Bacterial Growth Curves in 96-well microplates

Written/Edited by: Tiffany Lowe-Power

Materials

Consumables

1. 96 well microplate, clear, flat-bottom, not treated e.g. Corning #3370
2. BreathEasy Gas Permeable Sealing membrane #BEM-1

Reusables

1. Plate reader capable of incubating at 28C with shaking and A 600 readings
2. Plate sealing device e.g. 3M P.A.-1

Experimental Design

• Sign up for plate reader at “Plate Reader Sign up” Google calendar. Try to schedule long experiments (>24 h) to run during the weekend.
• Use your Plate Layout Template to plan your experiment. Include 1-3 technical replicates of appropriate blank(s) per plate.
• Consider appropriate negative controls (e.g. “no carbon” control when testing carbon sources or “no bacteria” control when testing new media with components that can oxidize and change colors)
• For ‘normal’ 96-well plates, use 200 ul volume. For half-area 96-well plates, use 50 ul volume.
• Starting cell density for plate reader experiments: Growth curves should be performed in the dynamic range of the instrument (e.g. at time 0, wells with bacteria should have net positive A600 readings after blank subtraction). Because microplates have a smaller pathlength where the light passes through the sample, A600 in the plate reader is not equivalent to OD600.

Loose Methods

Note this protocol assumes use of typical 96-well plates. Half-area well or 24 well plates will require appropriately scaled volumes

Use multichannel P20 and P200 where possible

1. Streak out relevant strains on CPG agar and incubate at 28C for 2 d.
2. Starting from single colonies, start 3 biological replicate cultures in 1 ml CPG broth in individual XXX mm test tubes. Incubate overnight at 28C with 180 rpm shaking.
   • Doing a low culture volume ensures most Ralstonia will reach stationary phase by the next morning. Empirically, starting the growth curves from stationary phase cultures provides high consistency between biological replicates.
3. Harvest cells (>13 xg, 1 min). Wash cells 3x in di H2O.
4. Resuspend cells in 1 ml test media or di H2O. Measure OD of 1:10 dilution. Use Cell Density workbook to adjust OD600 of each replicate to 0.5.
5. Add test media (180 ul) to wells.
6. Add 20 ul (OD600=0.5) bacterial suspension to wells (starting OD=0.05, which corresponds to ~XXX blank-subtracted A600 on plate reader)
7. Seal plate with a BreathEasy film.
8. Read in plate reader with program that incubates with shaking at 28C, and measures A600 at periodic intervals (e.g. 30 min or 1 h) for appropriate length of time.