Protease activity assay

Direct environmental plating

Gram negative populations:

1. 5 g of processed soil is weighed out into a plastic weigh-boat.
   • Record the weight of the soil sample and empty weigh-boat.
2. Place the dry soil into 50 ml cold sterile DI-H~2~O
3. Place flask on orbital shaker at ~150 rpm for 1 h.
   • NOTE: It is best to shake the soil solution in the cold.
     • If a refrigerated orbital shaker is not available, the flask can be shaken for 20 min, then refrigerated for 30 mins, and repeat until soil solution has been shaken for 1 h 20 mins.
       • Note: This shake-refrigerate method requires longer total shaking time.
4. Once shaking is finished, serial dilute 10-fold soil solution up to 5 times, for a final 10^-5^ dilution.
5. Plate 100 ul of dilutions 10^-5^, 10^-4^, 10^-3^ on to skim milk agar.
6. Incubate plates at 28^o^C for 2-3 days.
   • Incubation timing can be adjusted to meet growth rates of isolates if known
7. After 2-3 days, observe colonies for clearing zones around colonies.
8. Measure diameter of all observed clearing zones.

Gram-positive and spore forming populations:

1. 5 g of processed soil is weighed out into a plastic weigh-boat.
   • Record the weight of the soil sample and empty weigh-boat.
2. Place the soil sample in the chemical fume hood, covered by sterile cheese cloth, for 36 h to dry.
3. Place the dry soil into 50 ml cold sterile DI-H~2~O
4. Place flask on orbital shaker at ~150 rpm for 1 h.
   • NOTE: It is best to shake the soil solution in the cold.
     • If a refrigerated orbital shaker is not available, the flask can be shaken for 20 min, then refrigerated for 30 mins, and repeat until soil solution has been shaken for 1 h 20 mins.
       • Note: This shake-refrigerate method requires longer total shaking time.
5. Once shaking is finished, serial dilute 10-fold soil solution up to 5 times, for a final 10^-5^ dilution.
6. Plate 100 ul of dilutions 10^-5^, 10^-4^, 10^-3^ on to skim milk agar.
7. Incubate plates at 28^o^C for 2-3 days.
   • Incubation timing can be adjusted to meet growth rates of isolates if known
8. After 2-3 days, observe colonies for clearing zones around colonies.
9. Measure diameter of all observed clearing zones.

Individual isolate

1. Inoculate a single isolated colony to isolate appropriate liquid rich media.
2. Incubate for 48 h at 25^o^C with shaking.
3. After incubation adjust culture to OD~600nm~ 0.1
4. Place 4-5 ul drops of adjusted culture in four quadrants of a skim milk agar plate, spaced equidistant from each other.
   • NOTE: Unless unavoidable, do not inoculate different isolates onto the same plate.
5. Incubate plates at 28^o^C for 2-3 days.
   • Incubation timing can be adjusted to meet growth rates of isolates if known
6. After 2-3 days, observe colonies for clearing zones around colonies.
7. Measure diameter of all observed clearing zones.

Citations:

• Verma et al 2016. Production of Thermostable Organic Solvent Tolerant Keratinolytic Protease from Thermoactinomyces sp. RM4: IAA Production and Plant Growth Promotion
• Frarjzadeh et al 2012 Plant Growth Promoting Characterization of Indigenous Azotobacteria Isolated from Soils in Iran
• Caravaca and Roldan 2007. Interactions between a plant growth-promoting rhizobacterium, an AM fungus and a phosphate-solubilising fungus in the rhizosphere of Lactuca sativa
• Grobelak et al 2015 Using plant growth-promoting rhizobacteria (PGPR) to improve plant growth
• Ahmadzadeh and Tehrani 2009 Evaluation of fluorescent pseudomonads for plant growth promotion, antifungal activity against Rhizoctonia solani on common bean, and biocontrol potential