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Monday, April 8, 2013

Media & pH Optimization For E. Coli in Shake Flask



E. coli is Gram-negative, facultative anaerobic and non-sporulating bacteria. It can live on a wide variety of substrates. Optimal growth of E. coli occurs at 37°C, but some laboratory strains can multiply at temperatures of up to 49°C. Growth can be driven by aerobic or anaerobic respiration. Optimization of a particular parameter for any culture in any culture condition can be done by keeping all the other parameters constant. For optimization of the pH and the media for E. coli in shake flask conditions, all the other parameters such as temperature, shaker speed, media volume
per volume of flask, inoculum percentage etc should keep constant. E. coli grows well in neutral pH and hence media with pH 6.9 and 7.8 are used for optimization of the pH. LB broth, Nutrient broth and Tryptose soya broth are some best known media for E.coli and hence these are tried to optimize the media. The optimal pH and media are selected on the basis of the optical density given by the cultures at specific intervals.

List of Materials:

LB broth, Nutrient broth and Tryptose soya broth of pH 6.9 & 7.8, Conical flasks,E.coli culture, Test tubes, Test tube stand, Pipettes and Bunsen burner.

List of Equipments:

pH meter, vortex mixer, Spectrophotometer, Laminar air flow chamber and Shaker incubator.

Procedure:

Day 1: Media preparation and Inoculation

  1. The components of the media for 150 ml are weighed and dissolved in 100 ml and equally distributed into 2 conical flasks.
  2. The pH of the media in two flasks is adjusted to 6.9 and 7.8 respectively.
  3. The volume of media in each flask is made up to 75 ml with purified water.
  4. The flasks are autoclaved at 121°C, 15 lbs pressure for 30 minutes.
  5. The flasks are inoculated with 3 ml pre-inoculum of E.coli inside the laminar air flow chamber.
  6. The flasks are incubated at 37°C in the shaker incubator at 200 rpm.

Day 2: Observation

  1. 2 ml of the culture broth from both flasks are taken into separate test tubes and the pH and OD of the culture are checked at intervals of 16 hrs, 19 hrs, 20 hrs and 21 hrs after inoculation.

Observation & Inference


It was found that the LB broth of pH 6.9 is optimum for growth of E. coli in shake flask conditions from the above observations.