Phage culture

I. Liquid
Culture
Method
1. The designated host bacteria are cultured at an appropriate temperature by liquid culture, generally cultured for 16 to 24 hours
2. the
phage body
progenitor 100 sl is evenly mixed with the host bacteria suspension liquid for 300 sl and remained in place for 15 minutes to infect it.
3. The mixture is connected to
test tubes
containing 10 ml of fresh liquid
trag medium
, which oscillates and cultures for about 8 to 24 hours at appropriate temperatures, and the culture time depends on the phage.
4. Transfer the culture fluid into the
centrifuge tube
and centrifuge at 10,000 rpm (5,000 g) for 10 minutes to precipitate the host bacteria.
5. Remove the supersalt liquid from the new tube and remove the residual bacteria with a
filter at a aperture of 0.22
m, i.e. without the phage progenitor of the host bacteria. However, because the phage original is transparent, it is not possible to directly judge its proliferation effect with the naked eye, so its effective price should be determined to confirm the effectiveness of proliferation culture.II. Double-layer
agargum
culture method
1. The designated host bacteria will be cultured at an appropriate temperature by liquid culture method, generally cultured for 16 to 24 hours.
2. Mix the phage progenitor 100 sl evenly with the host bacterial suspension 300 sl and leave to infect for 15 minutes.
3. Add the mixture to a 0.7% agar medium cooled at 5 ml to 45 degrees C and spread evenly on the poured 1.8% agar medium plate immediately after mixing.
4. After the plate solidified and moved to the appropriate temperature of the culture box, the general culture of 8 to 24 hours, the culture time depends on the phage.
5. Another control group without phages was produced at the same steps as above.
6. The cultured plate was compared with the control group for its clarity, and the tablet containing phages was generally clarified, while the control group containing only host bacteria was cloudy.
7. After placing the tablet containing the phage at -20 degrees C for 4 to 5 hours, remove and thaw at room temperature.
8. The liquid produced by thawing is moved into the centrifuge tube and centrifuged at 10,000 rpm (5,000 g) for 10 minutes to precipitate the host bacteria.
9. Remove the supersalt liquid from the new tube and remove the residual bacteria with a filter of 0.22 m aperture, i.e. without the phage proton of the host bacteria.III. Surface agar culture method
1. The designated host bacteria will be cultured at an appropriate temperature by liquid culture method, generally cultured for 16 to 24 hours.
2. Spread the host bacterial suspension 3 ml evenly on a 1.8% agar culture base plate and leave to rest for 2 hours.
3. Suck out the remaining host bacterial suspension and apply 0.3 ml of phage progenitor fluid evenly to the plate.
4. Culture at appropriate temperatures for about 16 to 24 hours, the culture time depends on the phage.
5 ml of fresh liquid medium to the plate and scrape the bacteria off the surface of the plate with an L-shaped stick.
6. Move the scraped liquid into the centrifuge tube and centrifuge at 10,000 rpm (5,000 g) for 10 minutes to precipitate the host bacteria.
7. Transfer the supersalt fluid to the new tube and remove the residual bacteria with a filter of 0.22 m aperture, i.e. without the host bacteria phage