IGEM:Caltech/2007/Protocols/Ecoli liquid culture

Materials

• LB broth (or appropriate media) with antibiotic
• culture tubes or Erlenmeyer flasks
• assorted pipetmen
• assorted pipet tips
• inoculation loops
• 1.5 ml microfuge tubes
• pipet
• pipet aid
• bunsen burner
• striker
• 60% glycerol
• cryogenic vials
• spectrophotometer cuvettes
• vortexer
• 37°C culture shaker
• SpectraMax M2 spectrophotometer

Method

• Comments on sterile technique

Materials, such as tips and glassware, and many solutions are sterilized by autoclaving. Solutions that cannot be autoclaved are sterilized by filtration. Benchtops and equipment such as pipetmen should be kept clean by wiping with 70% ethanol. When transferring sterile solutions between containers we use sterile technique. Sterile technique requires that the containers, material, and transfer equipment are sterile and that contaminating species are not introduced into the material during the transfer. Glass materials are kept sterile by flaming before and after opening them to the environment. Bunsen burners provide the flame at your bench workspace. A gas line going into the burner is turned on and a striker is used to ignite a flame from the burner. The strength of the flame is tuned by controlling the amount of gas and air going into the burner. When transferring sterile liquid between two sterile containers, as is required in the following procedures, both containers are flamed to maintain sterility. First, the cap on the receiving container is removed quickly and both the opening and cap of this container are passed over the flame. The cap is placed loosely back on the receiving container. Then, the cap on the container containing the sterile material is removed quickly and both the openings and cap of this container are passed over the flame. The desired volume is then transferred from one container to the other using the appropriate container. Before turning off the flame and closing the containers both openings and caps are flamed once again. Make sure to always turn off your flame when not in use as sometimes flames can go out while the gas line is still open allowing gas to flow into the laboratory through the Bunsen burner, which creates a very dangerous situation.

• Inoculating liquid cultures with bacterial colonies

1. Using sterile technique, transfer an appropriate volume of LB broth + appropriate antibiotic into a sterilized culture flask or test tube using a pipet and electronic pipet aid.
2. Using sterile technique, inoculate a fresh bacteria colony from solid media into the liquid media with a sterile inoculation loop (or pipet tip). Note that for optimal results the colony should be freshly grown on the solid media.
3. Place the test tube in the 37C shaker and monitor growth as needed.

• Inoculating liquid cultures with cells in liquid media

1. Using sterile technique, transfer an appropriate volume of LB broth + appropriate antibiotic into a sterilized culture flask or test tube using a pipet and electronic pipet aid.
2. Measure the OD600 of a stock culture. Remove the culture from the 37C shaker. If the OD600 is expected to be greater than 1.5 make a dilution of the culture. To make a dilution, using sterile technique transfer 900 µl of LB broth into a 1.5 ml microfuge tube with a L1000 pipetman.
3. Using sterile technique, transfer approximately 100 µl of the stock culture into the microfuge tube with a L200 pipetman and vortex briefly to mix.
4. Transfer 1 ml of the diluted culture (or the undiluted stock culture if dilution was not necessary) to a spectrophotometer cuvette with a L1000 pipetman. Transfer 1 ml of LB broth to a second spectrophotometer curvette with a L1000 pipetman. Make sure that there are no bubbles or lint in the light paths, wipe the cuvettes with a kim wipe as needed.
5. Measure the OD600 of the stock culture on the SpectraMax M2 spectrophotometer. This method is described in further detail in a separate protocol handout. Blank the instrument with the cuvette containing just the LB Broth. Record the OD600 in your laboratory notebook.
6. Use the formula provide above along with the volume of the fresh media and desired starting OD600 of the culture to determine the volume of the stock culture required for inoculation.
7. Using sterile technique, transfer the calculated volume of the stock culture to the fresh media using an appropriately sized pipetman (or pipet aid) and pipet tips (or pipet).
8. Place the test tube in the 37C shaker and monitor growth as needed by measuring the OD600 as described above.

• Making freezer stocks from liquid culture

1. Grow cultures in the 37C shaker until the appropriate OD600¬ is reached. For strains harboring plasmids make the freezer stocks from cultures in late log phase (~0.7 OD600 for E. coli and ~1-1.5 OD for yeast) to avoid plasmid loss. Strains not harboring plasmids can be grown to higher ODs.
2. Using sterile technique, add 0.5 ml of 60% glycerol to appropriately labeled cryogenic vials. Transfer 1.5 ml of the cell culture to the vials. This can be done by transferring 750 µl of the culture with a L1000 pipetman into the vial and repeating this transfer with a fresh tip.1 For each culture make at least two separate freezer stock vials. Tighten the vial caps and shake the vials to disperse the glycerol in the solution. If making stocks of E. coli cells, shock freeze cells in liquid nitrogen before placing them into an appropriately labeled freezer box. If making stocks of yeast shock freezing is not necessary.