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Microbial Growth Protocols


Find more protocols and selection guides in the Molecular Biology Guide.

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

Prepare LB medium by weighing appropriate powder medium and adding to water in a sterile flas
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 37°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 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
 
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  

 

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 47°C; add appropriate amounts of X-gal and IPTG solutions
Add serially diluted M13 bacteriophage stocks 100 µL 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 37°C
 Pale blue plaques of M13 bacteriophage appear on a lawn of bacterial growth


The following are the bacterial broth components offered by Sigma-Aldrich:

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

Materials

     

 References

 

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