Experiment 7 Screening of Pseudomonas sp for Pesticide Tolerance Aim Principle To screen and identify Pseudomonas sp isolates capable of tolerating and degrading pesticides using growth and tolerance assays in the presence of specific pesticide concentrations The ability of Pseudomonas sp to tolerate and degrade pesticides can be evaluated by culturing them in a medium containing specific concentrations of pesticides as the sole carbon source The growth of the bacteria in the presence of pesticides indicates their tolerance and potential biodegradation capabilities The evaluation is based on monitoring bacterial growth and comparing it to control conditions without pesticides Pseudomonas sp are well known for their metabolic versatility including their ability to degrade various organic compounds which makes them promising candidates for bioremediation particularly in environments contaminated with pesticides Materials 1 Bacterial culture o Pure cultures of Pseudomonas sp isolates 2 Pesticides 3 Media o Select a pesticide commonly found in contaminated sites e g chlorpyrifos atrazine or malathion o Luria Bertani LB broth and agar for initial growth o Minimal salt medium MSM for pesticide degradation composition per liter of distilled water Na2HPO4 6 0 g KH2PO4 3 0 g NH4Cl 1 0 g MgSO4 7H2O 0 5 g CaCl2 0 1 g FeSO4 7H2O 0 05 g 4 Equipment o Autoclave for sterilization Incubator 37 C o o Shaker incubator o UV Vis spectrophotometer for OD600 readings o Sterile pipettes spreaders and loops o Sterile test tubes flasks and petri dishes 5 Reagents o Pesticide stock solutions prepare in sterile water or solvent o Sterile distilled water o Ethanol or acetone if required to dissolve pesticides Methods 1 Preparation of Inoculum Subculture Pseudomonas sp isolates on LB agar plates and incubate at 30 C for 24 hours Pick a single colony from the LB plate and inoculate it in 10 mL of LB broth Incubate overnight at 30 C with shaking at 150 rpm After overnight incubation adjust the bacterial suspension to a standardized optical density OD600 0 5 which corresponds to approximately 10 CFU mL 2 Preparation of Minimal Salt Medium MSM Prepare MSM and sterilize by autoclaving at 121 C for 15 minutes Cool the MSM and aseptically add the pesticide of choice at varying concentrations e g 10 ppm 50 ppm 100 ppm and 200 ppm 3 Screening for Pesticide Tolerance o Label sterile 250 mL flasks for each pesticide concentration 10 ppm 50 ppm o o o 100 ppm 200 ppm and include a control without pesticide Inoculate each flask with 1 mL of the prepared Pseudomonas sp inoculum 10 CFU mL Incubate the flasks in a shaker incubator at 30 C and 150 rpm for 5 7 days Include flasks with MSM containing no pesticide control inoculated with the bacterial culture o Also set up uninoculated controls with MSM and pesticide to monitor abiotic degradation 4 Growth Monitoring At intervals e g 24 48 72 hours measure the bacterial growth by taking 1 mL aliquots from each flask and determining the optical density OD 600 using a spectrophotometer Plot growth curves OD 600 vs time to determine the pesticide tolerance of the Pseudomonas sp isolate 5 Interpretation of Results Tolerance Inhibition Significant bacterial growth high OD600 in the pesticide containing flasks compared to the control indicates tolerance to that concentration Little or no growth low OD600 suggests that the pesticide is inhibiting the bacteria at that concentration Reduction in pesticide concentration from initial levels in the media confirms Degradation optional biodegradation by the bacterial isolate Result The Pseudomonas sp isolates demonstrated growth in MSM with pesticides at the concentration of 10 ppm which can be considered tolerant to the pesticides