First, the purpose:
1. Understand the meaning of phage titer and its measurement principle.
2. Learn how to check phage.
3. Master the operational skills of measuring phage titers using a two-layer agar plate method.
Second, the principle:
Phage is a type of virus that is parasitic on microorganisms such as bacteria and actinomycetes. Its individual form is extremely small, and its number cannot be measured by conventional microbial counting. When the phage infects the bacteria, it will quickly cause the sensitive bacteria to cleave, releasing a large number of progeny phage, then they will spread and infect the surrounding cells, and finally the suspension containing the sensitive bacteria will gradually become clear from the turbidity, or contain sensitive bacteria. A plaque visible to the naked eye appears on the plate - plaque. Understanding the characteristics of phage, rapid inspection, separation, and titer determination play an important role in preventing phage contamination in production and scientific research.
The sample may be fermented liquid, air, sewage, soil, etc. (As for the object that cannot be sampled and inspected, the surface of the cotton that has been soaked with sterile water may be used as an inspection sample). For easy isolation, the culture can be first propagated to increase the number of phage in the sample.
The phage test using bioassay takes about 12 hours, so it is impossible to judge whether there is phage contamination in time. A quick check can be used to determine if there is phage contamination to take the necessary control measures. According to the normal fermentation (culture) liquid after centrifugation, the protein content of the supernatant is small, and it remains clear after heating; while the fermentation (culture) solution infecting the phage is centrifuged, and the supernatant contains self-lysing bacteria. The active protein that escapes in the medium undergoes protein denaturation after heating, so that the Tyndall effect occurs under light irradiation without clearing. This method is simple and rapid, and the judgment of the phage contaminated by the fermentation broth is also accurate. However, it is not suitable for the diagnosis of lysogenic bacteria and thermophilic phage, and it is often not applicable to the primary seed culture medium with less phage infestation.
The titer of the phage is the number of particles of the infective phage contained in the 1 mL sample. The titer is generally determined by a two-layer agar plate method. Since a phage produces a plaque on an agar plate containing a specific host bacterium, the phage titer can be calculated based on the number of plaques present in a certain volume of the phage culture solution. The plaque formed by this method has the same shape and size, and has high definition, so the counting is relatively accurate, and thus it is widely used.
Third, the material:
1. Species:
Sensitive indicator bacteria (E. coli), E. coli phage (separated from gutter or septic tank sewage).
2. Medium:
Double meat paste peptone culture medium, upper layer meat paste peptone semi-solid agar medium (containing agar 0.7%, tube-packed, mL per tube), lower layer of meat paste peptone solid agar medium (containing agar 2%), 1% peptone Water medium.
3. Instruments and appliances:
Sterile test tubes, petri dishes, flasks, pipettes (1, 5 mL), thermostatic water baths, centrifuges, 721 spectrophotometers, etc.
Fourth, the process:
Phage check
2. Determination of phage titer
Fifth, the method:
1. Phage inspection:
1 sample collection
Put 2~3g soil sample or 5mL water sample (such as gutter sewage) into the sterilized triangle bottle, add 3~5mL of sensitive indicator bacteria (E. coli) in the logarithmic growth phase, and add 20mL twice broth protein 胨Culture medium.
2 proliferation culture
Incubate at 30 ° C for 12 to 18 h to allow the phage to proliferate.
3 centrifugal separation
The culture solution was centrifuged at 3000 rpm for 15 to 20 minutes, and the supernatant was taken and diluted with pH 7.0, 1% peptone water to 10 -2 to 10 -3 for phage examination and potency measurement.
4 Bioassay:
Double layer agar plate method;
Pour down the agar, melt the lower medium, and pour the plate (about 10 mL / dish) for use.
Pour the upper agar to melt the upper culture medium. When the upper culture medium to be melted is cooled to about 50 °C, add 0.2 mL of sensitive indicator bacteria (E. coli) to each tube, and the sample liquid to be tested or the above phage proliferation solution is 0.2 to 0.5 mL. After mixing, pour into the upper plate and pour it.
Constant temperature culture: The observation was carried out at a constant temperature of 30 ° C for 6 to 12 hours.
Observations: If phage is present, translucent sterile round plaques appear in the upper layer of the bilayer medium.
 Single layer agar plate method  The lower medium was omitted, the amount of agar in the upper medium was increased to 2%, and after cooling, it was cooled to about 45 ° C. The indicator bacteria and the sample were added as in the above method, and the plate was quickly poured after mixing. The results were observed after incubation at 30 ° C for 6 to 16 hours.
5 Centrifugal heating method (quick check)
The normal culture medium of Escherichia coli and the abnormal E. coli culture medium infected with phage were centrifuged at 4000 rpm for 20 min, and the supernatants of the two fermentation broths (A1) were taken, and the OD650 optical density value was measured on a 721 spectrophotometer. Take 5 mL of the supernatant in a test tube, boil for 2 min (A2) in a water bath, measure the optical density value of OD650 of A2 solution, and record the result.
2. Determination of phage titer:
1 inverted plate
The lower layer of meat paste peptone solid medium which was cooled and cooled to about 45 ° C was poured into 11 sterile culture dishes, about 10 mL of medium was poured into each dish, and placed flat. After condensing, the phage dilution was indicated at the bottom of the dish.
2 diluted phage
According to the 10-fold dilution method, 0.5 mL of Escherichia coli phage was aspirated and injected into a test tube containing 4.5 mL of 1% peptone water, which was diluted to 10-1 and diluted to a dilution of 10-6.
3. Mix the phage with the bacterial liquid:
11 sterile empty tubes were labeled 10 -4 , 10 -5 , 10 -6 and control, respectively. Pipette 0.1 mL from the 10 -4 , 10 -5 and 10 -6 phage dilutions into the above-mentioned numbered sterile tubes, make three tubes in parallel for each dilution, and add 0.1 mL to the other two control tubes. The bacteria water was added to each tube, and 0.2 mL of Escherichia coli suspension was added to each tube. The bacterial tube was mixed with the phage solution by shaking the tube, and the mixture was incubated for 5 minutes in a 37 ° C water bath to allow the phage particles to fully adsorb and invade the cells.
4. Inoculate the upper plate:
Add 11 mL of the upper layer of meat paste peptone semi-solid agar medium which was melted and kept at 45 ° C, respectively, into a mixing tube containing phage and sensitive bacteria solution, quickly homogenize, and immediately pour into the surface of the corresponding number of the bottom medium plate. Shake the plate while pouring it to spread the surface quickly. The cells were allowed to stand horizontally, and cultured at 37 ° C after solidification.
5. Observe and count:
Observe the plaque in the plate and record the result in the calculation formula: N=Y/V%×X
(N: efficacy value, Y: average plaque number / dish, V: sample size, X: dilution)
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