Microbial Growth Protocols

Culture of E. coli

LB Broth (Miller) is a granulated medium for the cultivation of E. coli on scales ranging from small cultures to fermentation. The granules ensure rapid and uniform dissolution in water, and prevent clumping of the medium and inhalation of the airborne powder.

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

Initiating a Starter Culture

1. Pick a single colony from a freshly streaked plate and inoculate a starter culture with 3 to 5 mL of media.
   
2. Use the appropriate antibiotic and incubate at 37 °C* for approximately 8 hours while shaking at 250-300 rpm.
   
3. Use the starter culture to inoculate an overnight culture. Dilute the starter culture 1:500 to 1:1000 in a large flask with the appropriate volume of media and incubate at 37 °C* for 12 to 16 hours while shaking at 250-300 rpm.

*This temperature is for standard E. coli. Some bacteria require different culture temperatures.

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