The specific operation is to inoculate an appropriate amount of virus into high-concentration sensitive cell culture or highly diluted virus cell culture, or treat virus cell culture with antiviral serum to establish synchronous infection, and draw virus characteristic curve with infection time as abscissa and virus titer as ordinate, that is, one-step growth curve.

One-step growth curve is divided into incubation period, cracking period and stable period.

Different stages of one-step growth curve

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incubation period

Refers to the time from the invasion of phage nucleic acid into host cells to the assembly of the first mature phage particles. It can be divided into two stages: 1, and the obscure stage refers to a period of time after the host cell is artificially lysed (with chloroform, etc.). ) In the early incubation period, the cells are in the stage of replicating phage nucleic acid and synthesizing protein capsid; 2. Intracellular aggregation period: that is, the late incubation period, which means that after the obscure period, if cells are lysed artificially, the lysate has been infectious for some time, which means that phage particles have been assembled in cells, which can be observed by electron microscope.

Cracking period

After the incubation period, the host cells were rapidly lysed and the phage particles in the solution increased rapidly. Phage or other virus particles cannot grow alone because they are only assembled by individuals. In addition, if the host cells suddenly rupture, theoretically, their rupture is instantaneous. But in fact, because the lysis of each cell in the host population can not be carried out synchronously, a long lysis period appears.

locking phase

It means that all infected host cells have been lysed and the number of bacteriophages in the solution has reached the peak. During this period, the average number of phage particles released by each host cell is the amount of lysis.

experimental method

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The experimental method of one-step growth curve is as follows: firstly, the suspension of sensitive bacteria in logarithmic growth period is mixed with an appropriate amount of phage. Generally, the mixing ratio of phage and bacteria is 1: 10 to prevent several phages from infecting a bacterial cell at the same time. After adsorption for a few minutes, a certain amount of phage antiserum was added to the mixed solution to neutralize the non-adsorbed phage. Then dilute the culture solution at high magnification to avoid secondary adsorption and infection. After culture, samples were taken regularly, the samples containing phage were mixed with sensitive bacteria, cultured on the plate, and the number of plaques was calculated. The results showed that the number of plaque did not increase in the initial period after adsorption (5 ~ 10 min), which indicated that the phage had not completed replication and assembly, and this period was called the incubation period of phage. After the incubation period (20 ~ 30 min after infection), the number of plaque in the plate suddenly increases linearly, indicating that the phage has been released from the host cell, which is called lysis period. The average number of new bacteriophages released by each infected bacterium is called lysis amount. When the host is completely cracked and the titer of phage in the solution reaches the highest point, it is called quiescent period.

conclusion

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Through one-step growth curve, we can get the latent period and cracking amount of the virus. The incubation period is the shortest time required for virus particles to adsorb cells and infected cells to release their offspring. The amount of lysis is the average number of progeny virus particles produced by each infected cell, which is equal to the number of all progeny viruses released by stable infected cells divided by the number of latent infected cells, that is, equal to the ratio of stable virus titer to latent virus titer.

Monopulse curve

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Experimental curve for quantitative description of growth law of virulent phage. The basic step is to infect high concentration host cells with diluted phage suspension to ensure that each cell absorbs no more than one phage. After a few minutes, the adsorption was stopped, diluted and cultured at the optimum growth temperature of the bacteria. Within a certain period of time, samples were taken every few minutes for titer determination. The curve drawn with titer as the ordinate and culture time as the abscissa is a one-step growth curve, which can reflect the three most important characteristic parameters of each phage: incubation period, lysis period and lysis amount.