Microbial Growth Protocols
Culture of E. coli
Materials required:
Suspension culture of E. coli
- Prepare LB medium by weighing appropriate powder medium and adding to water in a sterile flask
- Autoclave the broth and cool to room temperature
(Alternatively ready-to-use LB medium may be used)
- In a laminar flow chamber, transfer approximately 1 mL of overnight E. coli culture to the flask
- Seal the mouth of the flask with sterile cotton (non-absorbant) plugs; ensure the flask is not tightly sealed
- Incubate overnight at 37Ā° C with continuous shaking
Plating E. coli
- Prepare LB agar by weighing appropriate powder medium, agar and water to a sterile flask
- Autoclave the medium and cool just enough to be able to handle the flask
- In a laminar flow chamber, pour approximately 25-30 mL of the LB agar into sterile plates
- Allow the plates to set in the laminar flow chamber with lids slightly opened
(The plates may also be stored inverted at 4Ā° 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 ĀµL 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
- Invert and incubate the plates overnight at 37Ā° C
Advanced Protocols
Plating Bacteriophage M13
Materials required:
- Prepare a pure culture of E. coli by inoculating a single colony into 5 mL of LB or YT medium.
- Prepare ten-fold serial dilutions of M13 bacteriophage stock in LB or YT medium.
- Prepare LB agar or YT medium supplemented with 5mM MgCl2in sterile test tubes; equilibrate at 47Ā° C; add appropriate amounts of X-gal and IPTG solutions.
- Add serially diluted M13 bacteriophage stocks 100 ĀµL of pure bacterial culture; mix by gentle vortexing.
- Pour the LB agar or YT medium with X-gal and IPTG to tubes containing infected bacteria; mix by gentle vortexing.
- Transfer the contents to plate and swirl for even distribution of infected bacteria.
- Allow the plates to set; invert and incubate the plates at 37Ā° C.
- 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
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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
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