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[Home](https://www.sigmaaldrich.com/US/en)[Microbial Cell Culture](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-cell-type/microbial-cell-culture)Microbial Growth Protocols
# 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* cultureCabinet incubator (37 °C)
[LB agar EZMix™ (Product No. L7533)](https://www.sigmaaldrich.com/US/en/product/sigma/L7533)Water Bath (47 °C)
Appropriate selection antibioticCulture dishes
[IPTG (Isopropyl-β-D-thiogalactoside)
(Product No. I6758)](https://www.sigmaaldrich.com/US/en/product/sial/I6758)15 mL polypropylene culture tubes (sterile)
[X-gal (Product No. B9146)](https://www.sigmaaldrich.com/US/en/product/sigma/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](https://www.sigmaaldrich.com/US/en/product/sigma/Y0875) | [Select Yeast Extract](https://www.sigmaaldrich.com/US/en/product/sigma/Y0875) | Powder |
| [N4767](https://www.sigmaaldrich.com/US/en/product/sigma/N4767) | [N-Z-Amine® EKC](https://www.sigmaaldrich.com/US/en/product/sigma/N4767) | |
| [Y1626](https://www.sigmaaldrich.com/US/en/product/sigma/Y1626) | [Select Yeast Extract, EZMix™ powder](https://www.sigmaaldrich.com/US/en/product/sigma/Y1626) | EZMix™ |
| [T2559](https://www.sigmaaldrich.com/US/en/product/sigma/T2559) | [EZMix™ Tryptone](https://www.sigmaaldrich.com/US/en/product/sigma/T2559) | |
## 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 02 Aug 2016; cited 05 Aug 2020]. Available from: [http://www.scienceprofonline.org/microbiology/types-culture-media-for-growing-bacteria.html](http://www.scienceprofonline.org/microbiology/types-culture-media-for-growing-bacteria.html)
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- [Introduction to Microbial Media](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/microbial-cell-culture/microbial-media)
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