The soil sample was collected from a hole that dug about 10in deep on Sep 14th, 2021 for testing and analysis. The location was chosen because no grasses and weeds around corn lines were observed. Thus, I was doubted that the farmer used some kinds of herbicide to protect their crops from the invasion of grasses and weeds.
1. Week 0
Then, 1 gram of the soil sample was transferred to into a 14mL tube containing 6mL of LB media (lysogeny broth) to nourish the bacteria. The tube was then incubated at 30oC, 200 rpm for one week starting from 09/15/2021 to 09/22/2021.
To prepare for week 1
each of 1mL in week 0 tube was aspirated and pipetted into 4 new tubes as follows:
Tube 1: 1mL of the soil culture from week 0 into 4ml CSM and Paraoxon
Tube 2: 1mL of the soil culture from week 0 into 4mL CSM and Methyl Parathion
Tube 3: 1mL of the soil culture from week 0 into 4mL SSM and Paraoxon
Tube 4: 1mL of the soil culture from week 0 into 4mL SSM and Methyl Parathion
All the tubes were then incubated at 30oC, 120 rpm for one week
To prepare for Week 2
each of the 200 microliters in week 1 tubes was aspirated and pipetted into the 4 corresponding new tubes as follows:
Tube 1: 200 microliters of the sample from week 1 (CSM +Px) into 4ml CSM and Paraoxon
Tube 2: 200 microliters of the sample from week 1 (CSM + MP) into 4mL CSM and Methyl Parathion
Tube 3: 200 microliters of the sample from week 1 (SSM + Px) into 4mL SSM and Paraoxon
Tube 4: 200 microliters of the sample from week 1 (SSM + MP) into 4mL SSM and Methyl Parathion
All the tubes were then incubated at 30oC, 200 rpm for one week from 09/29/2021 to 10/06/2021.
As performed in week 2, the same soil inoculation technique was carried out for weeks 3, 4, and 5. After each transfer, all the tubes (week 2-5) were incubated at 30oC, 200 rpm for one week.
The yellow color which indicates the production of p-nitrophenol was lessen after each inoculation and incubation. Through weeks 2, 3, 4 and 5, CSM and SSM tubes that contained MP kept showing the negative result as the broth in those tubes remained clear and did not change to yellow.
Especially, the tube that contained CSM/Px that continuously showed evidence of OP degradation in week 1, 2, and 3 by the yellow color, eventually in weeks 4 and 5 indicated no more OPH activity. This indicates that the bacteria were not able to degrade OPs and the organophosphate compound inhibited the enzyme, acetylcholinesterase and therefore killing the bacteria.
