August 31

Spotting Test for Soil A 8.27.18

Research Question:

…………To find out how the presence of bacteriophages in the soil of oak trees would affect the pathological process of Oak Wilt Disease and if the variation in oak species has a correlation with the presence of bacteriophages.

Rationale:

………….To test if sample Soil A has the presence of bacteriophages, a spotting test will be administered to Soil A Direct Isolation & Enrichment. By adding drops of lysate from sample Soil A to a bacterial lawn on the agar plates, we can determine if bacteriophages are present in the lysate or not.

Experimental Procedure for Spotting Test for Soil A:

……………………. 1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% …………………………Ethanol and let it evaporate) on the workbench, prepare:

…………………………………………………….(1) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

……………………..2. Add 5 ml LB Broth, 45 ul 4.5mM CaCl2 (ag) to a new 50 ml conical tube (A) using …………………………Serological Tubes.

……………………………….4.5mM x 10ml = 1000mM x V, V=0.045ml=45ul

……………………..3. Add 5 ml of 2x TA to the solution in the new 50 ml conical tube (A) and pipette …………………………with Serological tube before pouring onto the agar plate, slightly swirl the plate …………………………to let it cover the whole plate evenly and set still. (labeled Group 5 Control …………………………8.27.18)

……………………..4. Add 13.5 mL LB Broth, 135 ul CaCl2 (aq) & 1.5 ml Arthrobacter to a different 50 …………………………mL conical tube (B).

……………………..5. Add 5 ml of 2x TA to 50 ml conical tube (B) and pipette with serological tube …………………………before pouring 10 ml on three plates, then discovers an error occurred during …………………………step 3. 4. & 5. , discard plates and restart.

……………………..6. Add 4.5 ml LB Broth, 0.5 ml Arthrobacter & 45 ul CaCl2 (aq) to a 50 ml conical …………………………tube (C) using micropipette.

……………………..7. Section a new Agar Plate into 3 sections, each label C (Phage Buffer as control), ………………………..(D) Direct Isolation & (E) Enrichment. (Plate labeled: JY5 Spotting test 8.27.18)

……………………..8. Add 5 ml of 2x TA to 50 ml conical tube (B) and pipette with serological tube …………………………before adding the solution on to a new Agar plate, slightly swirl the plate to let it …………………………cover the whole plate evenly and wait for 10 min.

……………………..9. Filter 2 ml Soil A Enrichment Lysate with filter paper (0.22um) to filter out ………………………….bacteria into an Eppendorf. (labeled: JY5 8.27.18)

…………………….10. Use micropipette to drop 1 ml of Phage Buffer, Direct Isolation Lysate & ………………………….Enrichment Lysate to the middle of each section and set still for 15 min before ………………………….placing in incubator.

Observations:

……………….During the adding of top agar, several bubbles formed on the plate, which is caused by adding the.solution too fast.

…………………As soon as 2x TA is added to the solution it starts to solidify, so right away when I tried to poke out the bubbles using pipette tips the bubbles wouldn’t pop, which I presume is due to the agar has already solidified.

Results & Data:

………………….The Spotting Test set up was successfully completed, after waiting for 48 hrs it will show whether there are bacteriophages in both Direct Isolation Lysate & Enrichment Lysate from sample Soil A.

…………………..The Spotting Test Plate is Labeled: JY5 Spotting Test 8.27.18.

Interpretations & Conclusions:

…………………..If preparing a solution more all at once, such as all in one conical tube for one group, ALWAYS double check the math before adding anything.

…………………..When adding the Top Agar to the Agar plate, always add at a moderate speed in case of any bubbles forming.

Next Step:

……………………If the spotting test on Soil A yields positive results than I am one step closer to the purification of bacteriophage. I will be doing Plaque Assay on sample Soil A in the next lab.

……………………In the next lab, I will need: