BTE258
Summer ‘24
LAB REPORT
Submitted By
Radita Khan
ID: 22336021
Section: 05
Submitted To
Md Tawsif Ur Rashid
Lecturer
MNS, BRAC University
Date: 15/09/24
Date of the experiment: 08.09.2024
Name of the experiment: Bacteriophage Double-Layer Agar Plaque Assay for Phage Titer
Determination.
Abstract:
A widely used method for determining the titer of bacteriophages, which measures the number of
infectious viral particles in a sample, is the bacteriophage double-layer agar plaque assay. This
technique is valuable in virology for quantifying phage concentrations and assessing their
infectivity. The objective of this experiment was to use the plaque assay to evaluate the titer of a
specific bacteriophage suspension.
In this method, a double layer of agar is prepared, with the bottom layer providing a solid base
and the top layer allowing for phage diffusion. Both bacteriophages and their host bacteria are
mixed into the soft agar, leading to the formation of visible plaques that indicate areas of
bacterial lysis caused by phage infection. After incubation, plaques are counted, with each plaque
representing one infectious phage particle. The dilution factor is then used to calculate the phage
titer in plaque-forming units per milliliter (PFU/mL).
The results showed that this assay effectively measures bacteriophages, providing important
information for research in virology and microbiology. Understanding phage titers is crucial for
applications such as phage therapy and ecological studies involving microbial populations.
Principle:
The Double-Layer Agar (DLA) technique, also known as the double agar overlay method, is a
common procedure used to measure bacteriophages, which are viruses that specifically infect
bacteria. This method involves two layers of agar: a solid base layer made of hard agar that
forms a gel and supports bacterial growth, and a soft agar overlay that contains a mixture of
diluted phage particles and a large number of host bacterial cells.
Hard Agar Layer (1.5% Agar)
The hard agar layer at the bottom of the Petri dish serves as a sturdy floor. It is made of 1.5%
agar, making it thick and strong. This hard layer has two important functions:
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1. Keeping Everything in Place:
The hard layer prevents the phages and bacteria from falling out of the dish, acting as a
container that keeps everything together.
2. Helping Phages and Bacteria Stick:
The hard surface allows phages and bacteria to attach to the agar easily, which is crucial for
phages to infect the bacteria.
Soft Overlay Layer (0.5% to 0.7% Agar)
The soft agar layer is poured on top of the hard layer and is made of 0.5% to 0.7% agar, making
it thinner and softer. This soft layer also serves two important functions:
1.Allowing Phages to Move Around:
The soft agar permits phages to move freely and spread out, helping them find and infect more
bacteria.
2. Keeping Bacteria in Place:
The soft agar holds the bacteria in their spots, even while the phages are moving around, which
helps create clear areas (called plaques) where phages have infected and killed the bacteria.
Importance of the Layers
The hard and soft agar layers work together to create an ideal environment for counting phages:
- The hard layer keeps everything in place and facilitates attachment for phages and bacteria.
- The soft layer allows phages to move around and infect bacteria.
When phages infect the bacteria, they produce clear plaques in the agar. By counting these
plaques, scientists can determine how many infectious phages are present in the original sample,
known as the phage titer. This measurement is crucial in phage research and therapy.
In summary, the hard and soft agar layers function like a sturdy floor and a soft cushion, enabling
phages and bacteria to interact effectively so that scientists can count the phages and gain
insights into their behavior.
Each Petri dish is prepared for different dilutions of phage stock, allowing researchers to expose
a dense culture of bacteria to various phage concentrations in this quantitative test. The DLA
plaque assay is based on the idea that when phages infect and reproduce within bacterial cells,
they cause the cells to burst or lyse. Each phage particle can infect a bacterial cell that is
sensitive to it, resulting in clear areas called plaques surrounded by healthy, uninfected bacterial
cells. Each plaque represents an infection caused by a single phage particle. By counting the
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number of plaques formed at different dilutions and considering the dilution ratio, researchers
can calculate the phage titer—the quantity of phages in the original sample—expressed in
plaque-forming units per milliliter (PFU/mL). This test allows for the precise measurement of
infectious phages in a sample.
Originally developed as a virological test to quantify bacteriophage infectivity, the plaque assay
has also been adapted for studying mammalian viruses. It has become one of the most popular
methods for isolating viruses, purifying them, and maximizing their amounts. The plaque assay
measures how well an infectious virus can create a “plaque” on a layer of cultured cells. A
plaque forms when a single viral particle infects one bacterial cell, multiplies inside it, and
eventually kills it. The newly replicated virus particles then go on to infect nearby cells, causing
them to die as well. When a single phage particle attaches to a susceptible cell, it enters the cell,
replicates, and releases new phage particles that can infect other bacteria nearby. Each plaque is
designated as a plaque-forming unit (PFU) and represents the lysis caused by an infection.
To calculate the number of phage particles in the initial stock culture, researchers count the
number of plaques that form on the seeded agar plate and multiply this number by the dilution
factor used for that plate. For reliable results, plates should contain between 30 and 300 plaques;
plates with more than 300 PFUs are considered too numerous to count (TNTC), while those with
fewer than 30 PFUs are considered too few to count (TFTC). The plaque assay differs from
colony counting primarily in that it involves spreading 100-400 phage particles over the agar
surface along with many host bacterial cells. In contrast to counting colonies where each pile
represents a colony formed from individual bacteria, plaques represent zones of destruction
against a background of intense bacterial growth. Each clear spot or plaque corresponds to one
phage from the sample applied to the plate.
By following this method carefully, researchers can effectively isolate and quantify
bacteriophages, providing important insights into their biology and potential applications in areas
such as phage therapy and microbiological research.
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Materials:
Cultures:
1. 3-hour nutrient broth cultures of Escherichia coli B and Escherichia coli phage.
Media:
1. Luria agar plates,
2. Luria soft agar tubes
3. Luria broth
Equipment:
1.Bunsen burner
2. Water bath
3. Thermometer
4. 1-ml sterile pipettes
5. Sterile Pasteur pipettes
6. Mechanical pipetting devices
7. Test tube rack
8. Glassware marking pencil
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Method:
1. Dilution of Phage Stock:
● A series of dilutions was performed to obtain a plaque count on plates of 100–250 pfu
(plaque-forming units).
● Saline, broth, or sterile water was used for the dilutions.
● A variety of phage dilutions, such as tenfold dilutions, was plated.
2. Plating of Phage:
● One soft agar tube was taken out of the 50°C water bath at a time.
● To the 3.0 ml of soft agar, 0.3 ml of bacteria and 0.1 ml of diluted phage were added.
● The agar tube was rolled between the palms for two or three seconds to mix the contents.
● The mixed agar was poured rapidly onto the warm base plate’s agar surface.
● The soft agar was spread quickly but gently across the surface of the base plate agar.
● The soft agar was allowed to set, and the plates were inverted and incubated at 37°C.
● Plaques varied in appearance depending on the phage but became apparent within 6 to 24
hours.
●
3. Counting and Calculating Titers:
● The viral titer, measured quantitatively and represented as plaque-forming units (pfu) per
milliliter, indicated the biological activity of the virus.
● After removal from the incubator, the plates were examined.
● Except for small, transparent patches known as plaques, hazy areas where bacteria had
proliferated were observed all over the plate.
● Each plaque, representing a patch of dead bacteria, indicated a single virus.
● The precise number of plaques was counted on plates that contained between thirty and
three hundred plaques.
● The titer (pfu/ml) of the viral stock was then calculated using the following formula.
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Counting and Calculating Titers:
1. Plaque Representation
- Each plaque symbolizes a single virus and is formed from defunct bacterial colonies
- The number of plaques on a plate typically ranges from 30 to 300. The exact count needs to be
determined for accurate calculations.
2. Titer Calculation Formula
To determine the titer of the viral stock in plaque-forming units per milliliter (pfu/ml), use the
following formula:
Titer (pfu/ml) = (pfu / µl) × (1000 µl / 1 mL) × dilution factor
3. Variables Defined
- pfu: The number of plaques counted on the plate with 20-200 plaques.
- µl: The volume of phage lysate plated, measured in microliters (µl).
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Figure – 4: Phage Plaques during overnight growth at a dilution of 10-´
Here, 297 plaques were found to have formed in an inadequate manner in figure 7.1.
Titer(pfu/ml) is therefore equal to (297/0.1) × 10000=297,00,000, or 2.97×10^7
pfu/ml.
Result: The titer of the original phage stock is or 2.97×10^7 pfu/ml
Discussion:
In this experiment, the effective infection of phages and the resulting destruction of bacteria were
shown by the formation of plaques. The titer of phages, measured in plaque-forming units per
milliliter (PFU/mL), can be calculated by counting the number of plaques at different dilutions.
The success of this assay depends on several important factors, such as choosing the right host
bacteria, having the best incubation conditions, and preparing the phage dilutions accurately.
Any mistakes made during these steps can greatly affect the results. For example, if the
bacterial-phage mixture is not evenly spread or if the dilution is incorrect, it can lead to
misleading plaque counts and inaccurate titer calculations.
The results of the experiment showed a consistent and repeatable number of plaques across
various dilutions, indicating that the methods used were correct and produced trustworthy
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findings. However, there are some limitations to consider. Differences in how quickly the host
bacteria grow or small changes in the agar concentration can affect the size of the plaques.
Additionally, this method only detects living phages that can infect host cells; any non-infective
phages may go unnoticed.
When fewer than 30 plaques appear, it may suggest that either the bacteria defended themselves
well against infection or that there were very few phages present. On the other hand, if more than
300 plaques are counted, it indicates that there are too many phages, which can also make
counting difficult.
The plaque assay is a reliable way to measure phage concentrations, but it is essential to maintain
standard conditions to ensure accuracy and reproducibility. This technique is valuable not only
for basic virology research but also for environmental microbiology, genetic engineering, and
phage therapy—areas where knowing precise phage amounts is crucial for success. Future
improvements could enhance the accuracy and efficiency of measuring phages by using
automated or more advanced detection methods. These changes would help streamline the
process and reduce human error during analysis. By refining these techniques, researchers can
get better measurements of phage activity and improve their use in various scientific fields.
Precautions:
1.A lab coat, and gloves were put on.
2. Sterile methods were utilized.
3. Caution was exercised when handling reagents, and safety data sheets were referred to.
