PART 2: ISOLATION AND CULTIVATION OF VIRUS-

BACTERIOPHAGES
I. OVERVIEW AND INTRODUCTION:
1.1. Introduce:
Bacteriophage is defined as a virus that infects bacteria. This virus is common and
needs a bacterial host, but it offers no danger to humans. Bacteriophages are various
organisms prevalent in food, drink, soil, water, and the ocean (Zurabov & Zhilenkov,
2021). In the early 20th century, Treating bacterial illnesses in humans and animals has
been suggested using bacteriophages (Cisek et al., 2017). In 1940, with the
development of antibiotics, phage therapy suffered a setback and became limited to
basic research (Chanishvili, 2012). Recently, there has been a resurgence of interest in
phage therapy, which is used as a novel therapeutic agent to treat pathogenic bacteria
(Golkar et al., 2014).
Frederick Twort produced the first discovery of bacteriophages in 1915, and Canadian
scientist Félix d'Hérelle carried out additional research on them in 1917. D'Herelle was
the first to investigate the use of bacteriophage therapy in treating human ailments.
Nevertheless, the research ran into contentious concerns and was rejected (Haq et al.,
2012). Phage therapy saw a resurgence in the early 1980s, mostly as a result of the rise
in multidrug-resistant (MDR) infections and the need to discover alternatives to
resistance chemical biology (Sulakvelidze et al., 2001). As a result, fresh research has
concentrated on the application of phage therapy in industry, veterinary medicine,
agriculture, rhinoplasty, and food safety in addition to treating human illnesses.
1.2. General structure:
The structure of bacteriophages is basic. They have a prismatic head with a protein
capsid around the genetic material inside of it. Through the neck or collar region, this
portion is joined to the extended shell, which is occasionally referred to as the tail.
Surrounded by protective coat proteins, the envelope forms an empty tube that allows
the insertion of phage DNA/RNA into the host cell. The tail fibers, which typically
number six and aid in joining the host cell, are attached to the baseplate at the base of
the sheath.
Different bacteriophage species can vary in size from 24-400 nm length and genome
length. Their DNA sequences vary significantly and can range in size from 18 to 400
kb. Each virion has a polyhedral head (capsid), primarily icosahedral, that covers the
genome. Heads consist of many copies of one or several different proteins and have a
very stable organization. The phage tail is attached to the capsid via a connector that
serves as an adapter between these two important components of the phage. The
connector is a heterooligomer composed of several proteins (Lurz et al., 2001; Orlova et
al., 2003). The connector performs several functions during the life cycle of the
bacteriophage. They are involved in packaging dsDNA into the capsid, and they then
perform a gatekeeper function: blocking the phage capsid exit, and preventing DNA
 leakage under high pressure, after the signal has passed. tail for knowing that the phage
is attached to the bacterium, the junction will open allowing the release of DNA into the
bacterium (Plisson et al., 2007).

Figure 2.1 Structure of a bacteriophage

(Nguồn: Doss et al., 2017)
The bacteriophage tail and its associated structures are important elements that
guarantee the entry of phage nucleic acids into the host bacterium during infection.
During infection, the tail is both a signal transmitter and a channel for DNA to enter the
host cell. There are two types of tails: constricted and non-contracted. Tails can be short
or long. Multiple copies of various proteins arranged in helical symmetry frequently
make up the long tail. External appendages, which are often base plates with several
filaments and a tip or needle that is specifically designed for bacterial membrane
receptors, belong to the posterior end of all types of tails (Leiman et al., 2004). The
base plate and tail fibers contribute to the binding of a phage to the bacterial outer
membrane, making this binding irreversible, as soon as the tail needle finds the
receptor. This occurs in many (reversed) relationships of bacteria during their limited
lifetime (São-José et al., 2006). Connection (attach irreversibly) of the bacteriophage
causes the opening of the junction and the release of the genome through the tail tube
into the bacterial cell.
1.3. Classification of Bacteriophages:
Bacteriophage classification is based on morphology, nucleic acid type, replication
mode, host organ, and disease type. The International Committee on Taxonomy of
Viruses (ICTV) has created an orderly system of classifying viruses. Bacteriophages are
found in many different forms: filamentous phages, lipid-encapsulated phages, and
lipid-containing phages in the seed coat (Figure 2.2A). They have a genome, DNA or
RNA, which can be single-stranded or double-stranded, and contain information about
the proteins that make up the particles, the additional proteins that the task of shifting
cellular molecular metabolism in their favor. The genome can be single or multimeric
and is located inside the phage capsid. The shape of bacteriophages is closely related to
their genome, and a large genome indicates a large capsid and thus a more complex
organization. Bacteriophages have been studied extensively
especially tailed phages (order Caudovirales) are classified according to the type of tail;
Family Siphoviridae has a long non-retractable tail, Family Podoviridae has a short tail
uncontracted and Myoviridae have complex constricted tails (Fig.2.2B).

Figure 2.2: Morphology of some phage families

A) Representation of prokaryote bacteriophage morphotypes (Pietilä et al., 2014). (B) Members of the
Caudovirales family (White & Orlova, 2019).
Classifying bacteriophages depending on their morphology: Phages that are
filamentous, isohedral, tailed, and even some that have lipid-containing shells or lipids
in their seed coat. As a result, the largest class of viral are called bacteriophages.
Electron microscopy analysis has been performed on over 5500 bacteriophages
(Ackermann, 2009) (Figure 2.3). Approximately 190 known polymorphic filamentous
phages, or 3.6% of all phages, have been divided into 10 subfamilies (Ackermann,
2009). These phages exhibit notable differences in their features and properties, which
appear to indicate distinct lineages of origin. Three families including a limited number
of identified individuals that require additional characterization define polymorphic
phages. Bacteriophages belonging to the family Plasmaviridae (dsDNA) have dsDNA
that is encased in a lipoprotein shell, which makes them resemble nucleoprotein
particles. The Guttavirus (dsDNA) bacteriophage group is represented by droplet-
shaped virus particles (Figure 2.3B, Arnold et al., 2000). Members of the
Fusseloviridae family have dsDNA inside a lemon-shaped cap with small spikes at one
end.
 Figure 2.3: Photograph of bacteriophages
(Source: Arnold et al., 2000)
Some bacteriophages have a spiral or filamentous organization. The Inoviridae
(ssDNA) family includes long, rigid, or flexible filamentous phages of variable length
and are classified according to grain length, coat structure, and DNA content.
Lipothrixviridae (dsDNA) phages are characterized by the combination of a lipoprotein
shell and a rod-like shape (Figures 2.3C). The family Rudiviridae (dsDNA) represents
bacteriophages that are hard straight rods without an envelope and closely resemble
tobacco mosaic viruses.

Figure 2.4: Bacteriophage family with tail.

(Source: White & Orlova, 2019)
Tailed phages are classified in the order Caudavirales (dsDNA) (Figure 2.3 D, E, F)
(Ackermann, 2009). Tailed phages can be found everywhere and represent 96% of
known phages and are separated into three major phylogenetically related families.
Bacteriophages with tails are divided into three families (Figure 2.4): A - Myoviridae
have a tail consisting of a sheath and a central tube (25% of phages have tails); B -
 Siphoviridae, long tail, no contraction (61%); C - Podoviridae family, short tail (14%).
Because tailed phages represent the largest population of bacteriophages, they are easy
to find and pure; they are the most studied family both biochemically and structurally.

Figure 2.5 Transmission scanning electron microscope image of VT232 of the family Siphoviridae (A)
and 5A belongs to the family Podoviridae (B)
1.4. Life cycle of Bacteriophage:
Typical intracellular parasitic viruses and bacteriophages infect their host cells through
parasitism (Álvarez & Biosca, 2017). Bacteriophages do not damage other types of
bacteria; they exclusively kill the target organism. They can be either lytic or lysogenic
depending on how they experience life. While bacteriophage lysogens incorporate their
genome into the host bacterial genome and replicate without killing the bacterial cell,
lytic phages multiply and kill host bacterial cells, making them more efficient against
target bacteria. By expressing host cell-expressed genes that are not expressed,
lysogenic bacteriophages can modify the bacterial phenotype and remain stable across
generations a phenomenon known as lysogenic conversion.
 Figure 2.6: The lytic and lysogenic cycles in bacteriophages
(Source:(Doss et al., 2017)
Bacteriophages attach to a bacterial host on a receptor found on the surface of the
bacteria and inject its genetic material into the cell. The host cell provides the molecular
building blocks and enzymes necessary to replicate the bacteriophage's genetic material
and create new bacteriophages. Phage-encoded proteins such as endolysin and holin
lyse the host cell from the inside. Holins are small proteins that accumulate in the
cytoplasmic membrane of animals’ hosts and allow endolysin to degrade peptidoglycan,
allowing progeny phage to escape. Then, in the external environment, the bacteriophage
can infect and kill all neighboring bacteria. Generating large numbers of new
generations of bacteriophages by lytic phages is one advantage of their use in phage
therapy.
However, lytic phages have a narrow host range and infect specific bacterial species.
This lack of a broad host range can be overcome by using a phage mixture. During the
latent cycle, temperate bacteriophages do not lyse cell host cells immediately; instead,
their genome is inserted into the host chromosome at specific locations. This phage
DNA in the host genome is called prophage, while the host cell containing the prophage
is called lysogen. The prophages are replicated together with the genome of the
bacterial host, establishing a stable relationship. The disadvantage of the use of latent
phages in phage therapy is that some phage populations insert their genome into the
host chromosome and may lie dormant or alter the host's phenotype (Doss et al., 2017).
The physiological cycle can continue indefinitely unless the bacteria are exposed to
signals of danger or adverse environmental conditions (Penadés et al., 2015). When the
latent cycle ends, expression of the phage DNA occurs and the lysogenic cycle begins.
Recently, bacteriophages have been found to infect Bacillus species based on the small
molecule called “arbitrium” to communicate and carry out lysis decisions – lysogeny
(Erez et al., 2017). The biological significance of this communication system is very
important and explains that when a single bacterium encounters a large number or
colony of bacteria, there are many hosts to infect, creating favorable conditions for
activating the lytic cycle. When the number of hosts becomes limited, it is more
beneficial for bacteriophages, the progeny are in a dormant state and enter a latent state.
These recent findings are worth pursuing further to determine whether the peptides
communicate whether the same is used by other phages or whether cross-talk is
apparent between different phages (Doss et al., 2017).
1.5. Bacteriophage application:
Currently, bacteriophages are widely used as birth control agents study in the food
industry and agriculture, wastewater treatment, and farming seafood (D’Accolti et al.,
2021). Studies indicate that the direct application of bacteriophages or phage mixtures
to foods can significantly reduce contamination with various foodborne pathogens
(Abuladze et al., 2008). A report of bacteriophage KIT03 being able to infect E.coli
O157:H7, Salmonella enterica serotypes Choleraesuis and Enteritidis, the results
reported that ɸKIT03 could kill the tested bacteria and prevent the growth of them
(Pham-Khanh et al., 2019). Tran Trung Tu and colleagues. (2018) researched and
selected bacteriophages (bacteriophages) capable of resolving E. coli from chicken
farms in Hau Giang, Soc Trang, Ben Tre, and Tien Giang provinces. The results were
thirty-five children with disabilities isolated from four provinces (48.6%). Several
studies describe the potential for bacteria-mediated control of different serotypes of S.
enterica by use as a therapeutic agent, biocontrol, antibiotic film, and as disinfectant.
has been widely reviewed (Endersen & Coffey, 2020); (Moye et al., 2018). (Van et al.,
2015) sequenced the whole genome of the filamentous bacteriophage RS66, which
infects the plant pathogen Ralstonia solanacearum. Truong Thi Bich Van and
colleagues. (2019) isolated two bacteriophage strains ɸCT2 and ɸVL27 with the
possibility of Ralstonia solanacearum bacterial infection on chili plants in Can Tho,
Soc Trang, and Vinh Long provinces. Under greenhouse conditions, ɸVL27 with the
actual inoculation method bacteria (108 PFU/mL) at the same time as bacteria (106
CFU/mL) were able to control Green wilt disease is best after all three-time points: 7,
14, and 21 days. Bacteriophage therapy was also conducted in vivo, demonstrating that
bacteriophages can control bacterial infections in fish, shrimp, and other aquatic
products in aquaculture (Matamp & Bhat, 2019). In addition, trials of phage therapy
against a number of bacterial pathogens in animal models have also been tested and
shown effective results (Kalatzis et al., 2018).
Globally, efforts to control Vibrio spp. with the application of bacteriophages in
aquaculture have been reported by researchers. (Y. Wang et al., 2017) demonstrated the
use of CWD therapy in controlling V. harveyi in greenlip abalone. (Le, Southgate,
O’Connor, Vu, et al., 2020) showed the application of bacteriophages in reducing the
mortality of oyster larvae caused by V. alginolyticus. (Le, Southgate, O’Connor,
Abramov, et al., 2020) have demonstrated the effectiveness of bacteriological treatment
in decontamination of Vibrio spp. in commercially produced microalgae that are used as
the main food source for oyster larvae during the hatchery process. In hatchery trials,
phage treatment of Penaeus monodon larvae infected with Vibrio harveyi resulted in
larval survival rates above 85%, suggesting that bacteriophages would be an effective
replacement for antibiotics (Karunasagar et al., 2007). Research on evaluating the
treatment effectiveness of bacteriophages against Vibrio parahaemolyticus bacteria on
white shrimp (Litopenaeus vannamei) showed promising results, after 24 hours of
phage inoculation, the water in the tank changed from cloudy to clear, the bacterial
population in the water decreased sharply from 4.6x104 CFU/ml down to 6.5x102
CFU/ml and after 2 days of phage inoculation, the bacterial population continued to
decrease to 3.3x102 CFU/ml (Truong Thi Bich Van et al., 2021). Another study by
(Ngoc et al., 2022) observed a change in colony morphology when phage was exposed
to bacteria (Figure 37). Φ21 changed the colony size. This result shows that
bacteriophages have the ability to inhibit and change host characteristics. This is the
basis for further research on the application of bacteriophages in preventing and treating
diseases in shrimp.
 Figure 2.7: Change in colony phenotype when phage enters Vibrio sp. B1.1

The application of bacteriophages is not limited to clinical trials to fight bacterial

infections. It can also be used in livestock protection to reduce animal mortality as well
as improve food safety. Importantly, these viruses are not toxic to humans and can
destroy bacterial biofilms thanks to biofilm-degrading enzymes, which also have been
shown to be more promising than antibiotics (Chan et al., 2018).
II. PRACTICE AND REPORT
1. Survey of the bacterial population (CFU/mL)
Purpose: Determine the number of colony-forming units per unit volume
Method:
Bacterial strains and culture conditions: The bacterial strain Salmonella enterica
subsp enterica serovar Enteritidis ATCC 49223 is grown in Xylose lysine deoxycholate
(XLD) agar medium and Tryptic soy broth (TSB) at 37ºC for 24 hours and shaking at
120 rpm.
Dilution
Step 1: Prepare a series of at least six 1.5 mL Eppendorf containing 900 µL of TSB
medium.
Step 2: Using a sterile micropipette, add 100 µL of bacterial suspension to the first
Eppendorf. Obtain a bacterial suspension at a 10-1 dilution.
Step 3: Mix the mixture well with a micropipette.
Step 4: Withdraw 100 µL of bacterial suspension at 10-1 dilution into Eppendorf No. 2,
mix equal to the volume in Eppendorf, we obtain a bacterial suspension in phase dilute
10-2.
Step 5: Repeat the procedure with all remaining Eppendorf until a bacterial suspension
at a 10-6 dilution is obtained.
Note: Pipette tips are renewed at different dilutions.
 Figure 2.8: Serial dilution method
(Source: Tankeshwar, 2016)

Counting bacteria:
Step 1: Use a micropipette to withdraw 100 µL of bacterial suspension at a dilution of
10-6 onto the surface of the XLD agar plate.
Step 2: Dip the glass spreader into ethanol, then burn the spreader on an alcohol lamp.
Step 3: Spread the sample evenly on the agar surface using a cooled glass spreader,
while carefully rotating the Petri dish underneath at a 45º angle.
Step 4: Incubate the plate at 37°C for 24 hours.
 Figure 2.9: Spreading method
(Source: Rijal, 2017)
Calculation
The number of colony-forming units (CFU) present in each mL of the original sample
was calculated according to the formula.
CFU/mL = number of colonies X dilution / initial sample volume
For example, suppose a plate with a dilution of 10 -6 gives a colony count of 35. Then,
the bacterial population in 0.1mL of the original sample is calculated as follows:
CFU/mL=35x106x10=3,5 x 108
*Multiply by 10 because use 0.1 mL=100 µL while pouring the agar plate
2. Determine the number of viruses
Purpose: determine the number of phages capable of forming plaques per unit volume.
Methods:
Prepare serial dilutions of phage suspension similar to the stepwise dilution method of
(Tankeshwar, 2016). The Eppendorf series was replaced with sodium chloride
magnesium sulfate (SM) buffer, diluting the phage to a dilute 10-8.
200 µL of Salmonella enterica subsp enterica serovar Enteritidis ATCC 49223 (106
CFU/mL) cultured overnight mixed with 5 mL of semi-solid TSA medium incubated at
56ºC and spread onto TSA agar plate using double layer agar-drop method method
(Kropinski et al., 2009). 5 µL of phage filtrate at different dilutions were dropped onto
the solidified TSA medium and incubated at 37ºC for 24 hours.
Calculation
Some bacteriophages are calculated according to the formula:
1 1
Phage titer¿ N × ×
DF V
N is the number of soluble spots in a particular plate, DF is the phage dilution factor for
that plate, and V is the initial phage stock volume withdrawn, in mL. Results are given
as spot formation units per milliliter (pfu/mL) (Luzon-Hidalgo et al., 2021).
REPORTS
1. Practice
Determine bacterial population, calculate CFU/mL, and describe colony morphology:
 A plate with a dilution of 10-6 gives a colony count of 145. Then, the bacterial
population in 0.1 mL of the original sample is calculated as follows:
CFU/mL=145x106x10=14,5 x 108
*Multiply by 10 because use 0.1 mL=100 µL while pouring the agar plate

Determine virus density, and calculate PFU/mL:

2. Question and result

1. What disease does Salmonella bacteria cause? Food poisoning?
 Salmonella bacteria can cause gastroenteritis, typhoid fever, and other
infections. Food poisoning, also called foodborne illness, is an infection or
irritation of your digestive tract that spreads through food or drinks.
2. What is Plaque?
 Plaque is a clear area on a lawn of bacteria or a monolayer of cells, where
viruses have destroyed the cells.
3. What are the advantages and limitations of the serial dilution method? Give
real-life examples of serial dilution.
Pros of serial dilution
- It helps to reduce a dense culture of cells to a more usable concentration.
- A specific amount of bacteria is reduced with every dilution.
- The number of colonies cultured from serial dilutions of the sample is counted
to estimate the concentration of an unknown sample. Then track the measured
counts to the unknown concentration.
- Cons of serial dilution
- It does not separate bacteria like a streak plate.
- Overnight incubation is required.
4. Describe the life cycle of bacteriophages.
 During infection, a phage attaches itself to a bacterium and introduces its
genetic material into the cell. After that, a phage usually goes through one of two
life cycles: lytic (virulent) or lysogenic (moderate). Lytic phages hijack the cell's
machinery to produce phage components. They then destroy or lyse the cell and
release new phage particles. Lysogenic phages incorporate their nucleic acid into
the host cell's chromosome and reproduce with it as a unit without destroying the
cell. Under certain conditions, lysogenic phages can be made to follow a lysis
cycle.
5. Present some applications of bacteriophages in medicine, the food industry and
agriculture, wastewater treatment, and aquaculture.
Application of bacteriophage:
- Agriculture: bacteriophages can also be used for plant disease diagnostics.
- Health sciences: bacteriophages are a promising approach in the fight against
antibiotic-resistant bacteria.
- Food industry: phages are useful for detecting and hindering the growth of
foodborne pathogens during and after food processing.
- Wastewater treatment: Phage-mediated bacterial mortality has the potential to
influence treatment performance by controlling the abundance of key
functional groups. Phage treatments have the potential to control
environmental wastewater process problems such as foaming in activated
sludge plants; sludge dewaterability and digestibility; and pathogenic bacteria;
and to reduce competition between nuisance bacteria and functionally
important microbial populations.
- Aquaculture: Therapeutic bacteriophages, commonly called phages, are a
promising potential alternative to antibiotics in the management of bacterial
infections of a wide range of organisms including cultured fish.
6. What are the advantages of phage therapy?
 Benefits of phage therapy: phages can be bactericidal, can increase in number
over the course of treatment, tend to only minimally disrupt normal flora, are
equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often
are easily discovered, seem to be capable of disrupting bacterial biofilms, and can
have low inherent toxicities.
7. Present some notes when using micropipettes in the practice of cultivating
bacteria and bacteriophages.
Remove the lid from the box containing the correct size micropipette tips. P-1000
tips may be blue or clear, while P-20 and P-200 tips are yellow or clear.
Attach the tip by inserting the shaft of the micropipette into the tip and pressing
down firmly (figure on right). This should produce an airtight seal between the tip
and the shaft of the micropipette.
Replace the lid of the tip box to keep the remaining tips sterile. Avoid touching
the tip (especially the thinner end), because the tips are sterile.
Depress the plunger of the micropipette to the first stop.
Immerse the tip a few millimeters below the surface of the solution being drawn
up into the pipette. Pipetting is most accurate when the pipette is held vertically.
Keep the angle less than 20 ̊ from vertical for best results.
Release the plunger slowly, allowing the tip to fill smoothly. Pause briefly to
ensure that the full volume of the sample has entered the tip. Do not let the
plunger snap up.
8. What is your opinion about the practical section on bacteria and
bacteriophages?
 This lecture provides us with lots of knowledge and skills about virus
experiments. It’s helpful.