Oct 06, 2022
Growth_of_Pseudomonas_fluorescence_SBW25_in_48-well_Plate_Format
- 1Argonne National Laboratory
Protocol Citation: Rosemarie Wilton 2022. Growth_of_Pseudomonas_fluorescence_SBW25_in_48-well_Plate_Format . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwewdl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 22, 2022
Last Modified: October 06, 2022
Protocol Integer ID: 70401
Abstract
This protocol provides a method for culturing Pseudomonas fluorescens SBW25 in 48-well microplates. The protocol is specific for use with a BioTek Epoch 2 plate reader, but other plate readers can be used with appropriate modifications. One advantage of the Epoch 2 instrument is the ability to run with the plate lid at a higher temperature than the well contents. This prevents condensation on the lid and resulting noise in the OD readings.
This protocol is suitable for many Pseudomonas strains.
I thank members of the ORNL team, Joshua Elmore and Quint Peabody V, for assistance during development of this protocol and tips on use of the Epoch2 plate reader. I thank the NREL team for the M9 Minimal Media recipe.
Materials
48-well cell culture microplates (Greiner Bio-One 677180)
Note: If using an Epoch2 microplate reader, the physical maximum plate height is 23.5 mm.
The Greiner plates (with lids) have a maximum height of 22 mm.
50 mL Bio-Reaction tubes (CELLTREAT 229475)
LB medium
- 10 g Tryptone
- 5 g Yeast Extract
- 10 g NaCl
Add 1 L deionized water
Sterilize by autoclaving at 15 psi, 121-124ºC for 20 minutes
M9 Minimal Medium (1L):
1. To 800 mL H2O, add the following in the order below, mixing in between:
- 0.1 mL 1M CaCl2
- 0.18 mL 100 mM FeSO4*
- 2.0 mL 1M MgSO4
- 100 mL 10X M9 minimal salts
2. Add carbon source, and add H2O to bring the final volume up to 1 L.
3. Filter sterilize.
*Optional. Reduces fluorescent siderophore secretion in Pseudomonas strains. A 100 mM stock of FeSO4 should be aliquoted quickly and frozen to prevent oxidation.
10X* M9 Minimal Salts (1L):
Ingredients: (g/L)
Disodium phosphate (anhydrous) 67.8
Monopotassium phosphate 30
Sodium chloride 5
Ammonium chloride 10
*A 5X stock could be prepared instead
1. Dissolve the above (or 112.8 g M9 Minimal Salts, 5X, Sigma M6030) in 1L of distilled water.
2. Autoclave in liquid cycle or filter sterilize.
Culturing Pseudomonas fluorescens SBW25
Culturing Pseudomonas fluorescens SBW25
DAY1: Overnight culture
Inoculate 5 mL LB, in a vented or loosely capped culture tube (e.g. CELLTREAT 50 mL Bio-Reaction tube), from a glycerol stock or with a single colony from a plate.
Grow overnight at 30 °C with shaking at 250 rpm .
DAY2: Four hour culture
NOTE: Pseudomonas cells typically have a long lag time if transferred from a stationary-phase overnight culture directly to minimal medium. The additional four hour culture period in fresh LB allows the cells to return to exponential growth phase, and minimizes the subsequent lag upon transfer to minimal medium.
Dispense 10 mL LB into vented or loosely capped 50 mL culture tube.
Inoculate with 250 µL overnight culture.
Incubate for four hours at at 30 °C with shaking at 250 rpm
Pellet cells by centrifugation, 4000 rpm for 00:05:00 .
Pour off supernatant and resuspend in 5 mL M9 salts.
Collect cells by centrifugation, and repeat M9 salts wash step.
Resuspend pellet in5 mL sterile M9 salts.
Remove a small aliquot to measure OD600 on a spectrophotometer.
Plate set-up and data collection
Plate set-up and data collection
Prepare M9 minimal medium containing desired carbon source (M9-C) and filter sterilize.
Dilute washed cells to a final OD600 of 0.05 to 0.1 OD with M9-C.
Dispense 600 ul/well to 48-well plate and cover with the supplied lid.
Each unique well condition or sample type should be performed in triplicate.
NOTE: If long incubation periods are anticipated (>24 hours), fill edge wells with M9-C and only use the 24 inner wells for culturing. This will minimize evaporation during long culture times.
Start up the Gen5 software on the Epoch2 system and place the plate in the drawer.
Enter the protocol shown below; run times and sampling intervals can be adjusted as needed.
48-well plate notes:
Notice that we have left the "Use Lid" box unchecked (even though we use the lid). This is because the Default 48 well plate plus lid is higher than the instrument max of 23.5 mm. To prevent error message, leave lid box unchecked or generate a custom plate description for the Greiner plate (System>Plate Types>View/Modify).
Adjusting the Well and Lid temperature:
The Epoch 2 allows the lid to be run at a higher temperature to avoid condensation. This is set in the Gradient box below. This should be set to 1 or 2ºC.
Adjusting the Shaking mode and speed:
Adjusting the Read step:
Edit the Read Speed to bring up the second window. Increase the delay time to 250 msec to allow wells to stabilize before reading.
Once protocol has been set up, start the run and monitor data collection over time. The run can be stopped at earlier timepoints without losing the data.