IGEM:Caltech/2007/Protocols/Plating

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Revision as of 21:52, 19 June 2007

Materials

  • LB agar plates + appropriate antibiotic
  • toothpicks or inoculating loops
  • assorted pipetmen
  • assorted pipet tips
  • glass beads
  • 37°C incubator
  • bunsen burner
  • striker

Method

  • Plating liquid cultures onto solid media
  1. Plate 50-200 µl of cells on a labeled LB agar plate with appropriate antibiotic. Note, that 200 µl and 50 µl are the largest and smallest volumes, respectively, that should be plated. Transfer the desired volume of the cell suspension onto the center of the plate with a L200 pipetman.
  2. Carefully add 15-20 sterilized glass beads onto the plate surface and shake the beads across the surface of the plate in a horizontal manner to spread the solution evenly across the plate. Carefully pour the used glass beads into the bottles on each benchtop through the funnel.
  3. Place the plate, agar side up, in the 37C incubator until desired colony growth is observed.
  • Restreaking cells for isolating colonies
  1. Cells are streaked on appropriately labeled plates using aseptic technique normally with an autoclaved toothpick or a sterile plastic loop. To obtain isolated colonies cells should be streaked in a pattern according to Figure 1. Avoid penetrating the agar media by using continuous gentle strokes and use a new toothpick (or loop) between streaking steps.
  • Restreaking cells for making temporary stocks of colonies
  1. When streaking multiple colonies to create a stock plate for the colony of interest, one may more efficiently use the solid media by streaking to isolation using 1/6 (or any other appropriate wedge size) of the plate (Figure 2). The colony you select after 12 – 24 hours of incubation should be morphologically identical to (color, shape, and approximate size) and well isolated from other colonies.
  2. BEWARE of satellite colonies, which will appear as significantly smaller colonies, or contamination! For example, when selecting for ampicillin resistant colonies, after sufficient incubation (> 48 hours), resistant colonies will have sufficiently lowered the surrounding ampicillin concentration by consumption to enable nearby non-resistant cells to grow.
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