Microbial Growth Protocols

Culture of E. coli

Materials required:

Suspension culture of E. coli

  1. Prepare LB medium by weighing appropriate powder medium and adding to water in a sterile flask
  2. Autoclave the broth and cool to room temperature
    (Alternatively ready-to-use LB medium may be used)
  3. In a laminar flow chamber, transfer approximately 1 mL of overnight E. coli culture to the flask
  4. Seal the mouth of the flask with sterile cotton (non-absorbant) plugs; ensure the flask is not tightly sealed
  5. Incubate overnight at 37° C with continuous shaking

Plating E. coli

  1. Prepare LB agar by weighing appropriate powder medium, agar and water to a sterile flask
  2. Autoclave the medium and cool just enough to be able to handle the flask
  3. In a laminar flow chamber, pour approximately 25-30 mL of the LB agar into sterile plates
  4. Allow the plates to set in the laminar flow chamber with lids slightly opened
    (The plates may also be stored inverted at 4° C for future use)
    (Alternatively, ready-to-use LB agar plates may be used)
    • Lightly scratch the surface of frozen E. coli glycerol stock with a sterile inoculating loop
    • Pick up E. coli colony from a plate with culture with a sterile inoculating loop
    • Add 10-100 µL of E. coli suspension culture and add one the LB agar plate
      • Streak the loop across the LB agar plate
      • Spread the culture all over the plate using a sterile glass spreader
  5. Invert and incubate the plates overnight at 37° C

Advanced Protocols

Plating Bacteriophage M13

Materials required:

  1. Prepare a pure culture of E. coli by inoculating a single colony into 5 mL of LB or YT medium.
  2. Prepare ten-fold serial dilutions of M13 bacteriophage stock in LB or YT medium.
  3. Prepare LB agar or YT medium supplemented with 5mM MgCl2in sterile test tubes; equilibrate at 47° C; add appropriate amounts of X-gal and IPTG solutions.
  4. Add serially diluted M13 bacteriophage stocks 100 µL of pure bacterial culture; mix by gentle vortexing.
  5. Pour the LB agar or YT medium with X-gal and IPTG to tubes containing infected bacteria; mix by gentle vortexing.
  6. Transfer the contents to plate and swirl for even distribution of infected bacteria.
  7. Allow the plates to set; invert and incubate the plates at 37° C.
  8. Pale blue plaques of M13 bacteriophage appear on a lawn of bacterial growth.

The following are the bacterial broth components offered by our company:

Materials
Loading

References

1.
Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
2.
Parija S. 2009. Textbook of Microbiology and Immunology. Elsevier India.
3.
Types of Growth Media Used to Culture Bacteria. [Internet].[updated 01 Aug 2016; cited 04 Aug 2020]. Available from: http://www.scienceprofonline.org/microbiology/types-culture-media-for-growing-bacteria

Social Media

LinkedIn icon
Twitter icon
Facebook Icon
Instagram Icon

MilliporeSigma

Research. Development. Production.

We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.

© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Reproduction of any materials from the site is strictly forbidden without permission.