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Counting phages: Serial dilution technique

Phage plaque assay

Bacteriophage serial dilution is a reliable and widely used laboratory technique that enables scientists to accurately determine the concentration of bacteriophages (phages) in a sample. The technique involves a series of dilutions in which a phage suspension is progressively reduced in volume, resulting in a decrease in the number of viral particles per unit volume.

By performing this dilution series, the number of phages can be counted by infecting a bacterial host and observing the number of infected bacteria, which directly reflects the number of phages in the original sample. This method is particularly useful in cases where the concentration of phages is high, as it reduces the number of particles to a level that can be accurately counted and measured.

After performing the serial dilution, the results are then multiplied by the dilution factor and volume to determine the original phage concentration. This method is highly accurate and provides scientists with a valuable tool for studying bacteriophages and their interactions with bacterial hosts.

Serial dilution of bacteriophage stock
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The serial dilution technique is commonly used for the following purposes:

  1. Phage titer determination: This involves measuring the concentration of phages in a sample and is often used to monitor the efficiency of phage propagation and purification processes.
  2. Phage quantification: Serial dilution is used to quantify the number of phages present in a sample, which is important for many phage applications, such as phage therapy, molecular biology research, and the production of phage-based products.
  3. Phage screening: The serial dilution technique can be used to screen phages for specific properties, such as host range, lytic activity, and potential for genetic manipulation.
  4. Phage viability testing: Serial dilution can be used to assess the viability of phages in a sample, which is important for ensuring the effectiveness of phages for specific applications.

The basic steps for performing a bacteriophage serial dilution are as follows:

  1. Preparation of phage suspension: A phage suspension is prepared by collecting a sample of phages and suspending them in a suitable medium.
  2. Dilution: The phage suspension is diluted in a series of steps, typically using a 1:10 dilution factor (see the image below).
  3. Infection of bacterial host: Each dilution is used to infect a bacterial host, usually through spot-plating or streak-plating on an agar medium.
  4. Incubation: The infected plates are incubated to allow the phages to replicate and the infected bacteria to become visible.
  5. Counting: The concentration of bacteriophages is determined by counting the number of plaques on a plate covered with a bacterial lawn. Each plaque represents a single viral particle that has multiplied clonally. There are several tools that can help with this particular activity one of them is One Petri developed by Michael Shamash, A Ph.D. student from McGill University.

Bacteriophage serial dilution can be performed using different dilution factors, depending on the purpose and sensitivity of the experiment. The most commonly used dilution factor is ten-fold, where each successive dilution is made with a 1:10 dilution factor. This dilution factor provides a good balance between dilution accuracy and ease of counting the number of infected bacteria.

Serial dilution of bacteriophage stock
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Other dilution factors that can be used include two-fold (1:2), five-fold (1:5), and fifty-fold (1:50). The choice of dilution factor depends on the initial concentration of phages in the sample, the sensitivity of the detection method, and the desired level of accuracy.

Summary in photo

Serial dilution of bacteriophage stock
Serial dilution of bacteriophage stock

In addition to dilution factor, bacteriophage serial dilution can be performed in different ways, including:

  1. Plate-based serial dilution: This method involves making a series of dilutions and spreading each dilution on a separate agar plate. The plates are incubated to allow the phages to replicate and infect the bacteria, and the number of infected bacteria is counted to determine the number of phages in each dilution.
  2. Tube-based serial dilution: This method involves making a series of dilutions in tubes and then transferring a small volume from each dilution to a separate tube. The tubes are incubated to allow the phages to infect the bacteria, and the number of infected bacteria is counted to determine the number of phages in each dilution.
  3. Continuous serial dilution: This method involves making a single dilution and then transferring a fixed volume from the dilution to a series of tubes or wells in a microtiter plate. The tubes or wells are incubated to allow the phages to infect the bacteria, and the number of infected bacteria is counted to determine the number of phages in each dilution.

Bacteriophage serial dilution is a laboratory technique used to determine the concentration of phages in a sample. This method is commonly used for phage titer determination, quantification, screening, and viability testing. The serial dilution technique involves diluting a phage suspension in a series of steps, infecting a bacterial host, incubating the infected plates, and counting the number of infected bacteria to determine the number of phages in each dilution and in the original sample.

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