Rationale: To conduct a spot test and plaque assay on the enriched sample on Soil Sample 2 and to gather the data from the procedures conducted last class

Materials:

Spot Test

• Lysate (direct and enriched)
• 2.0 mL of LB broth
• 2.5 mL of 2X Top Agar
• 22.5 µL of CaCl
• 0.5 mL Arthrobacter
• two agar plates
• pipettes
• microcentrifuge tubes
• syringe and filters

Plaque Assay

• 8 mL of LB broth
• 50 mL conical vial
• 1 mL enriched lysate
• 90 µL of CaCl
• 10 mL of top agar
• agar plate
• pipettes

Spot Test Procedure:

• Weigh the enriched sample in order to be put back in the centrifuge then set aside.
• Place the sample in a centrifuge to be spun at 3000 g for 5 minutes.
• Weigh the petri dish filled with soil and calculate the percentage of water.
• Pipette the supernatant out of the Falcon tube using a bulb pipette and determine the volume each component of the soil takes up.
• Serological pipette 2.0 mL of LB broth into a new vial labeled “TA 2.”
• Pipette 22.5 µL of CaCl into TA 2.
• Filter the now centrifuged enriched sample and label a microcentrifuge tube “Filtered Enriched 2” using a syringe and 0.22 µm filter.
• After filtering, add 0.5 mL of Arthrobacter to TA 2.
• Add 2.5 mL of 2X Top Agar to TA 2, stir immediately then pour the mixture in the agar plate labeled “Spot Test 2.”
• Allow the top agar to solidify for 10 minutes.
• After 10 minutes, add the direct, enriched, and phage buffer to spot test in the designated area in drops of 3-5 mL.
• Allow it to remain in an incubator for 48 hours.

Plague Assay Procedure: 

• Add 8 mL of LB to a 50 mL conical vial using a serological pipette
• Add 90 µL of CaCl to the conical vial
• Transfer 1 mL of enriched lysate from the tube labeled “FIltered Enriched 2” to a new microcentrifuge tube.
• Add 0.5 mL Arthrobacter to the 1 mL enriched and allow it to sit for 10 minutes
• Pipette 10 mL of top agar to the CaCl and broth
• Add Arthrobacter to a new vial and pipette 5 mL of TA with the enriched and Arthrobacter mixture
• Stir and pour onto the agar plate labeled “PA 2” and allow it to sit for 10 minutes.
• After 10 minutes, place the plate in an incubator for 48 hours.

Results and Analysis:

• The weight of Enriched Sample: 21.22 g
• The weight of Soil Sample 2 soil including the petri dish: 10.84
• Percentage of water in Soil Sample 2
  • (3.65/4.61)×100= 79.18%

• Components of Soil Sample 2
  • 8.5 mL / 10.5 mL is soil
    • (8.5/10.5)×100= 81%
  • 1.5 mL /10.5 mL is silt
    • (1.5/10.5)×100= 14%
  • 0.5 mL / 10.5 mL is clay
    • (0.5/10.5)×100= 5%

Soil Sample 2 Components without Supernatant

Soil Sample 2 without Water

Spot Test

(note: there are bubbles in the top agar when trying to find phage, be careful when trying to identify phages)

Plaque Assay with bubbles in Top Agar

While pipetting the filtered enriched into the microcentrifuge tube, a considerable amount of lysate did not come of the pipette.

Conclusion and Future Plans:

• We successfully completed the spot test and plaque assay procedures to see whether or not there are phages present in our soil samples. For the spot test, group 5 made one collective sample of top agar and evenly distributed it to three separate vials. We then poured the solution on the plate and then added our own samples of enriched and direct and the same phage buffer which was the third area on the plate. For the plaque assay, we also made one collective sample of top agar and separate it evenly. We then poured our Arthrobacter and filtered enriched sample onto the plate.
• In the future plan, I will check for phages in both of my tests. If there are phages present, I will go on to reconduct the test to check again and to also do a plaque assay on my direct sample.