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Bacteriophage Enrichment, Morphology and DNA Analysis from Soil Sample

Using Aseptic Techniques

Kapil C. Baskaran

Carthage College, Kenosha, Wisconsin


2015

Bacteriophage Enrichment, Morphology and DNA Analysis from Soil Sample


Using Aseptic Techniques
Abstract:
Soil sample collected in Carthage College was used to enrich bacteriophages using aseptic
laboratory techniques. Soil sample was mixed with M.smegmatic bacteria to infect and incubated
at 37C for 24 hours. Clear agar plates were observed due to phage infection, replication
followed by bacteria lysis of bacteria. Serial dilution of the bacteriophage sample was used to
determine the titer and plaque formation unit (pfu). A single plaque was used to enrich by spot
testing procedure. 10 L of enriched stock bacteriophage samples were transferred on an EM
grid and stained by uranyl acetate. Electron microscopy observation showed plenty of
bacteriophages on the EM grid. The phages were observed without spikes in the tail section.
Phage genomic DNA was isolated and restriction enzyme digested. Electrophoresis of phage
DNA restriction enzyme digest showed degradation, possibly due to nuclease contamination. The
objective of phage enrichment protocol, electron microscopy sample preparation and genomic
DNA preparation and restriction enzyme digestion were met.

Introduction:
Aseptic laboratory techniques were followed in the lab during the phage sample collection,
extraction and analysis. Working area was cleaned with 70% alcohol and gloves were worn
during experiments. Sterile pipets, test tubes, conical flasks were used to minimize
contamination. Agar plates were prepared aseptically to minimize environmental contamination
to agar plates.
Bacteriophages are a class of viruses that are parasites specific to bacteria live in water and soil
(References 1 and 2). Bacteriophages are smaller, ranging from 34 to 160 nm (References 3 and
4). The size of bacteriophage ranges are from 100 to 200 nm. Bacteriophages cannot replicate or
propagate outside their host bacteria. They are the most abundant life-form on earth and can
survive in almost any environment and are found inside and outside bacterial cells.
In 1910, French-Canadian microbiologist Felix dHerelle noticed a clear spots on a lawn of
bacteria known as plaques. In 1915, he added the plaque to turbid bacteria culture found the
culture very clear (Reference 5). Felix d Herelle used the term bacteriophage to describe the
phages that killed the bacteria. In 1915, another scientist, Edward Twort also was credited to the
discovery of phages that killed bacteria.
In 1960, IMax Delbrck, Alfred Hershey and Salvador Luria discovered the replication of
viruses and their genetic structure (Reference 6), which unraveled the structure of
bacteriophages. Bacteriophage contains a head where the genetic material resides, neck and tube
like tail. They use their tail to attach to bacteria and to inject the DNA into the host cytoplasm.

Filtration through membranes of 0.220.45 m pore size effectively purifies the bacteriophages
from debris and larger particles (References 7 and 8).
Bacteriophages replicate in two different ways called lytic lysogenic cycle. During lytic cycle the
bacterial cells are broken open (lysed) and destroys the bacteria. This phase is called temperate phage.
Their viral DNA integrates into host DNA and replicate along with it fairly harmlessly. The
bacteriophages remain dormant until host conditions deteriorate then the prophages become active and
lyse the host cell (Reference 9).

The objective of our work is to aseptically culture, enrich the bacteriophages from the soil
sample by infecting M.Smegmatis bacteria, and determine titer, morphology and DNA extraction.
The titer of the bacteriophage was determined through serial dilution in terms of Phage forming
unit (PFU) and the DNA was restriction enzyme digested and electrophoresed on agarose gel.
Enriched bacteriophage stock was prepared and mounted on EM grid. The morphology of the
bacteriophage was observed under the electron microscope.

Methods:
Aseptic Technique: The purpose of aseptic technique is to ensure sterile working area and avoid
contamination. For a successful laboratory experiment the work area must be sterile and clean.
70% alcohol is used to clean the work area. Gloves must be worn to avoid introducing
contamination. Bunsen burner is used to keep the area free of environment contamination. Sterile
pipets, tips, test tubes, water and agar was used in the experiment.
Phage enrichment and plaque streak:
1 gram of soil sample was added to 50mL flask. To the flask 10 mL of sterile water, 1.25 mL of
LB/glycerol broth, 1.25 mL of AD supplement, 125L of 100 mM CaCl2 and 1.25 mL of
M.Smegmatis bacteria culture was added. The flask was incubated at 37C, shaking at 220 rpm
for 24 hours. The content was spun at 3000 rpm for 10 minutes to pellet particulate matter. The
supernatant was stored at 4C until next class. Plaque streak was used to purify a single phage
population from samples with potentially mixed phage population. A negative control plate was
streaked across the plate following a second streak touching part of the first streak and a the third
streak, touching part of the second streak. Top agar with 0.5mL aliquot of M.Smegmatis mixed in
4.5 mL of TA was layered on top of the streak and was incubated overnight at 37C.
Serial dilution of phage preparation for a titer:
For each putative phage, serial dilution was prepared by arranging 4 micro-centrifuge tubes and
labeled them as 10-1, 10-2, 10-3 and 10-4. 90 L of PB was added to each micro-centrifuge tubes.
10 L of undiluted phage sample was added to 10-1centrifuge tube. After mixing well, 10 L
from 10-1 was transferred to 10-2 centrifuge and mixed well. This step was repeated to 10-3 and 104
labeled micro-centrifuge tubes.

0.5 mL of M.Smegmatis bacteria was added to each of the micro-centrifuge tubes with 4.5 mL of
top agar and plated and incubated the plates at 37C for 48 hours. After 48 hours the plates were
checked for plaques.
PFU Determination:
The plaque forming units (pfu) were calculated according to the formula given below.
Titer (PFU/mL) = ( pfu/#L) X (1000L/mL) X dilution factor*

For a 10-3dilution the dilution is 1000 X(1000-fold):the dilution factor is 1000 or 10-3

for example: 3 plaques were identified on a plate that received a 10mL L of a 10-4 dilution.

Pfu = 3pfu/10mL X 1000L/mL =300pfu/mL

Spot test to Phage purification:


Spot Test on the phage lysate was performed by serial dilution from 100-10-10. 90L of PB was
added into each of the centrifuge tubes. 10 L of the sample from the phage stock was added to
sample 1 and mixed well. 10 L of sample 1 was add to sample to sample 2 and 10 L of sample
2 to sample 3 until sample 10. 0 is a negative control sample with no phage.
Phage preparation for Electron microscopy Analysis:
A single phage plaque stock was prepared by plate elution method and the suspension was
aseptically transferred using 1.0 mL phage buffer lysate into a sterile micro-centrifuge tube. The
centrifuge was balanced and centrifuged for one hour at 4C at 10,000 x g. 100 L of phage
buffer was added and mixed well stored at 4C for one hour. 10 L of phage preparation was
placed on to the grid and allowed to absorb for 10 minutes. 10 L of sterile water was added to
wash followed by 10 L of Uranyl Acetate was added to the grid and allowed to stain for 2

minutes and removed by a wick and air-dried before observation under Electron microscopy
(Figure 3).

Restriction Enzyme Digestion of Phage DNA:


Enriched phage stock was used to prepare Phage DNA using Promega DNA isolation kit and
following restriction enzyme digestion. The restriction enzyme digested sample was loaded on
0.8% agarose gel containing ethidium bromide to a final concentration of 0.5g/mL along
markers on both sides of the agarose gel well (Figure 4).

Results:
Phage enrichment and phage titer:
The purification of phage plaque worked as expected with lots of plaque in the plate. Serial
dilution of plaque produced phages of distinct colonies at 10-3 and 10-4 dilutions. Single phage
plaque was isolated and allowed to infect M. smegmatis bacteria in subsequent phage infection.
Phage plaques were observed as seen in figure 1.

Figure 1: Phage titer determination

Figure 2: Spot test on the phage lysate

The sample contains phages that infected the bacteria and lysed them. The second phage dilution
sample labeled as -2 contained about 100 phage plaques on the agar plate after incubation at

37oC. Plate -3 contained about 20 phage plaques on the agar plate. Plate -4 contained about 4
phage plaques on the agar plate.
Electron microscopy Observation:

Figure 3a: Electron microscopy of Phage

Figure 3b: Single phage

The electron microscopy observation of morphology revealed the phages were the group
of Siphoviridae. There were plenty of them in the sample with a tail length 4.7 times the
capsid diameter. The phages appear to be fragmented and without spikes as shown in
figure 3.
Restriction Enzyme Digestion of Phage genomic DNA:

Figure 4a and 4b: Restriction Enzyme digestion of Phage genomic DNA


Phage DNA was loaded into lane 6. For both gels, our DNA was degraded due to nuclease
contamination while DNA extraction was performed.

Discussion:
Aseptic technique used throughout the experiment ensured sterile condition and no
contamination. The sample that was collected contains abundance of bacteriophages
showing that our college environment is rich in bacteriophages. They are able to adopt
well and grow abundantly. Titer determination experiment shows that the phages are
growing well and they are looking more like in a temperate stage.

The overall size of the plaque was observed to be less than 2 mm in diameter showing that there
are plenty of them in the sample. The final titer of the sample was determined to be 6 x 108
pfu/ml.The electron microscopy observation of morphology revealed the phages were the
group of Siphoviridae, with a tail length 4.7 times the capsid diameter. Although, the exact
size of the phage was not determined due to fragmentation and loss of tail spikes, it is
assumed to be well defined and grown in abundance as seen in the electron microscope
morphology.

The disconnection of tail spikes may be due to the preparation and purification of
bacteriophages. Though it looks like a small bacteriophage, its presence in soil and abundance in
environment clearly shows the bio-diversity of our environment. Finally, DNA extraction,
isolation and preparation and electrophoresis show that the DNA was fragmented due to
contamination of nuclease and may be due to over digestion of genomic DNA.

References Cited:
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soil. Appl. Environ. Microbiol, 41: 13891393.
2. Germida, JJ and Khachatourians, G. 1988. Transduction of Escherichia coli in soil. Can.
J. Microbiol, 34: 190193.
3. Ackermann, HW. 1998. Tailed bacteriophages: the order Caudovirales. Adv. Virus Res,
51: 135201.
4. Ackermann, HW and DuBow, MS. 1987a. Viruses of Prokaryotes, Vol. 1. General
Properties of Bacteriophages, Vol. 1, Boca Raton: CRC Press.
5. The bacteriophage, by Felix dHerelle, Science News 14:44-59 (1949).
6. "The Nobel Prize in Physiology or Medicine 1969". Nobel Foundation. Retrieved 200707-28.
7. Lanning, S and Williams, ST. 1982. Methods for the direct isolation and enumeration of
actinophages in soil. J. Gen. Microbiol, 128: 20632071.
8. Williams, ST, Mortimer, AM and Manchester, L. 1987. Ecology of soil bacteriophages.
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John Wiley and Sons.
9. Mason, Kenneth A., Jonathan B. Losos, Susan R. Singer, Peter H Raven, and George B.
Johnson. (2011). Biology, p. 533. McGraw-Hill, New York. ISBN 978-0-07-893649-4.