Microbial Growth Protocols

Culture of E. coli

Materials required:

Reagents	Equipment
Pure E. coli culture	Cabinet incubator (37 °C)
LB – Miller	Shaker incubator (37 °C)
LB – Miller Plates	Culture dishes, sterile
	Culture flasks, sterile

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:

Reagents	Equipment
Pure E. coli culture	Cabinet incubator (37 °C)
LB agar EZMix™ (Product No. L7533)	Water Bath (47 °C)
Appropriate selection antibiotic	Culture dishes
IPTG (Isopropyl-β-D-thiogalactoside) (Product No. I6758)	15 mL polypropylene culture tubes (sterile)
X-gal (Product No. B9146)
Magnesium chloride

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:

Bacterial Broth Components	Name	Format
Y0875	Select Yeast Extract	Powder
N4767	N-Z-Amine® EKC
Y1626	Select Yeast Extract, EZMix™ powder	EZMix™
T2559	EZMix™ Tryptone

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