Spot Test 8.27.18

Rationale)

To conduct a spot test using enriched and direct isolation lysate from Soil Sample A in the hopes of achieving plaque that can then be picked and the phage that caused it isolated, and if no plaque is found determine the next steps in regards to conducting a plaque assay and potentially collecting another soil sample.

Scientific Question)

Does the presence of arthrobacter phage vary amongst oak tree species found on Baylor’s campus?

Procedures)

1. Set up an aseptic zone using CiDecon, 70% ethanol, and an ethanol flame.

2. Retrieve the 50mL conical vial of “Soil A Lysate Pt2 Enriched” from the refrigerator. Under aseptic conditions use a .22μ filter syringe apparatus and syringe, filter about 1.25mL of “Soil A Lysate Pt2 Enriched” into a micro test tube labeled, “8.27.18 NMN Lysate for Spot Test”, put the remaining unfiltered lysate back into “Soil A Lysate Pt2 Enriched”

3. Retrieve plate with base agar and divide/label in three sections: Direct Isolation, Enriched, and Negative Control, also label the plate as Spot Test NMN 8.27.18, retrieve another plate with base agar and label as the “Top Agar Control 8.27.18 EAG, NMN, SS”.

4. Retrieve 50mL conical vial and label 8.27.18 “Top Agar for Spot Test Control”.

5. For the “Top Agar for Spot Test Control” use M1V1=M2V2 to calculate for the total amount of 1M CaCl2 required for the Top Agar solution to have the CaCl2 be 4.5 mM, this number ends up being 42.75μL, use a micropipette under aseptic conditions to transfer this amount to the 50mL conical vial labeled “Top Agar for Spot Test Control”.

6. Using a 10mL serological pipette transfer 4.5mL of LB broth to the 50mL conical vial labeled “Top Agar for Spot Test Control”  under aseptic conditions, unfortunately, the cap of the vial “Top Agar for Spot Test Control”  was moved out of the aseptic zone in which this procedure was suppose to be conducted.

7. Using a 10mL serological pipette transfer 5mL of top agar from its container to the 50mL conical vial “Top Agar for Spot Test Control” under aseptic conditions and seal with the cap.

8. Mix 50mL conical vial “Top Agar for Spot Test Control” by inverting the vial repeatedly, after doing so for about 15 seconds pour into the plate labeled “Top Agar Control 8.27.18 EAG, NMN, SS” under aseptic conditions. Cover the plate and let rest for 15 minutes before placing in the incubator.

9. Retrieve a 50mL and label “NMN Top Agar Spot Test 8.27.18”

10. Retrieve LB broth and 4.5mL to “NMN Top Agar Spot Test 8.27.18”  under aseptic conditions using a 10mL serological pipette.

11. Retrieve the red-capped vial of .5mL Arthrobacter and pour into “NMN Top Agar Spot Test 8.27.18” under aseptic conditions.

12. Add .45μL of 1M CaCl2 to vial “NMN Top Agar Spot Test 8.27.18”, as was derived by M1V1=M2V2 in order to achieve a final molarity 4.5mM of CaCl2, using a 20-200μL micropipette under aseptic conditions.

13. Add 5.0mL of 2xTop Agar to “NMN Top Agar Spot Test 8.27.18” using a 10mL serological pipette under aseptic conditions, cover “NMN Top Agar Spot Test 8.27.18” with the cap and swirl to incorporate for 30 seconds.

14. Pour the 50mL conical vial labeled “NMN Top Agar Spot Test 8.27.18” into the plate labeled “Spot Test NMN 8.27.18” under aseptic conditions, cover the plate and let solidify for 15 minutes.

15. After 15 minutes has passed and the plate “Spot Test NMN 8.27.18” has solidified use a 0-10μL micropipette to transfer 6μL of  Direct Isolation from the vial labeled “NMN Soil A Direct Isolation 8.24.18” onto the corresponding zone of the divided and labeled plate “Spot Test NMN 8.27.18”, repeat this with “8.27.18 NMN Lysate for Spot Test” and Phage Buffer Solution. All of this is done under aseptic conditions.

16. Let the plate rest for about 6 minutes and then place into the incubator. Clean the area and leave.

Observations)

I observed some air bubbles in my top agar which could be mistaken as plaques if not careful. I also observed that there were minor instances in which contamination could have occurred and affected the results, additionally the top agar we produced was much more golden in appearance compared to the base agar.

Conclusions/Next Steps)

The next time I do this I need to wait longer, closer to 15 minutes, for the samples to absorb onto the top agar instead of the 6 minutes I waited for. I also need to take greater care to avoid contamination of materials and equipment that should be kept in an aseptic zone as there were minor instances in which contamination could have occurred when I conducted the procedures. We also now have a spot test that can be used to determine if we have a potential phage we could isolate based on positive or negative plaque formation, which will be determined on 8.29.18. I will also conduct a plaque assay on 8.29.18 to provide further information about the presence or absence of phage in lysate derived from Soil Sample A.