One step multiplication curve for bacteriophages.

The single-step growth experiment of Ellis and Delbruck demonstrates the cyclic replication of the phage. These authors devised a method to demonstrate only a single step of the many steps of phage replication. Essentially they drastically diluted the mixture after attachment of phage to bacteria, so when the infected cells lysed, no new host cells could be found for a second round of infection. A number of modifications have been introduced since the original experiment was reported. For instance, instead of diluting the initial bacterium:phage mixture, antibodies specific for the phage attachment apparatus may be added to the mixture to ‘neutralize’ and thus render all of the unadsorbed phage unable to adsorb to any bacterium. image

How do you perform this experiment:

Bacteriophages are infected with a very large number of phage particles: The large number of phage ensures all bacteriem are rapidly infected. The high level of infection is called multiplicity of infection (MOI) and can be achieved with a phage to host ratio of 5 to 10 plaque forming units (PFU) per cell. Adsorption of virions to cells is allowed to proceed for suitable time : To replicate, a virus should induce its host to synthesize components that are necessary for the assembly of new virus particles. The virus accomplishes this process by first attaching to the host (adsorption) and then injecting its nucleic acid into the cell (injection or penetration). The viral DNA can stay free in the cell and be replicated as such, or it can be incorporated into the host chromosome and be replicated simultaneously with it. Viral proteins are next synthesized with the host’s machinery under the direction of viral DNA and the new virus particles are assembled mechanically. These particles can find their way out of the cell or lyse the cell and be released into the medium, ready to infect new cells.

The phage/cell mixture is then diluted synchronizing the infection or adding antivirus antiserum. This stop the absorption  of virions to cells that are infected and also prevent infection of new cells other than those that has been infected. Antivirus antiserum contains antibodies directed against the virus. It binds and occupy all the attachment sites of the viral particles so that no new bacteria cells can attach a virus. As the infection of bacteriophages is synchronised, the interaction of virus with the cell population can be seen as a single interaction between phage and the cell. In order to visualize  the infection over time, samples are removed at specified intervals and plated to quantitate the phage present in the culture.

At the start of the experiment, the plaque count is relatively constant over a time period because each infected bacterium will yield only one plaque. A rise in plaque forming units (pfu) to a plateau level occurs as bacteria are lysed and the newly synthesized phage are released into the medium. These phage particles fail to meet susceptible bacteria (due to the dilution of the adsorption mixture) and thus remain free in the culture fluid. The average number of phage released per bacterium is called the burst size and this value may be calculated from the data. The burst size varies in accordance with the specific virus, and may range from 10 to 100 for the DNA transducing phages to approximately 20,000 pfu for the RNA viruses. Plaque assay for bacteriophages are performed by mixing the phage into a layer of bacteria which are spread out as an overlay on the surface of an agar plate. As the plate is incubated, the bacteria grow and they become visible as a turbid layer on the plate. When a phage infects bacteria cells, a zone of Lysis or growth inhibition can occur. This produces clear zone in the bacterial lawn known as plaque. Each plaque originates from a single phage particle. If the number of phage particles was monitored during growth, a growth curve could be drawn which would be similar to that of the bacterial growth curve except in the last stage.

The phage growth curve starts with a latent or eclipse period (similar to the bacterial lag phase). During this phase, the infection, adsorption, injection and syntheses of new viral DNA and protein coat occur. The next phase is called the maturation or release stage (similar to the log phase in bacteria) when new phage particles are assembled and released. The cycle can then start over with the infection of new cells. In this manner, the shape of the curve would look step-wise and that is why the process is called “one-step phage growth curve”. image

 

Stages of one step multiplication curve:

Eclipse: or initial period can be defined as the time period taken for the appearance of first intracellular phages. No phage particles can be detected during this period as the phages are being uncoated and  phage DNA is being injected foe replication.

Synthetic period: During synthetic period intracellular particles are being produced. As in the Eclipse period, there are no phage particles released during the synthetic period.

Latent Period: The first two periods are combined in the third period known as latent period. The latent period is described as the time period prior to the release of infection particles or appearance of extra cellular phages.

In the latent period, attachment, entry, replication, transcription, translation and assembly of progeny phages occur.   

Rise Period: In this period lysis occurs and extracellular phages appear and they increase in number of concentration of bacteriophages rises.

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  • Burst Size: Average yield of infectious virus per cell is called burst size.
  • Burst Size = Final titre of virus / Initial viral titre.
  • There is much variation in bursts size between different kind of cells.
  • In one study with phage, a burst size of 170 was obtained when growing bacteria cells and value of 20 was obtained with resting bacteria cells. This is because rapid growing cells means, its cell machinery are active and are metabolically active than the resting bacteria cell. So yield of infectious virus increases with growing cells than resting bacteria cell.
  • Extra step, lysis from without (LO): LO described as an early lysis of bacteria induced by high-multiplicity virion adsorption and that occurs without phage production and leads to killing of bacteria. LO can be induced by adding chloroform, which break open the host cell and the intracellular phages are released. It is an artificial lysis. 

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