One step growth experiment is An experiment by which molecular events that are occurring during reproduction of virus can be observed.
- It reveals the fundamental nature of virus replication process.
- This process was first performed by Ellis & Delbruck in 1939 by using T2 bacteriophages.
- They also determined the plague counting method for the enumeration of bacteriophages.
- In this experiment, only a single or one cycle of virus growth is observed.
- Therefore, it is called as one step growth experiment.
- Excess number of host cells are allowed to infect with phage particles.
- This makes the infection synchronous. That means the simultaneous infection of large number of particles to the host cell is taking place.
- Observation made on such host cell culture is similar to observation made on single host cell infected by a phage.
- In the experiment, excess host cells are infected with phage particles at a ratio of 1:10.
- This is done to prevent the adsorption of more than one virus per cell.
- Mixture is incubated for a short period of time (5 min).
- This incubation allows the adsorption of phage particle on host cell.
- If the bacteria are in excess, all the phage particles will be adsorbed.
- Such mixture is then diluted to such an extent (1:1000) that the virus particles released after first round of replication cannot adsorb to uninfected cell.
- Thus, only one step of virus growth can occur.
- Samples of diluted mixture are then removed at regular time interval & used for plaque count.
- This gives a measure of infectious centers i.e. the infected bacteria & number of virus particles (i.e. No. of plaques per ml).
- When a log no, of plague forming units/ml is potted against time, a curve is obtained & it is termed as one step growth curve.
- This one step growth curve shows the various events that are occurring during the virus replication cycle.
• This curve gives three distinct phases -
1]. Latent Period
2]. Burst or Rise Period
3]. Plateau Period
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| One step growth curve of virus |
I]. Latent Period:
- It is the period from infection to cell lysis.
- During this, there is no release of new virus particles from infected cells.
- Therefore, the plaque count remains constant.
- T phage has latent period of 22 to 23 min at 37°C.
- This period can be divided into two phases as 'Eclipse' period & 'Intracellular Accumulation' period.
- Time from infection until intracellular accumulation of phages is called as 'Eclipse' period.
- T2 bacteriophage has eclipse period of about 11.5 min at 37°C.
- In this, gene expression, protein synthesis & genome synthesis occurs.
- The time from initiation to the end of intracellular accumulation of phages is called as 'Intracellular Accumulation' period.
- During this, phage proteins & genomes assemble into new phage particles.
- T2 bacteriophage requires the period of about 11 to 12 min. at 37° C for this period.
II]. Burst Period or Rise Period
- The time from initiation of infected host cell lysis to the end is called rise or burst period.
- At the end of latent period, each infected cell lyses & liberates a crop of new virus particles.
- During this phase, there is release of new viral particles from infected cells & therefore, plaque count increases rapidly.
- T2 bacteriophage has the rise period of about 10 min. at 37°C.
- Due to the asynchrony of infection the rise period is slightly extended.
III] Plateau Period :
- This period represents the end of all infected host cell lysis.
- The newly liberated phage particles fail to meet uninfected host cells due to high dilution.
- Therefore, during this phase, the plaque count remains constant.
- T2 phage enters in plateau in about 30 min. at 37°C.
- Burst Size - Burst size is defined as the number of virus particles produced from the infection of a single cell. The burst size is calculated using following formula -
- T2 phage has a burst size of less than 100 phages/cell.
- Burst size varies from 20 to 3000 virions/cell for different viruses.
