OBSERVATION OF VIRUS ON BACTERIA WITH PLAQUE

METHOD

By:
Name : Pratiwi Kusuma Kurniawati
SID : B1B017007
Entourage : III
Group :8
Assistant : Agung Wiriat Putra Pratama Hadi

VIROLOGY LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGI
PURWOKERTO
2019
 I. INTRODUCTION

A. Background

Viruses are the most abundant biological agents in nature that need to enter
living cells to successfully replicate. Although cells are protected by membranes,
viruses have developed elaborate mechanisms to overcome this defense
(Kolomeisky, 2017). Viruses are the smallest microorganisms ever known. Generally
it cannot be seen with an ordinary microscope, except for poxvirus. The size of the
virus varies from poxvirus which is approximately 300 x 250 x 100 nm to parvovirus
which is approximately 20 nm in diameter. Viruses will only experience replication
in living cells by becoming parasites at the gene level. Viral nucleic acid contains
important information to produce offspring by programming infected host cells to
synthesize specific viral macromolecules (Sujudi, 1993).
Viruses have very simple structure. A viruses is made of nucleic acid core and
protein coat called capsid which surrounds the core, a fully assemble particle, a
virion is capable of infecting the host. A virion always contains only a single kind
nucleic acid, DNA or RNA. The nucleic acid may occur as a single or double strands.
Plant viruses contain only single or double stranded DNA or single or double
stranded RNA. Bacterial viruses contain single or double stranded DNA or single
stranded RNA. The infectious property of a virion is due to its nucleic acid. A host
cell can syntesize complete virion if only free viral nucleic acid is injected within the
cytoplasm of a living host cell. The protein coat of the virus called capsid. It is made
of many identical protein sub units called capsomeres (Gupta, 2007). Viruses are
obligate intracellular parasites, it size is 20-200 nm, it chemical form and
composition vary, but only contain one type of nucleic acid, that is DNA or RNA
only. Virion consists of capsids wrapped in a capsid glycoprotein or membrane.
Viruses are usually resistant to antibiotics. Viruses are covered by a kind of
protective material consisting of proteins, lipids, glycoproteins, or a combination of
the three. The viral genome encodes both proteins used to load genetic material and
proteins needed in its life cycle. Viruses can infect lytic and lysogenic host cells.
Viruses that do not have a virus cover are called naked viruses. In addition, at the
bottom there is a spike or needle to insert the genetic material into the host cell
(Tortora et al., 2010).
 Bacteriophage is a virus that infects bacteria and is able to kill bacterial cells
directly or integrate viral DNA into the host bacterial chromosome. Bacteriophage
has a nucleic acid that surrounded by a protein sheath or capsid. Capsule is composed
of morphological subunits called capsomer. Capsomer consists of a number of
subunits or protein molecules called protomers. Phage has cube or helical symmetry.
Cube phases are regular solid objects, while helical phage are rod-shaped. In general,
bacteriophage is a polyhedral head, but its tail is shaped like a rod (Buana &
Wardani, 2014). Bacteriophages have high abundance in nature. Bacteriophages can
infect as much as 1023/second. Bacteriophages are considered as biological weapons
againts pathogenic bacteria. The anibacterial properties of bacteriophage are used in
the treatment of fag againts antibiotic-resistant bacteria in humans and animals. In
addition, bacteriophages are used in vaccination (Golec et al., 2014). Another benefit
of bacteriophage are bacteriophage is used as a biofilm to find out if there are
harmful bacteria such as Salmonella spp. in the water content of refill drinking
(Damayanti et al., 2016).
The benefits of bacteriophages according to Akin (2006) in the world of
agriculture include being used as:
1. Vector for the purposes of molecular cloning. It is well known that some
bacteriophages can be useful as the most effective molecular tools because of
their ability to replicate nucleic acids independently. The use of plasmid
vectors is very important for studying the use of certain genes.
2. The need to detect the presence of certain bacteria. For this matter,
bacteriophage utilization can be applied with its main function as plasmids or
vectors, with little modification of nucleic acids in phage-based vector /
plasmid, for example by adding or inserting certain genes that facilitate
detection, so this is very useful.
One of the most known and fascinating examples of bacteriophage is
bacteriophage T4, which infects Escherichia coli bacteria. The structure of this virus
has been well investigated. It consists of three main parts that are assembled
independently and then joined together to produce a mature phage There is a large,
multi-protein icosahedral capsid that encapsulates the viral DNA genome. This
capsid is coupled via a neck region to a long and narrow tail part, surrounded by a
protein sheath. It ends with a baseplate with attached long and short tail fibers, which
are responsible for the recognition of specific receptors on the host cell and for
binding to the cellular membranes (Kolemeisky, 2017). Another Examples of
bacteriophages include T1, T2, T5, T6, and P2. Three even numbered phages (T2,
T4, T6), have similar structures. The third capsid has an elongated icosahedral head
that envelops DNA (Campbell et al., 2012).
Viruses have two principal replication cycles, the lytic and the lysogenic
cycles. The lytic cycle is characterized by a rapid production of new viral particles in
the system upon the host’s lysis, whereas the lysogenic cycle allows the integration
of the viral genome as a prophage within the bacterium. This prophage will remain in
the lysogenic bacteria until an inducing event takes place, triggering the onset of
viral production. Through lysogeny, the host gains immunity to super infection by
closely related phages and acquisition of antibiotic resistance and/or toxin encoding
sequences through phage conversion. Such acquired traits increase prokaryotic
fitness under specific environmental conditions. Lytic infection is generally found in
productive environments, characterized by high host abundance and activity,
allowing for the fast production of numerous new viral particles. Lysogeny is
proposed to enable the survival of the host and pro-phage under unfavourable
conditions, such as lo prokaryote abundance and primary production (Maurice,
2013).
Phages essentially belong to two categories, virulent and temper- ate. The
virulent phage life cycle consists in replicating in, and then lysing their bacterial host.
Temperate phages can alternate between two life styles: they either lyse their host,
like virulent phages, or establish asymbiosis with it, the so-called lysogeny state. By
entering into the bacterial genome and expressing only a small subset of their genes.
Upon infection in a given bacterial population, the fraction of temperate phages
entering into lysogeny varies with the bacterium physiology, and is generally low,
compared to the fraction going into lysis. In the condition of lysogeny, the phage is
named “prophage,” and the bacterium hosting the phage is said to be lysogenic for
this phage. The study of naturally occurring signals triggering the shift back from the
symbiosis life style to the lytic one. Virulent phages perform only lytic cycles (steps
1 to 5), they adsorb to the bacterial surface (1) inject their DNA (2) replicate it (3)
produce capsid proteins that assemble together (4) and finally lyse their host (5).
Temperate phages can proceed similarly or shift to a lysogenic cycle (6), where they
enter a silent stage of prophage (usually integrated into the bacterial genome), and
are replicated passively by the bacterial machinery (7). Either a rare stochastic event
or a stress induces the prophage (8), which then comes back into a lytic cycle (De
Paepe et al., 2014).
The lytic cycle is considered to be the primary mode of viral reproduction.
The lytic cycle is a viral reproduction cycle that causes the destruction of infected
host cells. Lysis occurs when new intact viruses are formed. Viruses use host cells to
produce virus components. Viruses take 10-60 minutes to complete all stages until
whole new viruses emerge from the host cell. The lytic cycle consists of five phase:
attachment, penetration, synthesis, assembly, and discharge (Caroll et al., 2015).
1. Attachment phase (adsorption)
The viruses is attached to the infected host cell. Once attached to the surface
of the host cell. Viruses secretes a lysozyme enzyme that can penetrate the plasma
membrane of the host cell. This hole will be used to insert the virus genetic material
into the host cell cytoplasm.
2. Penetration phase
In this phase the virus will enter its nucleic acid into the host cell through a
hole that has been formed on the cell wall.
3. Synthesis phase
Nucleic acid virus that has entered into the host cell will inactivate host DNA.
Furthermore, there is a takeover if the work of the host cell so that the host cell will
replicate the viral nucleic acid and produce tha various components required to
form a whole new virus.
4. Assembling or assembly phase
Viruse components that have been formed then assembled to from a newa
virus. This phase is also called the maturation phase. In one host cell can form more
than 100 new viruses.
5. Release phase (lisis)
Hundreds of new viruses that have matured will then converge on the plasma
membrane of the lost cell and inject the lysosome enzymes that can destroy the
plasma membrane. The host cell will break (lysis) and die after the plasma membrane
is destroyed by the new viruses. Then free viruses will invade other cells and the lytic
cycle will recur in the newly invaded cells (Caroll et al., 2015).
The lysogenic cycle is a latent viral reproduction cycle because the period of
one lysogenic cycle can last very long within the host body. The duration of the cycle
in the lysogenic cycle is influenced by virulence or host cell resistance to infectious
virus particles. The lysogenic sycle consists of four phase, namely adhesion,
penetration, merging, and division (Caroll et al., 2015).
1. Attachment phase (adsorption)
Basically, the attachment and penetration phase of the lysogenic cycle are not
dissimilar to the attachment and penetration phase of the lytic cycle. The virus
attaches to the surface of the host cell and punctures the cell plasma membrane.
2. Penetration phase
Virus nucleic acid enters the host cell after passing through the attachment
phase and penetrates.
3. Merging phase (integration)
Furthermore, the nucleic acid will join the host chromosome and is called
profage. The host cell does not undergo lysis in this cycle, but it can still perform the
metabolism and reproduction of the cells as it was before it was infected, only now
that the host cell carries viral nucleic acid as part of ts chromosome.
4. Phase division
In this phase the genetic material of the virus is already integrated with the
host cel chromosome. Viruse utilize the process of cell division to multiply its
genetic material. So, the number of viruses will be more and more along with the
division of host cells. Under certain conditions, profage can break away from the
host chromosome and immediately enter the lytic cycle. The condition in question is
when the host cell loses virulence to virus particles or runs environmental stresses
such as radiation or high temperatures (Caroll et al., 2015).
One method that is used to determine the unit of viral infection is the plaque assay.
When virus particles start infection in the host cell layer that grows spread on the
surface of the medium, the lysis zone or inhibitory zone will appear so that a bright
area will appear in the host cell layer. This bright area is called plaque, which is
assumed that each plaque comes from one virus particle. Plaque is a window in the
host cell layer that lives spread on the surface of the agar media. Plaque can be seen
when virus particles (bacteriophages) are mixed with a thin layer of bacterial hosts
grown on agar media (Sihombing, 2002). The main advantage of plaque assay is the
attemp to quantify the amount of plaque present, the criteria are subjective which can
lead to errors in repeatibility of measurements, the plaque index is that the criteria are
easy to interpretation. The main disanvantage of plaque assay is the interpretation of
the different scores, the criteria are subjective which can lead to errors in repeatibility
of measurements, no attemp has been made to quantify the amount of plaque present
(Eaton & Philip, 2015).

B. Objective
The objective of this laboratory activity are to determine whether there is a
virus that lyses bacterial cells visible from the clear zone or the presence of a
plaque formed in the Luria Bertani medium that has been inoculated with samples
and bacetria Eshericia coli.
 II. MATERIAL AND METHOD
A. Material

The tools used in this laboratory activity are drugalsky, bunsen burner,
wrapper, measuring pipette, filler, micro-pipette, tip, syringe, filter 0,45 μm,
reacting tube, petri dish, Erlenmeyer flask, centrifuge and incobator.
The materials used in this laboratory activity are solid Luria Bertani medium,
Luria Bertani broth, feces sample, alcohol 70 %, Escherichia coli, and Phosphate
Buffer Saline (PBS).

B. Method

The method used in this laboratory activity are:

1. Sample preparation
5 grams sample of livestock manure the suspected contain the virusis put into
an Erlenmeyer flask containing 50 ml of aquadest.
2. Bacteriophage enrichment
a. For threatment method, a total of 5 ml Luria Bertani (LB) and 5 ml
inoculum of Escherichia coli is combined in 40 ml sample.
b. For control method, a total of 5 ml Escherichia coli is inoculated in 5 ml
Luria Bertani broth.
c. The culture is incubated for 24 hours at 37o C.
3. Isolation of bacteriophage and plaque enumeration
a. A total of 10 ml culture is poured into Eppendorf tube then centrifuge at
2000 rpm for 5 minutes.
b. Supernatan is collected with syringe then filtered with filter 0,45 μm to
sterilized bottle as bacteriophage filtrate.
c. Continously diluted was carried out up to 10-6 by transferring 0.1 ml of
bacteriophage filtrate to Eppendorf containing 0,9 ml PBS as a dilution of
10-1.
d. Every last dilution was taken 0,1 ml to be transferred to a new Eppendorf
containing 0,5 ml E. coli.
e. The last three dilutions are mixed with the medium Luria Bertani semi-
solid and poured into a cup.
f. Incubate 48 hours at 37o.
g. Plaque formation that occurs was observed when a plaque formation is
formed on a bacterial growth colony and it is suspected that there is a
virus that lyses bacterial cells
h. The amount of plaque formed is calculate :

PFU/ml =
 III. RESULT AND DISCUSSION

Table 3.1 Observation Table of Total Plaque Entourage III

Dilution
Group
10-2 (PFU’s/ml) 10-3 (PFU’s/ml)
2
1 12,3 x 10 26,67 x 103
2 11,83 x 103 6,6 x 103
3 0,33 x 103 61,6 x 103
4 1,6 x 102 8,3 x103
5 8 x 103 6,7 x 103
6 2,4 x 101 7,16 x 101
7 240 x 103 20 x 103
8 12x 103 0

Calculation :

10-2 dilution

PFU’s/ml = Plaque / (dilution x inoculated volume)

= 77 / (10-2 x 0,6)

= 12, 103 PFU/ml

10-3 dilution

PFU’s/ml = Plaque / (dilution x inoculated volume)

= 0 / (10-3 x 0,6)

= 0 PFU/ml

Based on the existing table, the result of the plaque calculation by group 1
on 10-2 and 10-3 dilution are 12,3 x 102 PFU/ml and 26,67 x 103 PFU/ml, group 2 on
10-2 and 10-3 dilution are 11,83 x 103 PFU/ml and 6,6 x 103 PFU/ml, group 3 on 10-2
and 10-3 dilution are 0,33 x 103 PFU/ml and 61,6 x 103 PFU/ml, group 4 on 10-2 and
10-3 dilution are 1,6 x 102 PFU/ml and 8,3 x 103 PFU/ml, group 5 on 10-2 and 10-3
dilution are 8 x 103 PFU/ml and 6,7 x 103 PFU/ml, group 6 on 10-2 and 10-3 dilution
are 2,4 x 101 PFU/ml and 7,16 x 101 PFU/ml, group 7 on 10-2 and 10-3 dilution are 2,4
x 105 PFU/ml and 20 x 103 PFU/ml and group 8 on 10-2 and 10-3 dilution are 12 x 107
PFU/ml and 0 PFU/ml. The variation of bacteriophage titer in each dilution is due to
the presence of a bacterial defense system against bacteriophage infection,
bacteriophage properties and dilution treatment. Based on the data, most all cow
feces sample in all groups are gave positive interpretation, only group 8 (our group)
in 10-3 dilution that give negative interpretation. Interpretation of positive results is
indicated by the formation of clear zones on the media with full rounds (circles).
Plaque is formed due to lyse of bacterial cells by bacteriophages. The more amount
of plaque formed, the bacteriophages contained in the sample can be said to have
high concentrations (Aryulina, 2007).

Figure 3.1 Result of plaque observation on 10-2 dilution

Figure 3.2 Result of plaque observation on 10-3 dilution

The absence of plaque formed shows a negative result, in other words there
are no bacterial cells lyse due to infection with the virus. This is in accordance with
Baer & Kehn-Hall (2014) that the inability of bacteriophage to lyse the host may
occur due to differences in environmental conditons, faster host growth, defense
systems against bacteriophage infectious naturally. The amount of plaque is also
influenced by the specificity of the bacteria itself. The absence of plaque can be due
to the fact that there is no virus in the cow dung sample, the virus in the lysogenic
phase, and the viral receptors in the sample do not match the inoculated waste. On
the media samples that have been inoculated with waste which have been
contaminated by animal feces and E. coli will form plaque, but in the control media
there is no plaque which is an important parameter of the presence of phages in the
lytic cycle. It is possible that the virus is a lysogenic virus so that the media does not
appear plaque because it does not occur in the process of thinning, or indeed in E.
coli bacteria that has been inoculated there is no virus, whereas for media inoculated
samples of E. coli enrichment there are plaque thus means the process of thinning is
possible because of the enrichment of E. coli bacteria so that in the media there are
many viruses and E. coli bacteria.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion
Based on the result and discussion be conclude that there are phages that
lyse bacterial host cell, specifically phages that infecting Escherichia coli. The
amount of plaque which calculated by grup 8 in 10-2 dilution is 12 x 103 PFU/ml,
while in 10-3 dilution is 0 PFU/ml.

B. Suggestion
The laboratory activity will be better if the student to do the method more
carefully and use the lab activity correctly, so the result will not failed. The tools
may checked before used so the tools can be used well during lab activity.
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Assays: Using Traditional and Novel Overlay Systems. Journal of Visualized
Experiments, 93, pp. 1-10.

Buana, E. O. & Wardani, A. K., 2014. Isolasi Bakteriofag Litik Sebagai Agen
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ATTACHMENT (PORTOFOLIO)
Group : 8
Entourage : III
QUESTION
Explain the stage of lysogenic cycle !
1. Absorption and infection phase
Phage attached to the surface of the host cell and cell wall destruction by the
enzyme lysozyme, then the genetic material from the virus is inserted into the
host cell using a spike.
2. Merging phase (integration)
The phase where viral DNA joins the host cell DNA and forms prophage.
3. Cleavage phase
When the host cell divides, each cell produced by division contains prophage.
The lysogenic phase can turn into a lytic phase when under favourable
conditions