Quantification of Root Colonizing Bacteria   

Abstract

Materials and Reagents

1. Mechanical pipette tips
   
2. 1.5, 2 ml Eppendorf tube
   
3. Razor Blades (LabSupplyOutlaws, catalog number: 12-640 )
   
4. Nunc^TM 96 Microtiter Plates (Thermo Fisher Scientific, Thermo Fisher^TM, catalog number: 262162 )
   
5. Disposable Tissue Grinder Pestle (Corning, Axygen^®, catalog number: PES-15-B-SI )
   
6. TissueLyser II .Gentech Biosciences (GENTECH BIOSCIENCES, catalog number: 85300 )
   
7. Lab Parafilm M (Labdirect, catalog number: PM992 )
   
8. 100 mm Petri dish (Corning, Falcon^®, catalog number: 351029 )
   
9. Square Petri Dish with Grid (SIMPORT, catalog number: D210-16 )
   
10. Stainless Steel Beads, 2.3 mm diameter (Bio Spec Products, catalog number: 11079123ss )
    
11. Seeds of Arabidopsis (Arabidopsis thaliana)
    
12. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524 )
    
13. LB Broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897 )
    
14. Magnesium chloride anhydrous, ≥ 98% (Sigma-Aldrich, catalog number: M8266 )
    
15. Sodium chloride BioXtra, ≥ 99.5% (Sigma-Aldrich, catalog number: S7653 )
    
16. Potassium hydroxide (Sigma-Aldrich, catalog number: 484016 )
    
17. Silwet^® L-77 (PhytoTechnology Laboratories, catalog number: S7777 )
    

Equipment

1. Tweezers with Sharp Tip (Labdirect, catalog number: 560.002.115 )
   
2. Top centrifuge (Eppendorf)
   
3. Vortex (VWR digital Vortex Mixer)
   
4. 28 °C shaker-incubator
   
5. Growth chamber (Percival Scientific or similar types. Set the growth conditions to 22 °C, 16 h light/8 h dark cycles)
   
6. TissueLyser II^© (QIAGEN)
   
7. Autoclave (TOMY SEIKO, model: LSX-500 or similar types)
   
8. Mechanical pipettes (Gilson, catalog numbers: F123601 (P200) and F123602 (P1000)
   
9. Analytical balance (0.1 mg resolution)

Procedure

1. Prepare the bacterial culture
   1. Revive the SA187 bacterial strains (or any other strain) on LB Agar media (or equivalent media) and incubate at 28 °C for 24 h.
      
   2. Inoculate 10 ml LB media with a single colony on a shaker incubator with 220 rpm at 28 °C for 16 h (overnight) (pre-culture).
      
   3. Take 100 µl of the O.N. culture to start a new fresh 2 ml culture and let the bacteria grow for 2-3 h.
      
   4. Prepare and autoclave ½ MS with 0.9% agar adjusted to pH 5.8 with 1 M KOH. Allow the medium to cool down to reach 37-40 °C before pouring the plates.
      
      
2. Bacteralized vertical plate culture of Arabidopsis seedlings
   1. In a 50 ml Falcon tube mix 50 ml of warm ½ MS medium with 0.1 ml of a bacterial suspension (OD₆₀₀ = 0.21) to obtain 10⁷ bacteria, dispense the media in square Petri dishes (Figure 1) and let the agar solidify. For control plates (no bacteria) add 0.1 ml of liquid LB.
      
      
      Figure 1. Schematic illustration of different steps for preparing the bacteralized vertical plates of Arabidopsis seedlings
      
      
   2. Sterilize Arabidopsis seeds following a standard protocol (de Zélicourt et al., 2018) or similar methods described elsewhere and sow the sterilized seeds. Keep the plates and seeds at 4 °C in the dark for 2 days.
      
   3. Transfer the plates to a growth chamber and keep them vertically for 5 days in long day conditions: 16 h-light /8-h dark cycle at constant 22 °C.
      
   4. Transfer the colonized seedlings to ½ MS plates with or without 100 mM NaCl and incubate for 10 days or as desired.
      
      
3. Bacterial Quantification: Colony Forming Units
   1. With forceps and razor blades, separate root and shoot and combine three roots and/or three shoots in one 1.5 ml tube, record the fresh weight.
      
   2. Physically grind using the plastic cone grinder first without MgCl₂ and then add 500 μl 10 mM MgCl₂ supplemented with 0.02% Silwet L-77 and vortex vigorously (Figure 2).
      
      
      Figure 2. Schematic illustration of different steps for the quantification of bacteria in Arabidopsis plants
      
      
   3. Use TissueLyserII with Stainless Steel Beads to homogenize the plant material.
      
   4. Proceed with serial dilutions by removing 10 μl and add to 90 μl of sterile distilled H₂O in 96 microtiter plates.
      
   5. Spot 10-μl samples (3 replicates) from each dilution on LB agar plates. Allow spotted samples to dry, cover the plates, and wrap them with Parafilm.
      
   6. Incubate the plates at 28 °C for 24 h or until the colonies are clearly visible. Count colonies from each dilution, record the colony forming units (CFU), and calculate CFU/mg plant material.
      Note: All work shall be done under sterile conditions.

Data analysis

Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor The number of colonies per ml reported should reflect the precision of the method and should not include more than two significant figures.
The CFU/ml can be calculated using the following formula:

CFU/ml = (number of colonies x dilution factor)/volume of culture plate

CFU value could also be divided by the root/shoot fresh weight to get CFU/mg plant material.

Acknowledgments

The authors would like to express their gratitude to all members of the Hirt lab and the technical support team. This publication is based upon work supported by the King Abdullah University of Science and Technology (KAUST), base fund (No. BAS/1/1062-01-01) for HH.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

1. Andrés-Barrao, C., Lafi, F. F., Alam, I., de Zelicourt, A., Eida, A. A., Bokhari, A., Alzubaidy, H., Bajic, V. B., Hirt, H. and Saad, M. M. (2017). Complete genome sequence analysis of Enterobacter sp. SA187, a plant multi-stress tolerance promoting endophytic bacterium. Front Microbiol 8: 2023.
   
2. de Zélicourt, A., Synek, L., Saad, M. M., Alzubaidy, H., Jalal, R., Xie, Y., Andrés-Barrao, C., Rolli, E., Guerard, F., Mariappan, K. G., Daur, I., Colcombet, J., Benhamed, M., Depaepe, T., Van Der Straeten, D. and Hirt, H. (2018). Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production. PLoS Genet 14(3): e1007273.
   
3. Lafi, F. F., Alam, I., Geurts, R., Bisseling, T., Bajic, V. B., Hirt, H. and Saad, M. M. (2017). Draft genome sequence of Enterobacter sp. Sa187, an endophytic bacterium isolated from the desert plant Indigofera argentea. Genome Announc 5(7): e01638-16.