Phage examination and potency determination

Summary

Examination and potency determination of phage: It is easy to understand the characteristics of phage, rapid examination and isolation, and potency determination, which is important for preventing phage contamination in production and scientific research work.

Operation method

Phage examination and potency determination

Principle

Phages are a class of viruses that exclusively parasitize microorganisms such as bacteria and actinomycetes, and their individual forms are so tiny that it is impossible to measure their numbers by conventional microbial counting methods. When virulent phage infects bacteria, it will rapidly cause sensitive bacteria to lyse and release a large number of progeny phage, which will then spread and infect the surrounding cells, and ultimately cause the suspension containing sensitive bacteria to gradually become clear from turbidity, or a naked eye-visible empty spot - phage plaque - appears on the plate containing sensitive bacteria. Understanding the characteristics of phage, rapid examination, isolation, and potency determination play an important role in preventing phage contamination in production and scientific research. The sample can be fermentation broth, air, sewage, soil, etc. (As for the objects that cannot be sampled but need to be examined, the surface can be swabbed with cotton moistened with sterile water as the sample for examination). For easy isolation, the phage can be cultured by proliferation first to increase the number of phage in the sample. Phage examination using the bioassay method takes about 12 h, and thus does not allow timely judgment of whether there is phage contamination. A quick check can roughly determine whether there is phage contamination so that necessary preventive measures can be taken. According to the normal fermentation (culture) liquid after centrifugation of the bacteria precipitation, the supernatant protein content is very small, and it is still clear after heating; while the fermentation (culture) liquid infested with phage after centrifugation of the supernatant contains active proteins escaped from the lysogenic bacteria, and protein denaturation occurs after heating, and thus it does not appear clear under the light irradiation with the Tyndall effect. This method is simple, fast and accurate in determining the phage contamination of fermentation broth. However, it is not suitable for the diagnosis of lysogenic bacteria and warm phage, and it is often not suitable for the primary seed culture broth with less phage infestation. Phage potency is the number of infectious phage particles in 1mL of sample. The determination of potency is generally done using the double agar plate method. Since one phage produces one phage spot on an agar plate containing a specific host bacterium, the potency of a phage can be calculated based on the number of phage spots that appear in a certain volume of phage culture solution. The morphology and size of the phage spots formed by this method are more consistent, and the clarity is high, so the counting is more accurate, and thus it is widely used.

Materials and Instruments

E. coli Phage E. coli
Peptone Culture Solution Peptone Semi-Solid Agar Medium Peptone Solid Agar Medium Peptone Water Medium
Test tubes Petri dishes Triangular flasks Pipettes Constant temperature baths Centrifuges Spectrophotometers

Move

1. Phage examination:

① Sample collection

Put 2--3g soil sample or 5mL water sample (e.g. gutter sewage) into a sterilized triangular bottle, add 3--5mL of sensitive indicator bacteria (E. coli) bacterial solution of the logarithmic growth period, and then add 20mL of two-fold broth peptone culture solution.

② Proliferation culture

Cultivate the phage at 30℃ for 12--18h, so as to proliferate the phage.

③ Centrifugal separation

Centrifuge the above culture solution at 3000rpm for 15-20min, take the supernatant and dilute it to ^10-2-10-3 with 1% peptone water at pH 7.0 for phage examination and potency determination.

④ Bioassay:

Double layer agar plate method;

Pour the lower layer of agar, melt the lower layer of medium, pour the plate (about 10mL/dish) for use.

Pour the upper layer of agar

Melt the upper medium, when the melted upper medium is cooled down to about 50℃, add 0.2mL of the bacterial solution of the sensitive indicator bacteria (E.coli), 0.2-0.5mL of the sample solution to be examined or the phage proliferation solution mentioned above in each tube, and then immediately pour it into the upper plate and spread it out after mixing.

Constant temperature culture: 30℃ constant temperature culture for 6--12h to observe the result.

Observation result: If there is phage, translucent and sterile round empty spot phage plaque appears in the upper layer of double-layer medium.

Single-layer agar plate method

Omit the lower layer of medium, increase the amount of agar in the upper layer of medium to 2%, melt it and cool it to about 45℃, add the indicator bacteria and test samples as the above method, mix it and pour the plate quickly. incubate it at a constant temperature of 30℃ for 6--16h and observe the results.

⑤ Centrifugal separation and heating method (rapid examination)

Take normal culture medium of E.coli and abnormal culture medium of E.coli infected with phage, centrifuge at 4000rpm for 20min, take the supernatant of two groups of fermentation broths respectively (A1), partly determine the OD650 optical density value on 721 spectrophotometer, and in addition, take 5mL of each supernatant in a test tube, put it into a water bath and boil it for 2min (A2), detect the OD650 optical density value of the A2 solution, and record the results. The results were recorded.

2. Determination of phage potency:

① Pour plate

After melting and cooling to about 45 ℃ of the lower layer of meat paste peptone solid medium dumped in 11 sterile petri dishes, each dish about 10mL of medium, flat, to be condensed at the bottom of the dish phage dilution.

② Dilution of phage

According to the 10-fold dilution method, aspirate 0.5mL of E. coli phage, inject into a test tube containing 4.5mL of 1% peptone water, i.e., diluted to ^10-1, and sequentially diluted to ^10-6 dilution.

3. Phage mixed with bacterial solution:

Eleven sterilized empty test tubes were labeled 10-4, 10-5, 10-6 and control. Suck 0.1mL from 10-4, 10-5 and 10-6 phage dilution in the above numbered sterile test tubes, do three tubes in parallel for each dilution, add 0.1mL sterile water in the other two control tubes, and add 0.2mL E. coli bacterial suspension in each tube, oscillate the tubes to make the bacterial and phage solution mix uniformly, and put them into a 37℃ water bath to keep warm for 5min to let the phage particles fully adsorb. The phage particles were fully adsorbed and invaded into the bacterial cells. 4.

4. Inoculate the upper plate:

Melt and keep warm at 45℃, and add 5mL of top layer of Peptone semi-solid agar medium to the mixing tube containing phage and sensitive bacterial liquid, rub it together quickly, and pour it onto the surface of the corresponding numbered bottom layer of medium plate immediately, and shake the plate while pouring it to make the surface spread rapidly. Leave it horizontally, solidify and set it at 37℃ for incubation.

5. Observe and count:

Observe the phage spots in the plate and record the result in Calculation formula: N=Y/V%×X

(N: validity value, Y: average number of phage spots/dish, V: sampling volume, X: dilution)

Caveat

(1) The medium should be prepared with tap water, not with non-ionized water or distilled water, otherwise the contaminated phage spots can never be cultivated.(2) If 0.02% magnesium ion is added to tap water to prepare phage detection medium, the effect is more significant than purely using tap water, and it is easy to detect phage spots and fast.(3)Mg2+MnMnMn 2+are indispensable factors for phage multiplication.Cu2+, andFeCu 2+, Fe 2+have an inhibitory effect on phage.Na+, andK+K +, K +, K +, K +, K +, K +, K +, K +PO43-had no effect.

Common Problems

This method is simple, rapid and accurate in determining phage contamination in fermentation broth. However, it is not suitable for the diagnosis of lysogenic bacteria and warm phage, and it is often not applicable to the primary seed culture broth with less phage infestation.

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