Amylase 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 starch 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, flood plates with 1% iodine solution and allow to incubate for 10 mins.
8. After 10 mins, decant iodine solution
9. Observe colonies for clearing zones around colonies.
10. 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 starch 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, flood plates with 1% iodine solution and allow to incubate for 10 mins.
9. After 10 mins, decant iodine solution
10. Observe colonies for clearing zones around colonies.
11. 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 starch 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, flood plates with 1% iodine solution and allow to incubate for 10 mins.
7. After 10 mins, decant iodine solution
8. Observe colonies for clearing zones around colonies.
9. Measure diameter of all observed clearing zones.

Citations:

• Yassin et al 2021. Screening and Characterization of Thermostable Amylase-Producing Bacteria Isolated from Soil Samples of Afdera, Afar Region, and Molecular Detection of Amylase-Coding Gene
• Lal and Cheeptham 2012. Starch Agar Protocol