8/31/18

Rational:

To check and see if the plaque assay has any plaques and to comfirm the results from the spot test.

Procedure:

• Took the plaque assay and control plates out of the incubator at 2:30 (46.5 hrs in the incubator)
• Observed no contamination on the control plate for group 2
• The arthrobacter lawn looked rough, but there was no signs of a plaque

Future Steps:

By next lab I will get a new soil sample. Recording pictures, data, and observations about the tree that the soil sample was taken from. On Wednesday I will filter the soil sample to prepare for  new spot test and plaque assay.

Fig.8 – The plaque assay had a grainy texture, but no signs of a plaque were seen

*Note the soil sample was not collected on 9/5, but on 9/7. Also the filtration was not done on 9/5.

8/29/18

Spot Test Soil A Results and Plaque Assay Soil A

Rational:

To confirm the results of the spot test on soil A with a plaque assay of soil A. This will give results if the sample was contaminted and will confirm results found in the spot test.

Procedure:

• Cleaned the lab desk with CiDecon and 70% ethanol
• Labeled the plate plaque assay soil A with enriched
• Labeled conical vial team 2 TA
• Added 22.5 ML CaCl2 using a micropipette
• LB broth splashed out preventing us from knowing how much was added- conical vial was thrown out
• Labeled new vial team 2 TA
• Added 22.5 ML CaCl2 using micropipette
• Added 2.0 mL LB broth using a serological pipette
• In order to have 5 mL for each team member from the vial and 1 mL for the control plate we added 67.5 ML more CaCl2 with a micropipette and 6 mL LB broth with 10 mL pipette
• Labeled tube with arthrobacter (0.5 mL arthrobacter) arthrobacter with enriched soil A
• Added 10 ML lysate to the tube with a micropipette
• Set a timer for 15 min
• Added 10 mL of TA
• Added 5 mLmof TA mixture to the tube with the lysate (some spilled) (KEA and my tubes were mixed up so the plates were re-labeled)
• Added 1 mL of TA mixture without lysate to the control plate
• Put plates in the incubator at 26 C at 4:00 8/29
• Cleaned lab desk with CiDecon and ethanol

Results:

*results from spot test on 8/27                                                                                                                                   Contamination was found on the control plate as well as my plate so plate                           

fig.4 – shows where contamination and bubbles                  fig.7 – shows the plate after incubating for 46 hrs.               are found on the plate

fig.5 – shows where contamination was found                     fig.6 -show the dots of contamination on the control plate  on the control plate

Analysis and Interpretation:

The results from the spot test indicated that there was no bacteriophage present in the soil A, however the contamination observed prevents this from being confirmed in the spot test.

Future Plans:

I plan to check the plaque assay for plaques during the next lab. If no plaques are present then a new soil sample will be collected. The new soil samplewill then have LB broth added and filtered. Then a new spot test and plaque test will be done (not all in one lab). If a plaque is observed the plate will be saved and a new test to confirm the presence will be done. If there is contamination the test will be done over trying to prevent contamination again.

8/27/18

Spot Test Soil A

Rational:

This experiment will test for the presence of bacteriophage the specifically infect arthrobacter in the soil sample A. A spot test will be used in order to test this.

Procedure:

• Cleaned lab table with CiDecon and ethanol
• Used a 2 Mm filter on the enriched soil A to filter it out in order to get 1.5 mL filter sterile (FS) enriched lysate soil A in a microcentrifuge tube
• Labeled agar plate with FS lysate soil A, direct isolation soil A, and negative control
• Top agar- 0.5 mL arthrobacter sp. , 4.5 mL LB broth, 5.0 mL 2X TA, and 45.0 ML CaCl2 in a tube (CaCl2 calculations- (1,000 mM)*V1=(4.5 mM)*(10,ooo ML) V1= 45.0 ML CaCl2)
• For control CaCl2 is 42.75 ML (rounded to 42.8 ML) (calculations- (1,000 mM)*V1=(4.5 mM)*(9,500 ML) V1= 42.75 ML)
• 45.0 ML of CaCl2 was added to TA mixture
• Added 4.5 LB broth to TA mixture using serological pipette using 5 mL tip
• Added 0.5 mL arthrobacter under the clean hood using a micropipette
• Added 5.0 mL TA using serological pipette with a 10 mL tip
• Added TA mixture to plate and waited 10 min for agar to solidify
• Added 10 mL of direct isolation to plate
• Added 10 mL of FS lysate
• Added PB (phage buffer) to negative control quadrant on the plate
• Incubated plate at 26 C from 4:30 8/27 – 2:30 8/29 (46 hrs)
• Cleaned lab desk with CiDecon and ethanol

*see results on next entry

Future Plans:

During the next lab I will check the results of the spot test. If I observe a plaque I will save the plate and do a plaque assay for the direct isolation and direct lysate for soil A. If there is no plaque I will throw away the plate after recording data and do a plaque assay with enriched lysate soil A to confirm this. If the plate was contaminated then I will do a plaque assay with enriched lysate soil A.

*Note: there were a few bubbles between the negative control and the direct lysate (see fig 3 and 4       

fig.3 – shows where the bubbles are located       fig.2 – the plate after TA was added ( you can see where some of the      on the plate                                                                bubbles are located)

8/22/18

Filtration Soil A

Rational:

The soil sample will be filtered out in order to get a sample containing only bacteriophages in the broth. This will allow me to test for the presence of bacteriophage that infects arthrobacter since the bacteriophage will eventually be placed in and environment containing only arthrobacter (no other bacteria).

Procedure:

• Cleaned the lab desk with CiDecon and 70% ethanol
• Used burner to set up an aceptic zone and added 40 mL of broth to the soil sample A (weight 60.07 mg)
• Shook the tube for 15 min to break up the soil sample A
• The soil sample was then matched with another sample with a similar weight and centrifuged for 5 min at 3,000 G
• The liquid at the top of the sample was then filtered through a top filter
• The filtered lysate was then separated into a direct isolation sample of 8.3 mL and an enriched sample of 10 mL (with arthrobacter added)
• Lab desk was cleaned with CiDecon and ethanol

Future Steps:

I will do a spot test and later a plaque assay using the direct isolationa and enriched lysate in order to test for the presence of bacteriophage that infect arthrobacter in the soil sample A

fig.1 – shows the layers seen in the soil sample                                                                                                                          after the sample was centrifuged