Alyssa Ference

Materials and Methods

Growth of Bacterial Cultures, Media, and Reagents

Gordonia terrae/Microbacterium foliorum cultures were maintained on Peptone-Yeast-Calcium
(PYC) media (1.5% peptone; 0.5% yeast extract; 4.5 mM CaCl 2; 10 μg/mL cycloheximide; with
1.5% agar for solid media). Liquid cultures were grown to saturation with shaking at 250 rpm at
30°C for approximately 2-4 days and stored for a maximum of 14 days at 4°C before use. Phage
buffer for Gordonia/Microbacterium phages was prepared as 10 mM Tris (pH 7.5), 10 mM
MgSO4, 68 mM NaCl, 10% glycerol, and 4.5 mM CaCl2. PYC Top Agar for
Gordonia/Microbacterium phage growth was prepared as 1X PYC liquid media with 0.3% agar.

Enrichment Isolation of Bacteriophages

5 mL of dirt samples were collected in a 50 mL conical tube from different areas to be
tested in the lab. Host bacteria was then picked; either G. terrae or M. foliorum. Two
samples were assigned G. terrae and one was assigned M. foliorum sample. Using a
serological pipet, 2 mL of the host bacteria chosen was pipetted into each of the three
samples. PYCa media was then added to the soil samples until the meniscus was at 20
mL. The tubes were then vortexed so that they it was mixed together. The tubes were
then placed in a shaker rack at room temperature at 250 rpm for 4 days. The samples
were processed by spinning 1 mL of the enriched sample in a microcentrifuge tube at
10,000 rpm for 1 minute 30 seconds. The supernatant was passed through a 0.22 um
syringe filter. The liquid phage samples were collected 48 hours later. Bacterial lawns
were prepared by adding 3 mL PYCa top agar to 250 µL of bacteria and left to dry. The
plates were divided into three equal sections for each sample and labeled. 10 µL of each
sample were transferred onto the correctly labeled section of the agar plates. The plates
were incubated at 30° C for 48 hours. After 48 hours, the plates were checked for
clearings. Samples showing the clearings were purified.
Purification of Bacteriophages
The positive sample was serial diluted in phage buffer to 10-4 concentration in phage
buffer. Just 10 µL of phage buffer were put in the Negative control test tube and 10 µL
of undiluted phage sample were put in the 100test tube. The test tubes contained 250
µL of G. terrae. The test tubes then sat for 10 minutes at room temperature while the
top agar was prepared. 3 mL of top agar were pipetted into each test tube and
immediately pipetted from the test tube into the serological pipette and onto the agar
plates. The plates were then incubated at 30° C for around 4 days then refrigerated for
around 48 hours. The plate contained a plaque was picked using a P20 micropipette tip
and mixed into 100 µL phage buffer. The serial dilutions and the purification process
were then repeated as described above. The plates were incubated at 30° C for 48
hours. After 48 hours, plates were analyzed and the serial dilution and purification
process were performed at least 2 more times until all plates had the same morphology.