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Quantification of Root Colonizing Bacteria
Published: Jul 20, 2018 DOI: 10.21769/BioProtoc.2927 Views: 5283
Original research article
The authors used this protocol in:
Mar 2018
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Abstract
Here we describe a simple method to quantify the number of viable bacteria (e.g., Enterobacter sp. SA187) that colonize the root system of Arabidopsis thaliana.
Keywords: Arabidopsis thaliana
Colony forming unit
Endophytes
Background
Colonization of roots by inoculated bacteria is an important step in the interaction between beneficial bacteria and the host plant. Enterobacter sp. SA187 is an endophytic bacterium that has been isolated from root nodules of the indigenous desert plant Indigofera argentea (Andrés-Barrao et al., 2017; Lafi et al., 2017). SA187 promotes the growth of the model plant Arabidopsis thaliana under diverse abiotic stresses such as salinity, drought or high temperature, demonstrating an important potential for application as Plant Growth Promoting Bacteria (PGPR) to improve abiotic resistance and yield of crops in arid lands. SA187 can colonize both roots and shoots of A. thaliana on ½ MS agar plates or in soil (de Zélicourt et al., 2018). To follow the fate of inoculant strain SA187 on the non-host plant A. thaliana, we applied a routinely cultivation-dependent method. This protocol has been successfully used to mounter the bacterial number colonized both root and shoot of the A. thaliana plant under different abiotic stresses (de Zélicourt et al., 2018).
Materials and Reagents
- Mechanical pipette tips
- 1.5, 2 ml Eppendorf tube
- Razor Blades (LabSupplyOutlaws, catalog number: 12-640 )
- NuncTM 96 Microtiter Plates (Thermo Fisher Scientific, Thermo FisherTM, catalog number: 262162 )
- Disposable Tissue Grinder Pestle (Corning, Axygen®, catalog number: PES-15-B-SI )
- TissueLyser II .Gentech Biosciences (GENTECH BIOSCIENCES, catalog number: 85300 )
- Lab Parafilm M (Labdirect, catalog number: PM992 )
- 100 mm Petri dish (Corning, Falcon®, catalog number: 351029 )
- Square Petri Dish with Grid (SIMPORT, catalog number: D210-16 )
- Stainless Steel Beads, 2.3 mm diameter (Bio Spec Products, catalog number: 11079123ss )
- Seeds of Arabidopsis (Arabidopsis thaliana)
- Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524 )
- LB Broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897 )
- Magnesium chloride anhydrous, ≥ 98% (Sigma-Aldrich, catalog number: M8266 )
- Sodium chloride BioXtra, ≥ 99.5% (Sigma-Aldrich, catalog number: S7653 )
- Potassium hydroxide (Sigma-Aldrich, catalog number: 484016 )
- Silwet® L-77 (PhytoTechnology Laboratories, catalog number: S7777 )
Equipment
- Tweezers with Sharp Tip (Labdirect, catalog number: 560.002.115 )
- Top centrifuge (Eppendorf)
- Vortex (VWR digital Vortex Mixer)
- 28 °C shaker-incubator
- Growth chamber (Percival Scientific or similar types. Set the growth conditions to 22 °C, 16 h light/8 h dark cycles)
- TissueLyser II© (QIAGEN)
- Autoclave (TOMY SEIKO, model: LSX-500 or similar types)
- Mechanical pipettes (Gilson, catalog numbers: F123601 (P200) and F123602 (P1000)
- Analytical balance (0.1 mg resolution)
Procedure
Category
Microbiology > Microbe-host interactions > Bacterium
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