CN101775444A - Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii - Google Patents
Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii Download PDFInfo
- Publication number
- CN101775444A CN101775444A CN 201010118057 CN201010118057A CN101775444A CN 101775444 A CN101775444 A CN 101775444A CN 201010118057 CN201010118057 CN 201010118057 CN 201010118057 A CN201010118057 A CN 201010118057A CN 101775444 A CN101775444 A CN 101775444A
- Authority
- CN
- China
- Prior art keywords
- enterobacter sakazakii
- phage
- enterobacter
- nutrient solution
- real time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a method for rapidly determining the titer of bacteriophage of Enterobacter sakazakii, comprising the following steps of: A. preparing a bacteriophage concentration gradient suspension; B. preparing an Enterobacter sakazakii suspension; C. sequentially adding an Enterobacter sakazakii culture solution, the Enterobacter sakazakii suspension in the step B and the acteriophage concentration gradient suspension in the step A in a micropore plate; and D. scanning and determining in real time, scanning and determining a growth curve in a dynamics determining mode in real time under a certain culture temperature and wave length by utilizing a microplate reader. By using the determining method, the titer of the bacteriophage of the Enterobacter sakazakii can be simply, rapidly and efficiently determined.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of rapid assay methods of titer of bacteriophage of Enterobacter sakazakii.
Background technology
Enterobacter sakazakii is a kind of peritrichous that has, and can move no gemma, amphimicrobian gram negative bacillus, can cause meningitis, enterocolitis, microbemia etc., especially have stronger pathogenicly for infant and hypoimmunity crowd, mortality ratio is up to 50%-75%.Enterobacter sakazakii extensively exists in physical environment; and owing to wide, high temperature resistant, the anti-drying of its growth temperature range, have advantage such as mycoderm protective layer, so in a single day this bacterium has polluted the foodstuff production environment; cause the pollution of whole process of production easily, and be not easy to eradicate.At present, physics and chemical mode sterilizations such as main employing sterilization reagent, high temperature or ultraviolet.But these modes pollute environment easily, and using in the food-processing environment has certain limitation, and for this bacterium with mycoderm protection of Enterobacter sakazakii, some chemical reagent, sterilization effect is unsatisfactory.In this case, comprise the biological control of phage, because of it is little to environmental influence, with strong points and obtain more and more widely application.
Phage is a viroid, and volume is small, only just can see under electron microscope, is a kind of life of acellular structure, only enters host cell and just has vital signs, and have host specificity.Bacteriophage of Enterobacter sakazakii, be virulent phage, during host cells infected, use host's the DNA of metabolism machine reproduction phage and the protein of synthetic phage, be assembled into new phage, and discharge by the cracking of host cell, and then go to infect near bacterium again, and with they cracking, so repeatedly, thus thorough kill bacteria.And to estimate the effect of its kill bacteria, just must measure tiring of phage.
The measuring method of phage titer normally adopts the double-layer plate method to carry out at present.This method, operation steps is many, and when titer of bacteriophage of Enterobacter sakazakii is measured, can't draw significantly plaque of zone, tires thereby can't draw accurately.
Therefore, this area needs a kind of method quick, efficient, easy and simple to handle, carries out titer of bacteriophage of Enterobacter sakazakii and detects.
Summary of the invention
The object of the present invention is to provide a kind of method of rapid detection titer of bacteriophage of Enterobacter sakazakii, use this method, can measure tiring of bacteriophage of Enterobacter sakazakii apace, thereby be further used for Enterobacter sakazakii pollution in the biological control foodstuff production environment.
In order to realize purpose of the present invention, the invention provides a kind of method of rapid detection titer of bacteriophage of Enterobacter sakazakii, described method comprises utilizes microplate reader working power mode determination that the Enterobacter sakazakii nutrient solution that adds the different concns phage is carried out real time scan, to measure the growth curve of Enterobacter sakazakii.
In one embodiment, the method for rapid detection titer of bacteriophage of Enterobacter sakazakii of the present invention comprises the steps:
A. prepare phage concentration gradient suspension;
B. prepare the Enterobacter sakazakii bacteria suspension;
C. add Enterobacter sakazakii bacteria suspension among Enterobacter sakazakii nutrient solution, the step B and the phage gradient suspension in the steps A in the microwell plate successively;
D. real time scan is measured, and utilizes microplate reader, and the working power mode determination carries out the growth curve that real time scan is measured Enterobacter sakazakii.
Determine tiring of phage according to growth curve of Enterobacter sakazakii and the concentration of phage.
Real time scan in the aforesaid method is determined under certain culture temperature and the wavelength and carries out.
In a preferred embodiment, the real time scan in the described method is determined under 25 ℃～40 ℃ the temperature, more preferably carries out under 36 ± 1 ℃ temperature.
In a preferred embodiment, the real time scan in the described method is determined at 600 ± 50nm, more preferably carries out under the scanning wavelength of 600nm.
According to the preferred embodiments of the invention, in the method for the invention, described real time scan is determined under certain culture temperature and the wavelength, the working power mode determination is measured, wherein said culture temperature is 25 ℃～40 ℃, more preferably 36 ± 1 ℃, and described scanning wavelength is 600 ± 50nm, more preferably 600nm;
In one embodiment, in the method for the invention, phage dilution gradient suspension dilutes with 10 times of gradients.
In one embodiment, in the method for the invention, cultivate Enterobacter sakazakii, the Enterobacter sakazakii bacteria suspension of preparation logarithmic phase to logarithmic phase.In the step C of aforesaid method of the present invention, the Enterobacter sakazakii nutrient solution can be the nutrient solution of any suitable cultivation Enterobacter sakazakii.In one embodiment, in the method for the invention, the Enterobacter sakazakii nutrient solution is the brain heart infusion nutrient solution.
In one embodiment, in the step C of aforesaid method of the present invention, the Enterobacter sakazakii nutrient solution is the brain heart infusion nutrient solution that contains the Enterobacter sakazaii colour development substrate.Preferably, described Enterobacter sakazaii colour development substrate is X-α-D-glucopyranoside.
In one embodiment, in the step C of aforesaid method of the present invention, be X-axis with the minute, be that Y-axis is made growth curve with the absorption value, and, determine tiring of phage according to the phage concentration gradient situation that precipitous sigmoid growth curve occurs.
In a preferred embodiment, the method for rapid detection titer of bacteriophage of Enterobacter sakazakii of the present invention comprises the steps:
A. ten times of dilution phage solutions obtain phage concentration gradient suspension;
B. Enterobacter sakazakii is cultured to logarithmic phase, Enterobacter sakazakii is seeded in the brain heart infusion nutrient solution, cultivated 6～8 hours;
The brain heart infusion nutrient solution that C. will contain Enterobacter sakazaii colour development substrate X-α-D-glucopyranoside adds microwell plate; The Enterobacter sakazakii bacteria suspension of the logarithmic phase among the step B is added microwell plate; Then the phage gradient suspension in the steps A is added microwell plate;
D. real time scan is measured, and utilizes microplate reader, and under certain culture temperature and wavelength, the working power mode determination is measured, and described culture temperature is 36 ± 1 ℃, and described scanning wavelength is 600nm;
According to the concentration of the phage that the rugged bacillus growth curve of precipitous type slope occurs, determine tiring of phage.
Among the step B of measuring method of the present invention, Enterobacter sakazakii is cultivated logarithmic phase, in this vegetative period, Enterobacter sakazakii is to phage-sensitive degree height, and experiment effect is obvious.Generally speaking, Enterobacter sakazakii is seeded in the brain heart infusion nutrient solution, 36 ℃ of cultivations, and 2～10 hours is logarithmic phase, wherein 6～8 hours for more preferably.
Among the step C of measuring method of the present invention, the Enterobacter sakazaii colour development substrate is X-α-D-glucopyranoside (X-α-D-glucopyranoside), because of Enterobacter sakazakii has specific alpha-D-glucose glycosides enzymic activity, therefore, in the Enterobacter sakazakii process of growth, this activity can make chromogenic substrate decompose generation blue-greenish colour product, thereby makes nutrient solution by the colourless blue-greenish colour that becomes, and the quantity of the glaucous depth and Enterobacter sakazakii is proportional within the specific limits.
In a preferred embodiment, in step C, the contrast of use comprises positive control, blank.Positive control is to replace phage with sterilized water; Blank is to replace the Enterobacter sakazakii bacteria suspension with sterilized water.
In the step D of method of the present invention, growth curve is an X-axis with the minute, is Y-axis with the absorption value.In one embodiment of the invention, adopted the dynamics analysis functionality of SkanItso software of the Multiskanspectrum microplate reader of Termo company to make growth curve.Growth curve can be divided into three types: precipitous type, osculant, high order smooth pattern.Precipitous type: lag phase is short, and logarithmic phase linearly rises basically, maintains high absorption value stationary phase, and longer duration; Osculant: lag phase is long, and logarithmic phase slowly rises, and maintains lower absorption value stationary phase; High order smooth pattern: entire curve is pressed close to X-axis linearly type or parabolic type, keeps lower absorption value always.Positive control and the test hole that does not contain phage are precipitous sigmoid growth curve, because positive control and do not contain that Enterobacter sakazakii can not be subjected to phage splitting in the test hole of phage, thereby growth along with the time, thalline is constantly bred, its glucosidase activity constantly acts on chromogenic substrate, thereby make the nutrient solution color constantly deepen, the absorption value that records constantly raises, and is precipitous sigmoid growth curve.The test hole that contains phage is osculant or high order smooth pattern growth curve.The phagocytosis scale of construction for a long time, a large amount of timely cracking of phage Enterobacter sakazakii, thereby make Enterobacter sakazakii can't breed at all, be not enough to produce enough glucosidase activity effect chromogenic substrates and make the nutrient solution variable color, so the absorption value that records can't raise, so growth curve is a high order smooth pattern.The phagocytosis scale of construction after a little while, some Enterobacter sakazakiis of phagocytosis physical efficiency cracking, but still there are some Enterobacter sakazakiis to be bred, make nutrient solution colour-change lentamente so can produce certain glucosidase activity effect chromogenic substrate, so the absorption value that records slowly rises, so growth curve is the osculant growth curve.Blank is not because add Enterobacter sakazakii, so should be a baseline.
In the measuring method of the present invention, it is as follows to calculate the principle of tiring.After with the phage gradient dilution, add in the test hole, initial several test hole pnagus medius concentration height, so its growth curve is a high order smooth pattern; Along with testing tube pnagus medius concentration constantly reduces, so its growth curve gradually becomes osculant; When phage is diluted to concentration when very low, do not had phage in the testing tube, so its growth curve becomes precipitous type gradually.In the test hole of gradient dilution, the extent of dilution of precipitous sigmoid growth curve test hole before appears, and be the minimum extent of dilution of phage to host's generation effect, then this test hole pnagus medius number is at least 1, and its concentration is about 1/300 μ L.Can calculate tiring of phage stoste in view of the above.
The invention provides a kind of method for quick of titer of bacteriophage of Enterobacter sakazakii, it is to suppress the growing state of Enterobacter sakazakii in the brain heart infusion nutrient solution by phage, and quantitatively bacteriophage of Enterobacter sakazakii tires.
The present invention is suitable for the rapid determination of titer of bacteriophage of Enterobacter sakazakii, as finding that phage titer is low, then needs to recover the activity of phage, thereby is used for food better, particularly the biological control of milk-product process for processing environment.
According to the measuring method of titer of bacteriophage of Enterobacter sakazakii of the present invention, can be with this method summary:---the logarithmic phase Enterobacter sakazakii is cultivated, and---phage and Enterobacter sakazakii add microwell plate---real time scan mensuration---and determine titer of bacteriophage of Enterobacter sakazakii the preparation of phage concentration gradient suspension.As find that phage titer is low, then need to recover the activity of phage, thereby be used for food better, particularly the biological control of milk-product process for processing environment.After with the phage gradient dilution, add in the test hole, initial several test hole pnagus medius concentration height, so its growth curve is a high order smooth pattern; Along with testing tube pnagus medius concentration constantly reduces, so its growth curve gradually becomes osculant; When phage is diluted to concentration when very low, do not had phage in the testing tube, so its growth curve becomes precipitous type gradually.In the test hole of gradient dilution, the extent of dilution of precipitous sigmoid growth curve test hole before appears, and be the minimum extent of dilution of phage to host's generation effect, then this test hole pnagus medius number is at least 1, and its concentration is about 1/300 μ L.Can calculate tiring of phage stoste in view of the above.
According to a particular of the present invention, measuring method of the present invention may further comprise the steps:
A. concentration gradient suspension preparation is carried out activation recovering with the bacteriophage of Enterobacter sakazakii virus strain, preparation phage dilution gradient suspension.Bacteriophage of Enterobacter sakazakii joined cultivated in the Enterobacter sakazakii bacteria suspension 16～24 hours, observe the bacteria suspension clarification, the phage activation recovering is described.Use bacterial filter to filter and cultivate mixed solution, remove the Enterobacter sakazakii cell, obtain phage solution.Use physiological saline, ten times of dilution phage solutions obtain phage concentration gradient suspension.
B. cultivate, Enterobacter sakazakii is cultured to logarithmic phase.Enterobacter sakazakii is seeded in the brain heart infusion nutrient solution, cultivated 2～10 hours.In the follow-up test, Enterobacter sakazakii is 6～8 hours to the most responsive incubation time of phage.
C. application of sample is prepared the brain heart infusion nutrient solution that contains the Enterobacter sakazaii colour development substrate, adds microwell plate; The Enterobacter sakazakii bacteria suspension of logarithmic phase is to microwell plate then; The phage gradient suspension that adds the finite concentration gradient at last respectively is to microwell plate; Handling 2～5 times for every kind repeats; The each detection needs to set contrast;
D. real time scan is measured, and utilizes microplate reader, and under certain culture temperature and wavelength, the working power mode determination is measured; Real time scan is measured: microwell plate is put into microplate reader, and after cultivating under the predetermined culture temperature, under certain wavelength, the working power mode determination carries out the sweep measuring Enterobacter sakazakii and has growth curve under the situation in phage;
According to the phage concentration gradient situation that precipitous sigmoid growth curve occurs, determine tiring of phage, in the test hole of gradient dilution, the extent of dilution that precipitous sigmoid growth curve test hole before occurs, be the minimum extent of dilution of phage to host's generation effect, then this test hole pnagus medius number is at least 1, and its concentration is about 1/300 μ L.Can calculate tiring of phage stoste in view of the above.
Use this detection method, can measure tiring of bacteriophage of Enterobacter sakazakii simply, fast and efficiently.
Description of drawings
Fig. 1 is titer of bacteriophage of Enterobacter sakazakii measurement result figure.
Embodiment
The present invention is further illustrated for mode by embodiment, but the present invention is not limited only to following examples.
Embodiment
Present embodiment provides the representative instance of the method for rapid determination titer of bacteriophage of Enterobacter sakazakii of the present invention.
This method may further comprise the steps:
1. the preparation of phage:
The preparation of the bacteria suspension of Enterobacter sakazakii: (nutrient broth is available from Beijing overpass technology company limited product to be seeded to 10mL nutrient broth nutrient solution with Enterobacter sakazakii (from institute of China inspection section food safety microbial strains preservation administrative center), China, press product description preparation nutrient broth nutrient solution) in, under 36 ℃ ± 1 ℃, cultivated 8～12 hours.By spectrophotometric determination OD
600, OD
600Show that greater than 0.5 Enterobacter sakazakii is in logarithmic phase, stop to cultivate that the rugged bacillus of slope this moment is the highest to phage sensitivity.Draw Enterobacter sakazakii bacteria suspension 1mL, add in the nutrient broth nutrient solution of the double concentration of 99mL.
The preparation of phage: gather sample vegetatively from being fit to phage.In the present embodiment, from near the river water sampling Gaobeidian City, with serious pollution Chaoyang District, Beijing City sewage work, remove by filter impurity through absorbent cotton, get 100mL and add 100mL in the previous step and contain in the nutrient broth nutrient solution of double concentration of Enterobacter sakazakii, cultivated 24～36 hours for 36 ℃ ± 1 ℃.Draw and cultivate mixed solution 1mL, through sterile filters (0.22 μ m millipore filtration) filtration sterilization.Get filtrate 0.1mL, the double-layer plate of preparation Enterobacter sakazakii and phage, 36 ℃ ± 1 ℃ cultivation was observed plaque after 4～8 hours, can increase number of plates (5～10), thereby improved the probability that obtains plaque.The agar block of getting single plaque on the double-layer plate adds in the 5mL nutrient broth nutrient solution, and room temperature is placed after 1～2 hour 4 ℃ and spent the night.Drawing the 1mL bubble has the nutrient broth nutrient solution of plaque agar block, adds the bacteria suspension 0.1mL that contains the logarithmic phase Enterobacter sakazakii according to the preceding method preparation, adds nutrient broth nutrient solution 9mL, cultivates 24～36 hours for 36 ℃ ± 1 ℃.Observe nutrient solution and become clarification by muddiness, will cultivate mixed solution according to the preceding method filtration sterilization.Repeat above-mentioned steps 2 times, promptly obtain the phage of purifying.4 ℃ of preservations of packing are standby.
If after preservation for some time, phage is active to descend, and bacteriophage of Enterobacter sakazakii can be joined and cultivate in the Enterobacter sakazakii bacteria suspension 16～24 hours, observes the bacteria suspension clarification, and the phage activation recovering is described.According to the preceding method filtration sterilization, obtain the purifying phage solution of activation recovering.
2. phage concentration gradient solution preparation:
Draw the 1mL phage solution to the 9mL sterile saline, preparation 10
-1Dilution phage solution; Draw 1mL10
-1Dilution phage solution prepares 10 to the 9mL sterile saline
-2Dilution phage solution; Repeat above-mentioned steps, can prepare a series of ten times of dilution phage gradient dilution solution that differ.
3. the preparation of logarithmic phase Enterobacter sakazakii bacteria suspension:
Prepare the Enterobacter sakazakii bacterial strain according to preceding method, picked at random 4 strains, be numbered IQCC10403.7,10403.14,10403.17 and 10403.18 by Chinese inspection section institute food safety microbial strains preservation administrative center (IQCC) coding rule, it is the representative bacterial strain of Enterobacter sakazakii, all functions with Enterobacter sakazakii prepare their logarithmic phase bacteria suspension.
4. application of sample:
Preparation contains the brain heart infusion nutrient solution of 0.1% (W/W) Enterobacter sakazaii colour development substrate (X-α-D-glucopyranoside buys in Beijing road and bridge technology limited liability company), adds 240 μ L in each micropore.
It is about 100～1000CFU/mL that Enterobacter sakazakii bacterial strain IQCC10403.7,10403.14,10403.17 and 10403.18 in the step 3 is carried out concentration dilution, adds 30 μ L in each micropore.
Add phage gradient suspension 30 μ L in the step 2 respectively to micropore.
4. real time scan is measured:
After adding sample, cover the microwell plate lid.Put into scanner.The scanner culture temperature is made as 36 ℃, opens dynamics research software, detected 1 time at the 600nm place every 10 minutes, be 8 hours detection time.Make the absorption and the time plot in every hole.
Determine titer of bacteriophage of Enterobacter sakazakii:, determine tiring of phage according to the phage concentration gradient situation that precipitous sigmoid growth curve occurs.
The result:
As shown in Figure 1, A1～H1 is a blank, the positive contrast of A12～H12, and all the other join the result who cultivates in the 4 strain Enterobacter sakazakiis for phage.A2, B2 suppress Enterobacter sakazakii IQCC10403.7 growing state for phage stoste, and A3, B3 are 10
-1Phage solution suppresses Enterobacter sakazakii IQCC10403.7 growing state, and the like, A4, B4～A11, B11 are respectively 10
-2～10
-9Phage solution suppresses Enterobacter sakazakii IQCC10403.7 growing state; C2, D2～C11, D11 are respectively stoste～10
-9Phage solution suppresses Enterobacter sakazakii IQCC10403.14 growing state; E2, F2～E11, F11 are respectively stoste～10
-9Phage solution suppresses Enterobacter sakazakii IQCC10403.17 growing state; G2, H2～G11, H11 are respectively stoste～10
-9Phage solution suppresses Enterobacter sakazakii IQCC10403.18 growing state.Phage stoste is through gradient dilution, until 10
-4～10
-6Extent of dilution, Enterobacter sakazakii are the high order smooth pattern growth curve, illustrate that the growth of 4 strain Enterobacter sakazakiis all has been subjected to inhibition when phage concentration is higher.When phage is diluted to 10
-5～10
-7The time, precipitous sigmoid growth curve appears in Enterobacter sakazakii.10
-4～10
-6Be the minimum extent of dilution of phage to host's generation effect, this test hole pnagus medius number is at least 1, and its concentration is about 1/300 μ L, and 3.33/mL; Analogize in view of the above, the test hole pnagus medius concentration that adds phage stoste is 3.33 * 10
4～10
6Individual/mL; Because 30 μ L phages add test hole, final volume reaches 300 μ L, and is diluted 10 times, and therefore, the concentration of phage stoste should be 3.33 * 10
5～10
7Individual/mL.As seen, this strain phage that is separated to all has 4 strain Enterobacter sakazakiis and suppresses the growth effect preferably, and its order of magnitude of tiring can reach 10
5～10
7Individual/ml.Because tiring of phage is to measure according to its lytic effect to the host, its lytic effect is also relevant with host's characteristic, and therefore, tiring of providing is a value range.
More than employed isolating 4 strain Enterobacter sakazakii IQCC10403.7,10403.14,10403.17,10403.18 be not finish the present invention the biomaterial that must use, use all Enterobacter sakazakiis all can realize effect of the present invention.
Comparative example
The present inventor also uses the double-layer plate method to measure the phage titer of the phage of above preparation to the Enterobacter sakazakii of above preparation, to compare with method of the present invention.
The double-layer plate method concrete operations of being adopted are as follows:
5～10ml LB inoculation of medium Enterobacter sakazakii, cultivated 8～12 hours, until logarithmic growth (OD in mid-term
600Greater than 0.5).
The preparation brain heart infusion agar, the sterilization back is fallen dull and stereotyped, preparation bottom substratum.Each dull and stereotyped about 10～15ml substratum (is unfavorable for bacterial growth very little; The too many plaque that influences is observed).
Preparation top layer brain heart infusion agar divides to be filled in the sterile culture pipe, every pipe 2700ml, and 45 ℃ of water-baths are standby, and (temperature is too high, influences phage and bacterial activity; The too low culture medium solidifying of temperature is too fast, and is easily inhomogeneous, influences the result and observes).
With brain heart infusion nutrient solution 10 doubling dilution phages.New application of sample rifle head is all used in each dilution instead, uses aerosol to intercept the rifle head to avoid crossed contamination.
The Enterobacter sakazakii bacteria suspension branch of logarithmic phase is filled in the little finger of toe pipe every pipe 200 μ l.The phage adding of doubling dilution is contained in the centrifuge tube, only add a kind of diluent 100 μ l in the centrifuge tube, quick vortex, incubated at room 1min～5min.
Be transferred in the top layer brain heart infusion agar of 45 ℃ of water-baths amounting to 300 μ l mixed solutions, the vortex mixing is poured onto bottom platform immediately fast, and light and slow swing plate makes top layer substratum uniform distribution.
Cool off dull and stereotyped 5min, be inverted overnight incubation for 37 ℃.
Plaque count is that about 100 plate is as the criterion with the plaque number, and the plaque number multiply by extent of dilution can obtain every phage titer, the pfu of unit.
The double-layer plate method of more than carrying out, each experiment repeats more than 3 times.
The result difference that the double-layer plate method obtains is bigger, and repeatedly plaque is not observed in experiment, and the phage that the experimental result of observing plaque is also measured well below the method for utilizing microplate reader used in the present invention is to the order of magnitude of tiring of the rugged bacillus of slope.
The double-layer plate method has higher requirements to operation, and inexperience, unskilled operator are difficult to obtain rational experimental result.
Compared with prior art, the advantage that has of the present invention is: can measure titer of bacteriophage of Enterobacter sakazakii simply, fast and efficiently.
Though specific embodiments of the present invention is described, those skilled in the art will appreciate that under the prerequisite that does not depart from scope of the present invention or spirit and can carry out multiple change and modification to the present invention.Thereby, this invention is intended to contain all these changes and modification of dropping in appended claims book and the coordinator scope thereof.
Claims (10)
1. the method for a rapid determination titer of bacteriophage of Enterobacter sakazakii is characterized in that, said method comprising the steps of:
A. prepare phage concentration gradient suspension;
B. prepare the Enterobacter sakazakii bacteria suspension;
C. add Enterobacter sakazakii bacteria suspension among Enterobacter sakazakii nutrient solution, the step B and the phage gradient suspension in the steps A in the microwell plate successively;
D. real time scan is measured, and utilizes microplate reader, and the working power mode determination carries out real time scan and measures growth curve.
2. method according to claim 1 is characterized in that, the real time scan among the described step D is determined under 25 ℃～40 ℃ the temperature and carries out.
3. method according to claim 2 is characterized in that, the real time scan among the described step D is determined under 36 ± 1 ℃ the temperature and carries out.
4. method according to claim 1 and 2 is characterized in that, the real time scan among the described step D is determined under the scanning wavelength of 600 ± 50nm and carries out.
5. method according to claim 4 is characterized in that, the real time scan among the described step D is determined under the scanning wavelength of 600nm and carries out.
6. method according to claim 1 is characterized in that, the Enterobacter sakazakii among the described step B is the Enterobacter sakazakii that is cultured to logarithmic phase.
7. method according to claim 1 is characterized in that, the Enterobacter sakazakii nutrient solution among the described step C is the brain heart infusion nutrient solution.
8. method according to claim 7 is characterized in that, the Enterobacter sakazakii nutrient solution among the described step C is the brain heart infusion nutrient solution that contains the Enterobacter sakazaii colour development substrate.
9. method according to claim 8 is characterized in that, described Enterobacter sakazaii colour development substrate is X-α-D-glucopyranoside.
10. method according to claim 1 is characterized in that, said method comprising the steps of:
A. ten times of dilution phage solutions obtain phage concentration gradient suspension;
B. Enterobacter sakazakii is cultured to logarithmic phase, Enterobacter sakazakii is seeded in the brain heart infusion nutrient solution, cultivated 6～8 hours;
The brain heart infusion nutrient solution that C. will contain Enterobacter sakazaii colour development substrate X-α-D-glucopyranoside adds microwell plate; The Enterobacter sakazakii bacteria suspension of the logarithmic phase among the step B is added microwell plate; Then the phage gradient suspension in the steps A is added microwell plate;
D. real time scan is measured, and utilizes microplate reader, and under certain culture temperature and wavelength, the working power mode determination is measured, and described culture temperature is 36 ± 1 ℃, and described scanning wavelength is 600nm;
According to the concentration of the phage that precipitous type Enterobacter sakazakii growth curve occurs, determine tiring of phage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010118057 CN101775444A (en) | 2010-03-05 | 2010-03-05 | Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010118057 CN101775444A (en) | 2010-03-05 | 2010-03-05 | Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101775444A true CN101775444A (en) | 2010-07-14 |
Family
ID=42512030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010118057 Pending CN101775444A (en) | 2010-03-05 | 2010-03-05 | Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101775444A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063732A (en) * | 2020-09-17 | 2020-12-11 | 扬州大学 | Rapid quantitative detection method capable of specifically identifying survival cells of enterobacter sakazakii and primers thereof |
| CN113008852A (en) * | 2021-02-23 | 2021-06-22 | 集美大学 | Detection method of vibrio phage titer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1890366A (en) * | 2003-11-19 | 2007-01-03 | 雀巢技术公司 | Isolated phages and their use as disinfectant in food or for sanitation of factory environment |
-
2010
- 2010-03-05 CN CN 201010118057 patent/CN101775444A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1890366A (en) * | 2003-11-19 | 2007-01-03 | 雀巢技术公司 | Isolated phages and their use as disinfectant in food or for sanitation of factory environment |
Non-Patent Citations (4)
| Title |
|---|
| 《Appl Microbiol Biotechnol》 20071231 Steven Hagens等 Application of bacteriophages for detection and control of foodborne pathogens 第513－519页 1－10 第76卷, 2 * |
| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20060630 P. Mudgal et al Quantifying the Significance of Phage Attack on Starter Cultures: a Mechanistic Model for Population Dynamics of Phage and Their Hosts Isolated from Fermenting Sauerkraut 第3908－3915页 1－10 第72卷, 第6期 2 * |
| 《Journal of Virological Methods》 20051231 Helena Fridholm et al Rapid and reproducible infectivity end-point titration of virulent phage in a microplate system. 第67－71页 1－10 第128卷, 2 * |
| 《微生物学报》 20081004 赵贵明等 阪崎肠杆菌噬菌体的分离及其生物学特性 第1373－1377页 1－10 第48卷, 第10期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063732A (en) * | 2020-09-17 | 2020-12-11 | 扬州大学 | Rapid quantitative detection method capable of specifically identifying survival cells of enterobacter sakazakii and primers thereof |
| CN113008852A (en) * | 2021-02-23 | 2021-06-22 | 集美大学 | Detection method of vibrio phage titer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1957089B (en) | Measuring contamination | |
| CN101893589B (en) | Sterility test method and totally closed bacteria collection ampoule incubator used thereby | |
| Yu et al. | The bioavailability of the soluble algal products of different microalgal strains and its influence on microalgal growth in unsterilized domestic secondary effluent | |
| CA2330814A1 (en) | Bacteriophage assay | |
| CN109082396A (en) | Bacterium and its application in control of plant disease is quenched in a kind of DSF colony induction signaling molecule | |
| JP6975701B2 (en) | Bacillus subtilis isolation method, Bacillus subtilis, microbial preparation containing Bacillus subtilis, medium set for Bacillus subtilis isolation | |
| Simandjuntak et al. | Isolation and identification of thermophilic bacteria, producer of amylase enzyme, from Lake Linow, North Sulawesi | |
| CN101775444A (en) | Method for rapidly determining titer of bacteriophage of Enterobacter sakazakii | |
| CN103773709B (en) | Bacillus subtilis with effect of efficiently dissolving phosphorus and application of bacillus subtilis | |
| CN105441350B (en) | Produce the bacillus subtilis with a variety of enzymatic activitys and its application | |
| CN103509784A (en) | Screening method of mineralized microorganisms for self-repairing of concrete cracks | |
| CN105296678B (en) | A kind of biomembrane control method that colony induction signaling being quenched based on photocatalysis | |
| CN104313182A (en) | Detection method of phage | |
| CN105154353B (en) | A kind of bacillus subtilis and its application in greenhouse soil remediation | |
| CN102399731A (en) | Bacillus thuringiensis for preventing and controlling leptinotarsa decemlineata | |
| CN109439582A (en) | Raw bacillus megaterium and its application in Bo chrysanthemum | |
| Fuchs et al. | An improved protocol for harvesting Bacillus subtilis colony biofilms | |
| CN110184322A (en) | Method and device for collecting and culturing bioaerosol sample | |
| Wimpenny et al. | The use of two-dimensional gradient plates in determining the responses of non-sulphur purple bacteria to pH and NaCl concentration | |
| CN219239658U (en) | Detection device for evaluating disinfection effect of disinfectant | |
| CN102827770A (en) | Color developing method for screening amylase producing strain | |
| CN108588170A (en) | Culture medium for screening calibrating aflatoxin B1 degradation bacteria and screening calibration method | |
| Di Ning et al. | Optimization of PCR conditions for soil microbial diversity study. | |
| CN101974606A (en) | High-throughput biosensor array chip, construction method thereof and application | |
| Compost | El-Hayah |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Free format text: FORMER OWNER: YUAN FEI Effective date: 20130326 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yuan Fei Inventor after: Chen Ying Inventor after: Zhao Guiming Inventor after: Zhao Yongsheng Inventor after: Yang Hairong Inventor before: Yuan Fei |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: YUAN FEI TO: YUAN FEI CHEN YING ZHAO GUIMING ZHAO YONGSHENG YANG HAIRONG |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20130326 Address after: 100123 Beijing, Gaobeidian, North Road, a No. 3, No. Applicant after: Chinese Academy of Inspection and Quarantine Address before: 100123 Beijing, Gaobeidian, North Road, a No. 3, No. Applicant before: Chinese Academy of Inspection and Quarantine Applicant before: Yuan Fei |
|
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100714 |