Materials and Reagents

Note: All reagents used were from the specified manufacturers (catalog numbers indicated). Nonetheless, the same reagents from different manufacturers are expected to produce similar results.

Spatula
Weighing boat (Sparks Lab Supplies, catalog number: BAL1822 )
Gloves (Sparks Lab Supplies, catalog number: SAF5534X20 )
Sterile micropipette tips (100-1,000 µl) (Abdos Labtech, catalog number: P10102 )
Sterile micropipette tips (2-200 µl) (Abdos Labtech, catalog number: P10101 )
Microcentrifuge tube (1.5 ml) (Abdos Labtech, catalog number: P10202 )
Centrifuge tube (15 ml graduated) (Abdos Labtech, catalog number: P10402 )
Cuvette (LP ITALIANA, catalog number: 112117 )
2 mm laboratory test sieve (B5410/1986, Endecotts, catalog number: 200SIW2.00 )
96-well plate (TC microwell 96U) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 163320 )
90 mm Petri dishes (Abdos Labtech, catalog number: P10901 )
Silty-Loam soil
E. coli strains
1. E. coli COB583 (Somorin et al., 2016)
2. E. coli COB583ΔrpoS (Somorin et al., 2016)
Note: E. coli stock cultures were made in LB with 7% Dimethyl sulfoxide (v/v; Sigma-Aldrich) and kept at -80 °C pending use.
Luria-Bertani (LB) broth (Sigma-Aldrich, catalog number: L3022-1KG )
Agar No. 2 (Lab M, catalog number: MC006 )
MacConkey agar (Sigma-Aldrich, catalog number: M7408 )
Distilled water (Milli-Q) (EMD Millipore)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
LB broth (see Recipes)
LB agar plates (see Recipes)
PBS buffer (see Recipes)
MacConkey agar plates (see Recipes)

Equipment

250 ml conical flask (VWR, catalog number: 214-1132 )
-80 °C freezer
Styrofoam 15 ml tube holder
Bunsen burner
Orbital shaker (Gallenkamp)
Discovery comfort multichannel pipette (20-200 µl; HTL)
Discovery comfort multichannel pipette (5-20 µl; HTL)
Nichipet EX pipette (200-1,000 µl; Nichiryo)
Nichipet EX Pipetman classic pipettes (20-200 µl; Nichiryo)
Vortex mixer (Reax top; Heidolph)
Biomate 3 spectrophotometer (Thermo Fisher Scientific, catalog number: 335904 )
Note: This product has been discontinued.
Weighing scale (Sartorius, catalog number: BL120S )
Note: This product has been discontinued.
Centrifuge (Eppendorf, model: 5418 )
Labo autoclave (SANYO, model: MLS-3020U )

Procedure

Measurement of cell concentration by optical density (OD_{600 nm})
1. Streak out Escherichia coli COB583 wildtype and the rpoS deletion mutant (COB583ΔrpoS) from the stock culture kept in the -80 °C freezer onto LB agar (see Recipes) around a Bunsen burner and incubate them overnight (~16 h) at 37 °C.
2. Inoculate one colony of each strain with a disposable sterile inoculating loop into 10 ml LB in a sterile conical flask around a Bunsen burner and incubate them at 37 °C for 6 h. Perform this step in triplicate for each strain.
3. From the 6 h culture, take 100 µl and add it to 900 µl LB and determine the OD_{600 nm}.
4. Determine the volume of the 6 h culture required to give a starting OD_{600 nm} of 0.05 in a 25 ml LB as follows:
  C₁V₁ = C₂V₂
  C₁ = OD_{600 nm} of the 6 h culture; V₁ = volume of the 6 h culture
  C₂ = final OD_{600 nm} required; V₂ = final volume required
  V₁ = 0.05 x 25 ml/C₁
5. Make the cultures with starting OD_{600 nm} of 0.05 in 25 ml LB for each strain and their respective replicates in sterile 250 ml conical flasks and incubate the flasks overnight on a shaker at 37 °C.
6. On the following day, dilute the overnight cultures 1:10 in LB and determine the OD_{600 nm}.
7. Pipette 1 ml of each overnight culture into two separate sterile 1.5 ml tubes for each strain and their respective replicates.
8. Centrifuge the cultures at 9,000 x g for 10 min at room temperature (23-25 °C) and remove the supernatants with sterile pipette.
9. Wash the cell pellets by re-suspending them in sterile PBS (see Recipes) and centrifuge at 9,000 x g for 10 min. Repeat the washing step two more times.
10. Resuspend the washed cell pellets in 1 ml of sterile PBS by pipetting up and down and vortexing and dilute 1:2; 1:5; 1:10; 1:20; 1:100; and 1:1,000 in sterile PBS to make 1 ml in sterile microcentrifuge tubes.
11. Transfer each diluted cell suspension into a cuvette and determine the OD_{600 nm} of the diluted cell suspensions of the E. coli strains and their respective replicates in a spectrophotometer three times and determine the mean value.
12. Make serial dilutions of each cell suspension and spot 10 µl onto LB plates in triplicate and incubate the plates at 37 °C overnight.
13. Count the colonies and use the numbers derived to estimate the cell count (CFU ml^-1) and determine the mean cell count from the triplicate experiments.
14. Plot the mean cell count against the mean OD_{600 nm} of the respective dilutions of each strain in three independent experiments to obtain calibration curves from which desired cell numbers can be obtained. The standard curve of graph of the cell count versus OD_{600 nm} is presented in Figure 1.
  
  Figure 1. Standard curve of cell numbers versus OD_{600 nm} of COB583 (A) and COB583ΔrpoS (B)
Development of soil survival assay
1. Inoculate one colony each of COB583 and COB583ΔrpoS into 25 ml LB in triplicate and incubate overnight at 37 °C.
2. Dilute the resulting cultures 1:10 in LB and determine the OD_{600 nm}.
3. Add 1 ml of the overnight culture to two sterile Eppendorf tubes for each replicate of the two strains.
4. Wash the cultures as described above (steps A8 and A9).
5. Pool the resulting cell pellets and resuspend in 2 ml of sterile PBS in a 15 ml tube.
6. Make 1:10 dilution (1 ml) of the each washed cell suspension to determine the OD_{600 nm}.
7. Based on the OD600 nm, prepare a normalised cell suspension with OD_{600 nm} equivalent to 2 x 10⁸ CFU ml^-1 from each replicate.
8. Make serial dilution of each normalised bacterial suspension and spot 10 µl of each dilution on LB agar in triplicate. Allow the spots to air-dry and incubate the plates overnight at 37 °C. Count the colonies and determine the mean cell counts.
9. Sieve the soil to be used with 2 mm laboratory test sieve and weigh 1 g of the sieved soil into a 15 ml tube.
10. Take 50 µl of each normalised bacterial suspension for each strain and inoculate it into the soil (to give ~1 x 10⁷ CFU g^-1 of soil). The uninoculated control should have 50 µl of sterile PBS added to it. The experiment was set up in triplicate for each strain.
11. Cover the tubes with the lid and invert them 10 times by hand for proper mixing.
12. Leave the tubes for about 10 min at room temperature (23-25 °C).
13. Perform the recovery of the inoculated E. coli strains by adding 2 ml of sterile PBS to each 15 ml tube. Cover the tube with its lid, invert it three times and immediately vortex for 20 sec twice.
14. Leave the tube on the workbench and allow the larger soil particles in the resulting soil slurry to settle out (2-10 min, depending on soil texture) (Figure 2).
  
  Figure 2. Experimental set-up for the soil survival assay with the soil slurry just after mixing and after settling out
15. Take 20 µl from the supernatant of each soil slurry, place them in separate wells in a 96-well plate and make serial dilution (up to 10^-6) from them.
16. Take 10 µl of each dilution and spot onto MacConkey agar plates (see Recipes) in triplicate, and incubate plates overnight at 37 °C.
17. Count the colonies and use the numbers derived to estimate the mean recovered cell count (CFU ml^-1) from the triplicate experiments. The percentage recovery of cells was determined.

Data analysis

Cell count for each replicate is represented by the mean cell counts obtained from the three spots in each replicate. The mean and standard deviation of the cell counts from the normalised cell suspension (inoculum) and recovered cell counts were obtained from three independent replicates of each strain. The percentage of recovery ranges from 30-79% and the recovered cells from this procedure is presented in Figure 3.

Figure 3. Recovery of Escherichia coli from soil. Percentage recovery is provided above the recovered cells.

Notes

The optical density of the inoculum should be confirmed to be accurate before inoculating into soil.
Care should be taken when inoculating the soil to ensure the pipette tips do not contact the soil directly, as it prevents the release of the entire content of the pipette tip.
Care should be taken when spotting the cell suspension on agar plates so as not to puncture the agar with the pipette tips. This could vary the cell numbers arising from each spot.
The soil to be used should be checked and confirmed not to contain E. coli prior to the soil survival assay so that recovered E. coli will only be attributable to inoculated E. coli. Serial dilutions of 1 g of the soil sieved through a 2 mm mesh should be plated on MacConkey agar and incubated at 37 °C to determine this.
For long-term soil survival experiments, the tubes should be slightly capped to allow air exchange and incubated statically at the desired temperature. Multiple tubes should be set up for experiments requiring sampling at different time points since inoculated soils are destructively sampled (i.e., PBS was added and could not be reused at another time point) for E. coli recovery.
No significant difference was seen when soil slurry was left to settle for 2 and 10 min before aliquots were taken for serial dilution and plating for cell count determination.

Recipes

LB broth
Add 20 g LB powder to 1 L of distilled water and autoclave
LB agar plates
Add 20 g LB powder and 15 g agar No. 2 powder to 1 L of distilled water and autoclave. When cool to touch (~ 45 °C), pour about 25 ml of the molten agar into Petri dishes and allow to solidify
PBS buffer
Dissolve one phosphate-buffered saline tablet (Sigma-Aldrich) in 200 ml of distilled water and autoclave, giving a PBS buffer (pH 7.4)
MacConkey agar plates
Add 50 g MacConkey agar powder to 1 L of distilled water and autoclave. When cool to touch (~45 °C), pour about 25 ml of the molten agar into Petri dishes and allow to solidify

Acknowledgments

The protocol was adapted from a previously published work (Somorin et al., 2016). The authors thank Camila Thorne for her help and advice with setting up the soil survival assay. This work was supported by an NUI Galway College of Science Ph.D. Fellowship and Thomas Crawford Hayes research award to Y.S.

References

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