Jan 23, 2026
Isolation and Functional Screening of Native Plant Growth-Promoting Bacteria from Agricultural Soil
- Namrata Joshi 1
- 1University of Warsaw
- PathFood Published Protocols
Protocol Citation: Namrata Joshi 2026. Isolation and Functional Screening of Native Plant Growth-Promoting Bacteria from Agricultural Soil. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1dx5g5r/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: January 23, 2026
Last Modified: January 23, 2026
Protocol Integer ID: 240697
Keywords: bacteria from agricultural soil, selective recovery of bacillus, promoting bacteria, pseudomonas species, bacillus, pseudomonas species through heat, paenibacillus, functional screening of native plant growth, functional characterization of native plant growth, agricultural soil, native plant growth
Funders Acknowledgements:
PATHFOOD - Creating a Sustainable Functional Food Chain: A Pathway to EU Climate Neutrality and Improved Health
Grant ID: 101160011
Abstract
This protocol outlines a structured methodology for the isolation and functional characterization of native plant growth-promoting bacteria (PGPB) from agricultural soil. The procedure focuses on the selective recovery of Bacillus, Paenibacillus, and Pseudomonas species through heat-shock enrichment and specialized media.
Troubleshooting
Before start
Colony-forming units (CFU) were counted on plates containing 30-300 colonies.
CFU/g soil = Number of colonies×dilution factor×10/soil mass (g)
Bacterial abundance was calculated as CFU per gram of dry soil.
Isolation of Native Soil Bacteria
Sampling:
Agricultural soil samples were collected at a depth of 0-20 cm.
Samples were transported to the laboratory under sterile conditions and processed within 24 h.
Sample Processing:
Ten grams of soil were suspended in 90 mL of sterile saline solution (0.85% NaCl) and homogenized by vigorous shaking. Serial tenfold dilutions were prepared up to 10^-6.
Enrichment and Isolation of Bacillus spp. (Heat-Shock Method):
Selective enrichment for spore-forming bacteria was performed according to the heat-shock protocol described by Priest (1987).
- Transfer 1 mL of soil suspension into a sterile 1.5 mL microcentrifuge tube.
- Heat at 80°C for 10 min to eliminate non-spore-forming bacteria.
- Immediately cool on ice for 2 min.
- Plate 100 µL of undiluted or 10-1-10-4 dilutions onto Luria–Bertani (LB) agar.
- Incubate plates at 30°C for 48 h.
Enrichment and Isolation of Pseudomonas spp.:
Selective isolation of fluorescent Pseudomonas spp. was conducted using King’s B medium (King et al., 1954).
- Plate 100 µL of 10-3 and 10-4 soil dilutions onto King’s B agar.
- Incubate at 30°C for 24-48 h.
- Examine plates under UV light (365 nm) in a dark room.
Colony Enumeration
Colony-forming units (CFU) were counted on plates containing 30-300 colonies.
CFU/g soil = Number of colonies×dilution factor×10/soil mass (g)
Bacterial abundance was calculated as CFU per gram of dry soil.
Purification and Preservation
Eight to ten morphologically distinct colonies from each medium were selected.
Isolates were restreaked twice to ensure purity.
Pure cultures were preserved as glycerol stocks (800 µL overnight culture + 200 µL sterile 80% glycerol) and stored at -20 °C.
Screening for Plant Growth-Promoting Traits
Phosphate solubilization:
Use Pikovskaya or NBRIP agar (Cas(PO4)2) at 30 °C for 5 days. Positive indicator: Clear halo; PSI = (halo + colony)/colony.
Siderophore production:
Perform CAS assay (agar or liquid) at room temperature for ≤ 2 h. Positive indicator: Orange/yellow color change.
Indole-3-acetic acid (IAA) production:
Use Jensen broth + 0.2–0.5 g/L L-tryptophan; Salkowski reagent at 30 °C for 48 h. Positive indicator: Pink coloration or increased A_530.
Potassium solubilization:
Use Aleksandrov agar at 28–30 °C for 5–7 days. Positive indicator: Clear halo; SI = (halo + colony)/colony.
Ammonia (NH3) production:
Use Nessler’s reagent at 28–30 °C for 48–72 h. Positive indicator: Brown coloration; absorbance at 450 nm.
Taxonomic Confirmation of Bacterial Isolates
Taxonomic identification of bacterial isolates should be performed using partial to near-full-length 16S rRNA gene sequencing.
Growth Performance and Compatibility Assessment
Growth Rate Determination (Doubling Time):
Growth kinetics of selected isolates were evaluated to identify fast-growing candidates suitable for inoculum formulation.
Isolates were inoculated into LB broth to an initial optical density (OD_600) of approximately 0.02.
Cultures were incubated at 30 °C with shaking at 180 rpm.
OD_600 measurements were recorded at 0, 2, 4, and 6 h (or every 30-60 min using a microplate reader).
Data analysis:
The specific growth rate (μ) was calculated from the linear regression of ln(OD_600) versus time during the exponential phase. Doubling time (T_d) was computed as: T_d = ln2/μ.
Selection criterion:
Isolates with a doubling time (T_d) 3c 2 h were retained for further evaluation.
Cross-Streak Compatibility Assay:
Compatibility between candidate isolates was assessed to evaluate their suitability for co-inoculation.
Isolate A was streaked centrally on LB agar plates and pre-incubated for 12 h at 30 °C.
Isolate B was then cross-streaked perpendicularly to isolate A.
Plates were incubated for an additional 24 h at 30 °C.
Scoring of antagonism: 0 = No inhibition; -1 = Partial inhibition; -2 = Strong inhibition.
Selection criterion: Isolate pairs exhibiting no or minimal antagonism (scores 0 or -1) were considered compatible and selected for inoculum formulation.
Protocol references
Priest, F.G. (1987). Applied and Environmental Microbiology, 53, 1263–1266.
King, E.O., Ward, M.K., 26 Raney, D.E. (1954). Journal of Laboratory and Clinical Medicine, 44, 301–307.
Acknowledgements
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