to the sterilized media to identify cells that have the genetic element that is specific to them, like a plasmid transposon, or mutation in a gene through the antibiotic resistance cassette.
• X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) may be added to sterile medium when using the blue-white screen for plasmids bearing the alpha fragment of the beta-galactosidase gene (a-complementation analysis).
• IPTG (isopropyl-beta-D-thiogalactopyranoside) is sometimes added to induce expression of genes controlled by the lac promoter.
• Additionally to (or maybe due to) its prominence in the field of molecular biotechnology, LB has also been employed as a general-purpose culture medium for many facultative organisms.
• In the microbiology classrooms for undergraduates laboratory, LB is sometimes used as a growth surface while trying to determine the morphology of colony bacterial.

Reference

• 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 Protocols/LB Agar Plates.pdf
• iGEM Toronto 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.
• https://asm.org/getattachment/5d82aa34-b514-4d85-8af3-aeabe6402874/LB-Luria-Agar-protocol-3031.pdf
• https://www.protocols.io/view/Lysogeny-Broth-LB-fcrbiv6
• http://cshprotocols.cshlp.org/content/2016/1/pdb.rec090829.short