The examination of phage and the determination of effective

, Objective: To understand
meaning of phage esophageal price and its determination principle.
2. Learn how to examine phages.
3. Master the operating skills
determining the
of phages using a double-layer agar plate method.II, principle:
phage is a kind of virus that is specific to
microorganisms, such as bacteria and line-letting bacteria
, whose individual form is extremely small and cannot be measured by conventional microbial counting. When aggressive phages infect bacteria, they quickly cause sensitive bacteria to cleavage, releasing a large number of subphages, and then they spread and infect surrounding cells, eventually causing the suspension containing sensitive bacteria to gradually clear from turbidity, or on a plate containing sensitive bacteria visible to the naked eye - phage spots. Understanding the properties of phages, quickly checking, separating, and determining the effectiveness of bactericides plays an important role in preventing phage contamination in production and scientific research.
samples can be fermentation fluid, air, sewage, soil, etc. (as for objects that cannot be sampled but need to be examined, the surface can be wiped with sterile water-soaked cotton as a sample for inspection). In order to be easy to isolate the
culture
, so that the number of phages in the sample increased.
biometric method to carry out phage examination, about 12h, so it is not timely to determine whether there is phage contamination. A quick inspection can be used to approximate the availability of phage contamination in order to take the necessary prevention and control measures. Root According to the normal fermentation (culture) liquid centrifugal bacteria precipitation, the content of the liquid protein is very small,
heating
is still clear, and the fermentation (culture) liquid infected with phage after centrifugation, its upper liquid because it contains the active protein escaping from the self-lysed bacteria, after heating
protein
degeneration, so in the light exposure of the Tindal effect and unclear bright. This method is simple and fast, and the judgment of the fermentation liquid contaminated phage is also more accurate. However, the diagnosis of soluble bacteria and warm phages is not suitable for first-level seed cultures with fewer infested phages.
the effectiveness of phages, i.e. the number of particles containing infested phages in 1mL samples. The determination of the effective price is generally made using the double-layer agar plate method. Because on a agar plate containing specific host bacteria, a phage generally produces a phage spot, so according to a certain volume of phage culture appears phage spot number, calculate the effectiveness of phage. The form, size and clarity of phage spots formed by this method are more accurate, so the count is more accurate and therefore widely used.III, material:
1. Bacteria:
sensitive indication bacteria (E. coli), E. coli phage (separated from the gutter or manure tank sewage).
2.
Culture base
:
twice the meat paste protein culture, upper body paste proteinsemi-solid agar culture (containing 0.7% agar, test tube sub-pack, per tube mL), lower body paste proteinsolid agar culture (containing 2%) and 1% proteinwater culture.
3. Instruments and appliances:
sterile test tubes, petri dishes, triangular bottles, piped tubes (1, 5mL),
ringing thermostat
water bath pots,
centrifuges
, 721 d'photonometers, etc., process:
1. Phage examination
2. Bacterphage erythrobacterial property price determination 5, Method:
1. Phage examination:
(1) sample collection
2 to 3 g soil sample or 5mL water sample (e.g. culver sewage) into the sterilization triangle bottle, add a long-term sensitive indicator bacteria (E. coli) bacteria liquid 3 to 5mL, plus 20mL double broth proteinculture solution.
(2) proliferation culture
12 to 18h oscillation culture of 30 degrees C, so that phage proliferation.
(3) Centrifugation separation
the above culture solution at 3000 rpm centrifugation 15 to 20min, take the liquid, with pH7.0, 1% protein water dilution to 10
-2
to 10
-3
, for phage examination and physic determination.
(4) Biometrics:
double-layer agar plate method;
pour down layer agar, melt the lower culture base, reverse plate (about 10mL/dish) to be used.
Pour the upper agar
melt the upper medium, to melt the upper medium cooled to about 50 degrees C, each tube added sensitive indication bacteria (E. coli) bacteria liquid 0.2mL, the sample liquid to be examined or the above phage proliferative liquid 0.2 to 0.5mL, immediately poured into the upper flatbed after mixing.
thermostat culture: 30 degrees C thermostat culture 6 to 12h observation results.
observations: If there are phages, bright, sterile circular empty plaques appear on the upper layer of the double-layer media.the seloy-layer agar plate method omits the lower culture base, increases the amount of agar in the upper culture to 2%, melts and cools to about 45 degrees C, as the upper method adds the indicator bacteria and samples, and quickly reverses the plate after mixing. The results were observed after 6 to 16h culture at a constant temperature of 30 degrees C.
(5) Centrifugal separation heating method (quick check)
Take normal E. coli culture and abnormal E. coli culture with phage, 4000rpm centrifugation 20min, take two groups of fermentation liquid (A) 1), part of the 721 dicing photometric to determine the OD650 light density value, in addition to each take 5mL on the liquid in the test tube, boiling 2min (A2) in the water bath, detection of A2 solution OD650 light density value, recorded results.
2. Determination of the physiopic value of phages:
(1) inverted plate
will melt and cool to about 45 degrees C of the lower broth protein solid medium dumped in 11 sterile petri dishes, each dish about 10mL medium, flat, to be condensed at the bottom of the petri dish to indicate the bacteriophage dilution.
(2) Diluted phage
in accordance with the 10x dilution method, absorb 0.5mL E. coli phage, injected into a test tube containing 4.5mL1% proteinwater, that is, diluted to 10-1, diluted to 10-6 dilution degrees in turn.
3. Phage and bactericide mixture:
labeled 11 sterilized air test tubes at 10
-4
, 10
-5
, 10
-6
and control, respectively. 0.1mL from 10
-4
, 10
-5
and 10
-6
phage diluents, respectively, in the sterile test tubes numbered above, with three tubes in parallel for each dilution, in the other two The control tube added 0.1mL sterile water, and in each tube to add 0.2mL E. coli suspension, oscillating test tube so that the bacteria liquid and phage fluid mixed evenly, put 37 degrees C water bath insulation 5min, so that phage particles fully adsorbed and invade bacterial cells.
4. Inoculation of the upper plate:
11 melted and insulated in 45 degrees C of the upper meat paste proteinsemi-solid agar medium 5mL were added to the mixing tube containing phages and sensitive bacteria, quickly smoothed, immediately poured into the corresponding number of the base medium plate surface, side-to-side shaking plate to make it spread quickly surface. The water is calm and solidified and cultured at 37 degrees C.
5. Observe and count:
observe the phage spots in the tablet and record the results in the calculation formula: N-Y/V%×X
(N: effect value, Y: average phage spots/dishes, V: sampling, X:dilution