Lipase 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 lipid 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 lipid 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 lipid 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

• Gupta et al 2004. Bacterial lipases: an overview of production, purification and biochemical properties
• Ghosh et al 1996. Microbial lipases: production and applications
• Kouker and Jaeger 1986. Specific and Sensitive Plate Assay for Bacterial Lipases
• Margesin and Schinner 2000. Monitoring of bioremediation by soil biological activities
• Griebeler et al 2009. Isolation and Screening of Lipase-Producing Fungi with Hydrolytic Activity
• Park et al 2009. Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth‐promoting activities
• Wang et al 2018. Plant growth promotion and alleviation of salinity stress in Capsicum annuum L. by Bacillus isolated from saline soil in Xinjiang
• El-Deeb et al 2011. Characterization of endophytic bacteria associated with rose plant (Rosa damascena trigintipeta) during flowering stage and their plant growth promoting traits