Rationale: A spot test was performed in order to display the results of Soil Sample A. The results will how whether or not a bacteriophage or bacteriophages are present through existence of plaques on a bacterial lawn.

Scientific Research Question: Does the species of oak tree cause any discrepancy in number or type of phages found in soil samples? **Yet to be confirmed**

Procedure Performed in Lab:

1. Work site was cleaned using CiDecon (applied and wiped dry) and 70% ethanol (applied and wiped until half dry). An aseptic zone was established by setting up an ethanol burner near the center of the bench that the experiment and aseptic steps were completed close to.
2. Enriched lysate labelled as “HMB Arthro Added 8/22” was obtained along with a syringe and 22 microliter filter for the end of the syringe.
3. (Aseptic Step) 2 mL of enriched lysate was drawn into the syringe. 22 microliter filter was placed on end of syringe, then newly formed “Filter Sterilized Enriched Lysate” (abbreviated FSEL for labelling purposes) was created by moving the 2 mL of enriched lysate inside the syringe through the filter and into a microcentrifuge tube labelled “HMB FSEL 8/27/18” and “HMB” on lid. This step was done to remove existing bacteria from the enriched lysate while allowing possible bacteriophages to still pass through the filter and into the new tube.
4. Plate with agar was obtained. On the bottom, it was divided into 3 sections (enriched, direct, and negative control) and labelled “HMB Spot Test 8/27”.
5. One 50mL Conical Tube was obtained and labelled “HMB Top Agar 8/27/18”. A second Conical Tube was obtained and labelled as the negative control. Conical Tubes held components of Top Agar before the Top Agars (one with Arthro and one without) were poured out on experimental and control plates, respectively.
6. Concentration of CaCl2 was obtained for Experimental Plate containing Arthro:
   1. (1M CaCl2)(Volume)=(4.5mM)(10mL)
   2. (1000mM CaCl2)(Volume)=(4.5mM)(10000 uL)
   3. Volume= 45 microliters of CaCl2
7. Concentration of CaCl2 was obtained for Control Plate without Arthro:
   1. (1M CaCl2)(Volume)=(4.5mM)(9.5mL)
   2. (1000mM CaCl2)(Volume)=(4.5mM)(9500uL)
   3. Volume= 42.75 microliters of CaCl2
8. (Aseptic Step) Experimental Plate Containing Arthro (labelled”HMB Top Agar 8/27/18″): 4.5mL of LB Broth was added to 50mL Conical Tube. 0.5mL of Arthro was added by Lathan Lucas to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube.
9. (Aseptic Step) Control Plate without Arthro: 4.5mL of LB Broth was added to 50mL Conical Tube. 45uL of CaCl2 was added to 50mL Conical Tube. No bacteria was added to this tube.
10. (Aseptic Step) To finish the creation of Top Agar, 5mL of Top agar solution that had been warmed in an incubator was added to both the Experimental tube and Control tube. This step was done last because Top Agar solution hardens as it cools, so as soon as it had been added to both tubes and they had been gently swirled, the 50mL Conical Tube of LB Broth, Arthro, CaCl2, and Top Agar solution was poured over the plate labelled “HMB Spot Test 8/27” and the 50 mL Conical tube containing LB Broth, CaCl2, and Top Agar was added to the negative control plate.
11. Waited 15 minutes to allow for complete solidification of Top Agar.
12. (Aseptic Step) Micropipetted 10uL of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer into their respective sections as labelled on the bottom of plate.
13. Waited 15 minutes before placing plates into the incubator. Plates will be read on Wednesday to determine the presence of a phage or not.
14. Lab station was cleaned by disposing waste products into correct locations and storing tubes/equipment in designated places. CiDecon and 70% ethanol were used to clean the bench in similar manners as they were at the beginning of the procedure.

Observations:

• Filter Sterilized Enriched Lysate was yellow-gold in color after being moved through the 22uL filter.
• Filter was said to cause resistance in pushing lysate out of syringe. However, during experiment, only slight resistance was felt during expulsion. If there are adverse results, this could be a reason why.
• Top Agar was a dark golden color while kept in the Conical tube before being plated.
• Drops of Direct Isolate, Filter Sterilized Enriched Lysate, and phage buffer momentarily stayed on surface of Top Agar before being absorbed. This caused Direct Isolate drop to slide to a location relatively close to the border between it and the next section, which should be observed and considered when taking results.

Results:
Test is not completed, so results have yet to be obtained. On Wednesday (after 48 hours have gone by), it will be possible to discern whether or not a phage is present on the plate by the presence or lack of plaques.

Next Steps: In the lab on Wednesday, a plaque assay will be performed in addition to analyzing the plates that were prepared by the spot test procedure. This will serve to confirm the results that will be obtained at the beginning of the lab session on Wednesday.