26 September 2018 ✷ Spot Test #3, again

Rationale: A spot test will be performed to test for the presence of phage from the third soil sample collected because the data from the previous lab period was contaminated and thought to have used the incorrect type of bacteria.

Procedure

• The workspace was cleaned with CiDecon and 70% ethanol
• A tube with enough plate mix for two tests was mixed (fourth column) and a control plate mix was made (column 2), until it was discovered that there was not enough arthrobacter in the lab for two test plates. At this point, the single “control” mix became the tube of plate mix for the spot tests of two people and the plate had 5 circles for a negative control, two direct lysates, and two enriched lysates. The volumes and concentrations of their components can be seen below.

• component	volume (single)	concentration	volume (double)	concentration
  2X Top Agar	2.5 mL	1X	5 mL	1X
  LB Broth	2.1 mL	–	4.2 mL	–
  1M Calcium Chloride	23 µL	4.5 M	45 µL	4.5 M
  Arthro	400 µL	–	0	–

• The plates were poured immediately and were allowed to set for 10 minutes.
• The enriched sample was filtered last lab period and used for the spot test. 5 µL of the filtered enriched sample was added to the designated space on the plate for each group member. 5 µL of phage buffer was added to the plate as a control, and 5 µL of the direct sample was added to the plate for each group member.
• The plate was then incubated until Monday.

observations, results, data

There may be confusion with positive results in this test because two peoples’ samples were tested on the same plate due to lack of resources. However, because both lysates came from soil collected from the same tree, it is likely that if one is positive, both will be positive for phage.

interpretations. conclusion, next steps

One group member ran a plaque assay and two ran spot tests. If all 3 members receive negative results back, a new soil sample will need to be collected to find a phage. If positive results are seen, a plaque will be picked and a phage will be isolated.

Arthrobacterphage only infects arthrobacter, so the strain of bacteria accidentally used on Monday would need to be slightly different in order for the positive controls, which were remarkably strong titers. to be negative.