[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [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 Sorry, an unexpected error has occurred Response not successful: Received status code 500 ### 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) __Related Articles__ - [Streptococci - Overview of Detection, Identification, Differentiation and Cultivation Techniques](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/streptococci-overview-of-detection) - [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) - [Virus Cultivation](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/microbial-cell-culture/virus-cultivation) - [Yeast and Molds in Food and Beverages](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/microbial-cell-culture/yeasts) - [Microbial Detection Using Chromogenic and Fluorogenic Culture Media](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/bacteriology/overview-chromogenic-media) - [Media for Staphylococcus Aureus Detection](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/bacteriology/staphylococcus-aureus) - [Identification of Microorganisms Based on Color](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/cytology-and-microscopy/color-identification-microorganisms) - [EN ISO 11133 Microbiological Culture Media](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/en-iso-11133-water-for-the-preparation-and-performance-testing) __Related Product Categories__ - [Detection Substrates & Enzymes](https://www.sigmaaldrich.com/US/en/products/protein-biology/proteins-and-enzymes/detection-substrates-and-enzymes) - [Carbohydrates for Biochemical Research](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/carbohydrates) Top __Sign In To Continue__ To continue reading please sign in or create an account. Sign In__Don't Have An Account?__Register An unknown error has occured. - English - EN - Español - ES [Learn More](https://www.sigmaaldrich.com)