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The Process of Isolating Unique, The Phage

Kyliah Hughes
Phages Section 12
Dr. Dickson
December 9, 2015
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Introduction: The primary goal of this study was to capture and isolate a bacteriophage. A

bacteriophage (phage) is a virus that parasitizes a bacterium by infecting it and reproducing

inside it. These bacteriophages require a living host cell in order to replicate. Phages are now

acknowledged as the most abundant microorganisms on the planet and are also possibly the most

diversified. This diversity is mostly driven by their dynamic adaptation when facing selective

pressure such as phage resistance mechanisms, which are widespread in bacterial hosts [1 and 2].

The bacteriophages that were gathered for this study are from soil. From that soil sample,

several procedures were performed to begin the isolation of a single phage. These procedures are

as follows: Enrichment of Environmental samples, Direct Plating of Environmental Samples,

The Spot Test, Plaque Streaking, The Phage Titer, MTL, and Empirical Test. All experiments

were performed under aseptic technique. This technique involves use of a flame to create an

updraft to remove airborne microorganisms. In addition, the work area is wiped with a solution

of seventy percent alcohol. Complete top agar was used as the semi- solid medium for phage

infection in the study. Top agar is a mix of equal parts top agar and 7H9. To that CaCl2 is added.

If there is 20mLof top agar and 20mL of 7H9 then 400 um of CaCl2 will be added. The equation

used is C1V1=C2V2

Methods and Materials:

Enrichment and Plating: A soil sample was collected two inches below the ground at GPS

coordinates of (38.92484N, 77.02036W). One gram of sample was enriched with 45mL 7H9,

5ml CaCl2, and 5mL U102 in an Erlenmeyer flask, centrifuged, and then incubated at 30c for 48

hours. Remaining soil was submerged in phage buffer (PB), plated without enrichment and

incubated for 20 minutes at room temperature. 1mL of that solution was filtered using a 0.22um

filter. U102 was infected with 50ml of filtrate, plated and incubated.
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Enrichment Part Two: Similar to enrichment part one, enrichment part two was conducted to

isolate phage. First, centrifuged 50mL of the enriched soil mixture. The sample was stored at 4c.

After, a 10x serial dilution from 10-1 to 10-4 of filtrate in 1X, PB was performed. Then, the sample

was added to U102, and plated with 4.5mL of complete TA. Incubated at 30c overnight. The

HUph with U102 was used for the positive control and PB with U102 was used for the negative

control.

The Spot Test: The purpose of spot assay was to determine whether putative plaques contain

phages. First, previous plates were checked for plaques. Next, new plate was labeled. After, a

lawn of bacteria was made by mixing U102 and 4.5mL TA in a culture tube and plating it. After,

six micro centrifuge tubes, with of 100mL PB pipetted into them, were labeled A-D and

assigned a putative plaque for each tube A-D. Then the phage was spotted onto the plate.

The Plaque Streak Protocol: Purpose of this step was to purify a single phage population from

samples with potentially mixed phage populations. Three sterile wooden sticks were used to

streak the selected putative plaques. The method involved gently streaking back and forth across

a third of an agar plate. Then after discarding the stick, a second wooden stick is used to streak

the 2nd third of the plate beginning by overlapping just once over the end of the previous streak.

A new wooden stick is used to streak the last third of the plate following the same protocol.

Titer: The Purpose of this step was to determine the concentration of plaque forming units. First,

a potential plaque isolate was picked and mixed into 100ul PB and 10X serially diluted to 10-4.

Then, 10ul of each sample was inoculated into a correspondingly labeled tube of U102 and

incubated for fifteen minutes. Each tube was combined with 4.5mL of TA and plated.

MTL/ Spot Test :The purpose of this experiment was to isolate enough phage stock so that

enough phage titer can be empirically tested. First, the max web plate from the titer plates was
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selected. That plate was flooded with 7mL of PB and incubated overnight. Then, 5mL was drawn

from the plate. The fluid drawn was filtered using a 0.22um filter, then 0.5mL of that filtrate was

placed in a micro centrifuge tube. After, a plate was gridded and labeled 10-1 - 10-10.

A serial dilution to 10-10 was performed. An agar plate labeled with a grid corresponding to

dilutions and controls then covered with U102bacteria and TA. Then 5uL of each sample was

pipetted onto its corresponding grid.

Empirical Test: The purpose of this process was to determine the dilutions and volumes

required to form web patterns. First, the diameter of the plate and plaque was measured and the

area of each was found and the plate area was divided by plaque area to get the pfu max web

plate. Then the dilutions were calculated using 1/3, 1/6, 1/12, 1, 3,6, and 12. After, a serial

dilution was formed from the calculations. Then, the dilutions were placed in their corresponding

U102. They incubated for twenty minutes, then they were plated with TA.

Western Blotting: The purpose of western blotting was to get a more specific identification of

protein bands. The semidry method was used to transfer proteins to a membrane. The protein is

visualized using labeled antibodies. The antibody only bonds to the protein it has been raised

against. The membrane is washed but the primary antibody will save the gel. After that, the

secondary antibody bind to the primary antibody. Then, the secondary antibodies are labeled with

a compound which shows the presence of another chemical. When the membrane is washed the

protein will appear as a colored band on the membrane.

Results

Enrichment and Plating/Enrichment Part Two Directed plated samples did not yield enough phage to give
results. Positive control showed putative plaques. That was
expected for the positive plate to have bacteria growth.

Part Two: All the plates were lysed except for the negative
control and the 10-4 dilution. Dilutions 10-1 10-3
experienced little bacterial growth and no phage activity. In
contrast, the 10-4 dilution had a lawn of bacteria and large
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putative plaques.

Spot Test The first time running this test the plate did not solidify. In
the second trial there were lysed spots because the phage
worked against the bacteria. In this round 3.5mL of solution
was used.

Streaking On the first trial the plates were shifted. For the second trial
the room temperatures helped the highly concentrated phage
area to lyse over. On the third trial the phages where starting
to become singled out. On the fourth trial the phage was not
diluted and causing it to be more isolated than the last plate.
This whole streaking step was repeated eleven times. In the
last repetitions and previous ones 100mL of PB was added to
enhance the likelihood of isolating a phage.

In the first round the plates were basically completely lysed.

The titer that was taken from the 10-4 plate was

( 3010pfu
uL ) x(
1000 ul
1 mL )
x 10 -4
= 3x107pfu/mL

The dilutions yielded perfect plates in the second round.

From this round it was ok to continue on to MTL.
The titer from the 10-4 plate was

Titer ( 15310 uLpfu ) x ( 1000 ul

1 mL )
x 10 -4
=1.53x108

MTL/ Spot Test The titter could only be calculated from the 10-7 because the
rest of the grid was overly lysed.
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Empirical Test

Empirical Test Results The plates showed a few different sizes. This is because the
phage has two different morphologies.

Titer All the way up to the end of the semester Unique was being
titered, but the plates on which unique was plated did not
yeild enough plaques to calculate a titer.

SDS PAGE results There were not enough materials to yield any substaintal
results.

Western Blotting The antibody attached to the protien. When that happened
the protien was visible because it was darker then the other
bands..
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Conclusion: Isolating a phage was the main purpose for this study .From the results, isolating a

phage population was almost achieved. These results prove that these phages were and still are

highly lysogenic and it is going to take many attempts to completely isolate a phage population.

These results did not fit in to what was expected. It was not expected for this to be a highly

lysogenic strain of phage. In many but not all cases the growth of phage leads finally to a lysis of

the bacterial cells, a phenomenon which in dense cultures manifests itself to the naked eye as a

clearing of the bacterial culture [3]. In some of the steps the plates lysed completely or had so

many lysed regions that a plaque could not be singled out. Some of the results yielded that a

phage was being singled out, but when a plaque was picked from those plates and streaked on

other plates it proved otherwise. Those plates had experienced an insane growth of phages.

This experiment was important to understand the main purpose of bacteriophages and

how to single out a single population of phage to fight bacteria. Data is available which suggest

that those endogenous phages could play an important role in eliminating bacteria and regulating

the body ecosystem [4]. The fact that phages are self-replicating in the bacterial host is

considered to offer a great advantage over antibiotics as phages will continue to amplify

themselves at the site of infection as long as there are sensitive bacterial hosts to infect [5]. In the

near future, the phage will go through a series of analysis.

References
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1. Brssow H, Hendrix R. 2002. Phage Genomics. Cell 108:13-16.

2. Labrie S, Samson J, Moineau S. 2010. Bacteriophage resistance mechanisms. Nature Reviews

Microbiology 8:317-327.

3. Delbrck M. 1940. THE GROWTH OF BACTERIOPHAGE AND LYSIS OF THE HOST.

The Journal of General Physiology 23:643.

4. Grski A, Weber-Dabrowska B. 2005. The potential role of endogenous bacteriophages in

controlling invading pathogens. CMLS, Cell. Mol. Life Sci. 62:511-519.

5. Housby J, Mann N. 2009. Phage therapy. Drug Discovery Today 14:536-540.

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