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)