9.12.18 Soil Metadata and Spot Test

Rationale)

I will finish collecting metadata about Soil B and conduct a spot test with enriched lysate produced from Soil B, in the effort of determining if an isolatable phage is present in Soil B.

Procedure)

1. Set up an aseptic zone by cleaning the work surface with CiDecon and 70% ethanol, and lighting an ethanol flame.
2. Collect 50mL conical vial labeled “NMN 9.10.18 Soil B Enriched Lysate”  and add enough DI water using a pipette that mass of the vial registers 22.043g. Proceed to centrifuge the vial at 3000g for 5 minutes to pellet the arthrobacteria.
3. While the vial is centrifuging, determine the percent water of Soil B by taking the mass of the weigh dish labeled “NMN %H2O” which holds now dried sample of Soil B from Monday. The new mass of the soil is 3.197g and comparing that to Monday’s weight of 3.72g that means a total of .523g of water evaporated. And dividing the grams of water over the original soil mass and multiplying by 100 gives us a percent of water that is equal to 14.059%.
4. Observe the now settled middle-back falcon tube and use the markings to determine the total milliliters of the sand, silt, and clay in the sample and record. Sand: 2mL, Silt: .5mL, Clay: .5mL, dispose of the soil in the biohazard container and wash out the tube for later reuse.
5. Collect LB broth, a petri dish with base agar, and a 50mL conical vial that is labeled “Top Agar for Spot Test, Group 3 9.12.18”.
6. Label the plate by dividing it into four sections and write the initials of each Group 3 member in a section(NN, HB, SS), leaving one section blank to act as a control. On the rim of the plate label it “Spot Test 9.12.18”.
7. Using a 10mL serological pipette transfer 2mL of LB broth to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions, add .5mL of arthrobacter to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions as well.
8. Add 22.5 microliters of 1M CaCl2 using a 10-100 microliter pipette to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions.
9. Add 2.5mL of 2xTop Agar to the 50mL conical vial labeled “Top Agar for Spot Test, Group 3 9.12.18”, cap the vial and swirl for 10 seconds to mix. Pour the contents of the vial onto the previously labeled plate, shaking the plate slightly in order to distribute the top agar evenly. Wait 5 minutes for it to solidify.
10. Retrieve a .22 micron syringe filter and a 3mL syringe and under aseptic conditions filter enough of “NMN 9.10.18 Soil B Enriched Lysate” to fill a micro test tube, label the test tube “Filtered Lysate Enriched NMN 9.12.18”. Dispose of the vial and filter in the biohazard bag.
11. Add 10 microliters of “Filtered Lysate Enriched NMN 9.12.18” to the section of the now solidified plate in the section labeled “NN”. Also, add 10 microliters of Phage Buffer to the control section of the plate. All of this is conducted under aseptic conditions using a 2-20 micropipette.
12. Let the plate “Spot Test 9.12.18” sit for 15 minutes then store inverted in the incubator for 48 hours.
13. Clean the work area with CiDecon and 70% ethanol, and place any remaining equipment in the biohazard bag or the appropriate autoclave container.

Data/Observations)

The mass of the dried soil is 3.197g. The mass of the pre-dried soil was 3.72g. The mass of water in the soil is therefore .523g. The percent of water in Soil B is therefore 14.059%.

The volumes of Sand, Silt, and Clay are Sand: 2mL, Silt: .5mL, Clay: .5mL. Therefore the soil composition is: Sand: 66.66%, Silt: 16.66%, Clay: 16.66%.

I also observed that the pellet of arthrobacter was rather large, and left the lysate much clearer than it was before the vial was centrifuged. Additionally, it was relatively difficult to use the syringe to extract lysate from the 50mL conical vial which could increase the potential for contamination.

Conclusions/Next Steps)

From the data gathered it can be determined that the soil from which Soil Sample B has an average water percentage of 14.059%, additionally the next steps for collecting soil metadata will be to use a soil composition chart to determine the soil type for Soil B based upon the gathered percentages of sand, silt, and clay. In regards to the spot test, the next step will be to check on Friday for the presence of plaques in my section of the plate, which if positive will require the need to conduct a plaque assay with the same enriched lysate used for the spot test. Additionally, we will also begin the process of picking and isolating the plaques in order to isolate the phages that caused them. If the test comes back negative we will have to proceed to collect another soil sample, in the effort to find a phage that can be isolated and eventually sequenced.