CN103969207B - A kind of method of total number of bacteria in quick mensuration Pasteurella multocida culture - Google Patents
A kind of method of total number of bacteria in quick mensuration Pasteurella multocida culture Download PDFInfo
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- CN103969207B CN103969207B CN201410214797.5A CN201410214797A CN103969207B CN 103969207 B CN103969207 B CN 103969207B CN 201410214797 A CN201410214797 A CN 201410214797A CN 103969207 B CN103969207 B CN 103969207B
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Abstract
The present invention relates to a kind of method of total number of bacteria in quick mensuration Pasteurella multocida culture, it comprises the following steps: 1. cleaning mixture preparation；2. sample；3. sample treatment；4. detection: utilizing ultraviolet spectrophotometer, the OD value when 600nm wavelength of the sample after mensuration process, with step 1. middle cleaning mixture as reference；5. calculate: when OD value is in the range of 0.4 1.2, sample is (4129.4 × OD containing bacterium number600+509.31)×106CFU/mL, as OD value ＜ 0.4, measures after suitably being concentrated by sample, as OD value ＞ 1.2, measures after suitably being diluted by sample, and calculates containing bacterium sum.It is an object of the invention to provide a kind of method of total number of bacteria in quick mensuration Pasteurella multocida culture.It is an advantage of the current invention that: the simplest；The quickest；3. error is little.
Description
Technical field
The present invention relates to a kind of method of total number of bacteria in quick mensuration Pasteurella multocida culture.
Background technology
Fowl cholera, has another name called fowl hueppe's disease, fowl pasteurellosis or fowl and shakes the head pestilence etc., be by killing property more
The contagious disease encroaching on various birdss that pasteurellosis bacillus (Pasteurella multocida) causes.
The epidemic season of this disease is mainly late summer and autumn, main by digestive tract and respiratory tract infection.Various ages in days
The most susceptible, but multiple it is born in sexually matured birds.During the acute attack of this disease, the course of disease is very brief, dead soon,
Normal invisible morbidity i.e. death.During acute attack, sick carcass temperature rise, feather pine is random, down in spirits, solely
Occupy an a corner, close mesh sleepy.Mouthful, nasal secretions and cause dyspnea, attempt of shaking the head throw away larynx glue
Liquid.Typically cuing open inspection pathological changes is acute sepsis, is full of transparent orange yellow exudate, heart hat fat in pericardium
On have petechia.Liver, splenomegaly, the circular canescence that quality becomes fragile, surface is densely covered with a large amount of needle point big is bad
Dead point.Intestinal bleeding, congested and hemorrhage the most serious with small intestinal leading portion and colorectal mucosa, small intestinal back segment and caecum
Lighter.Intestinal contents is gel-shaped, and intestinal mucosa comes off, the ring-type enlargement of intestinal aggregate nodules, hemorrhage.Chronic
When making, sick fowl shows as becoming thin, dysentery, rhinitis, arthritis.The visible local arthroncus of the course of disease slightly elder,
Impact sick fowl walking.This disease is distributed widely in all over the world, and provisions fowl industry brings huge economic loss.
Count of bacteria is the basis that antibacterial carries out each side research.The method of plate culture count is the most frequently used side
Method.The principle of the method for plate culture count is that each antibacterial alive is under suitable culture medium and better growing conditions
Macroscopic bacterium colony can be formed by growth.Bacteria suspension serial dilution can be taken a certain amount of dilution
Liquid carries out flat board cultivation, according to the clump count turned out, calculates the viable count in bacteria suspension.The method is suitable for
The mensuration of the educable bacterium number of the work in sample, highly sensitive, higher to the competency profiling of operator,
The time expended is longer.
Summary of the invention
It is an object of the invention to provide antibacterial in a kind of quickly mensuration Pasteurella multocida culture total
The method of number.
The purpose of the present invention is achieved through the following technical solutions: one quickly measures Pasteurella multocida
The method of total number of bacteria in culture, it comprises the following steps:
1. cleaning mixture preparation: preparation is suitable for the fluid medium of Pasteurella multocida growth as washing
Liquid, room temperature preservation；
2. sampling: take the testing sample of certain volume；
3. sample treatment: sample 5000g is centrifuged 5min, sucking-off supernatant, add with remove supernatant after remaining
The isopyknic cleaning mixture of sample, blow and beat mixing gently, 5000g is centrifuged 5min again, sucking-off supernatant,
The isopyknic cleaning mixture of sample remaining after adding and go supernatant, blows and beats mixing gently；
4. detection: utilize ultraviolet spectrophotometer, the OD when 600nm wavelength of the sample after mensuration process
Value, with step 1. middle cleaning mixture as reference；
5. calculate: when OD value is in the range of 0.4-1.2, sample containing bacterium number be
(4129.4×OD600+509.31)×106CFU/mL, as OD value ＜ 0.4, surveys after suitably being concentrated by sample
Fixed, as OD value ＞ 1.2, measure after sample is suitably diluted, and calculate containing bacterium sum.
For prior art, it is an advantage of the current invention that:
1, simple.Compared with the method for plate culture count, eliminate the step of dilution, cultivation, artificial counting.
2, quick.Compared with the method for plate culture count, eliminate the time of antibacterial culturing.Fowl source killing property Pasteur
Bacillus forms naked eyes on flat board and is evident that bacterium colony about takes 16h-18h, and the method only needs 30min
Can obtain a result.
3, error is little.The method of plate culture count is higher to the competency profiling of operator, and different human users obtain
The data difference arrived is relatively big, repeatable poor.And the method utilizes ultraviolet spectrophotometer to carry out reading,
Favorable repeatability.
Accompanying drawing explanation
Fig. 1 is sample OD600Value and the linear relationship chart containing bacterium number.
Fig. 2 is sample segment OD in Fig. 1600Value and the linear relationship chart containing bacterium number.
Detailed description of the invention
Below in conjunction with embodiment, present invention is described in detail:
1, the selection of cleaning mixture
The cleaning mixture of Conventional bacteria selects sterile distilled water or normal saline, but Pasteurella multocida exists
Death rapidly in both liquid, so the liquid training of Pasteurella multocida growth can only be selected to be suitable for
Support base such as martin's bouillon, LB culture medium, pancreas peptone soybean broth, brain heart infusion broth etc..This technical side
The foundation of case selects martin's bouillon.
2, Pasteurella multocida CVCC44801 strain (purchased from National Veterinary Culture Collection),
Streak inoculation, in Martin's agar plate, cultivates 20h for 37 DEG C, and 10 single colony inoculations of picking are in 400mL horse
In fourth meat soup, in 37 DEG C of shaken cultivation casees, 200rpm shaken cultivation 5h.Now also in killing property bar more
In the exponential phase of family name bacillus, the viable count in culture fluid is total bacteria count.
3, culture fluid is taken out, water-bath 30min in 2 DEG C of-8 DEG C of cold water.
4, sample by table 1, sample 5000g is centrifuged 5min, sucking-off supernatant, adds the aseptic horse of equal-volume
Fourth meat soup, blows and beats mixing gently.5000g is centrifuged 5min, sucking-off supernatant again, adds in table corresponding whole
The aseptic martin's bouillon of volume, blows and beats mixing gently.With aseptic martin's bouillon as reference, measure different multiples
The sample after dilution or concentration OD when 600nm wavelength600Value, each sample is surveyed 3 times, is recorded it average
Value.
The dilution of table 1 culture fluid or concentration
5, sterile sampling 1mL, is carried out 10 times of serial dilutions with martin's bouillon, is taken 10-6、10-7、10-8
Dilution bacterium solution paving Martin's agar plate, each dilution factor 3 pieces of flat boards of paving, each flat board consumption is 200
μ L, 16h-18h after cultivating in 37 DEG C of incubators, the clump count on reading flat board, same dilution 3 pieces
Plate is taken the mean, and chooses in clump count dilution factor between 30-300 i.e. table 2 10-6Dilution factor, extrapolates
The viable count of every milliliter of culture fluid is 1105 × 106CFU/mL。
The clump count of each flat board after table 2 cultivation
6, with OD600Value for abscissa, with containing bacterium number divided by 106Value be vertical coordinate, draw curve, see figure
1, linearly dependent coefficient is 0.9934.When OD600 value is between 0.402 and 1.210, linear correlation system
Number is 0.9989, sees Fig. 2, and credibility is higher.
7, conclusion: work as OD600Value between 0.402 and 1.210 time, sample containing bacterium number and OD600The line of value
Property correlation coefficient is 0.9989, and regression equation is y=4129.4x+509.31, and i.e. sample containing bacterium number is
(4129.4×OD600+509.31)×106CFU/mL.OD when sample600Value not 0.402 and 1.210 it
Between time, accurate for guarantee containing bacterium number, it is impossible to directly utilize this formula and calculate.OD when sample600During ＜ 0.4,
Sample is suitably concentrated；OD when sample600During value ＞ 1.2, sample is suitably diluted, calculate containing bacterium after mensuration
Number.
Embodiment 1:
Pasteurella multocida CVCC44801 strain (purchased from National Veterinary Culture Collection),
Streak inoculation, in Martin's agar plate, cultivates 20h for 37 DEG C, and the single colony inoculation of picking is in 20mL Martin's meat
Tang Zhong, adds 50% glucose injection 2mL, cultivates 15h for 37 DEG C.Measure the tool containing bacterium number in this culture fluid
Body operational approach is as follows:
1, sampling: take 4mL culture fluid respectively and add in two 5mL EP pipes.
2, sample treatment: open TGL-15B type centrifuge (Anting Scientific Instrument Factory, Shanghai), by two EP
Pipe symmetry is put into, and 5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.
3, detection: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co., Ltd), regulation
Wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw
Sample 3mL after process, in glass cuvette, puts into sample position, records OD600Value is 0.648.
4, calculate: OD600Value, between 0.40 and 1.20, directly presses formula (4129.4 × OD600+509.31)
×106CFU/mL calculates, and the content of the Pasteurella multocida obtaining this culture fluid is
3.185×109CFU/mL。
Embodiment 2:
Pasteurella multocida CVCC44801 strain (purchased from National Veterinary Culture Collection),
Streak inoculation, in Martin's agar plate, is cultivated 20h, is washed lower bacterium colony with 8.5%NaCl, make containing bacterium for 37 DEG C
Suspension.Measure in this suspension as follows containing the concrete operation method of bacterium number:
1, sampling: take 4mL suspension respectively and add in two 5mL EP pipes.
2, sample treatment: open TGL-15B type centrifuge (Anting Scientific Instrument Factory, Shanghai), by two EP
Pipe symmetry is put into, and 5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.
3, detection: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co., Ltd), regulation
Wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw
Sample 3mL after process, in glass cuvette, puts into sample position, records OD600Value is 1.834.
4, sample reprocessing and detection: because OD600Value ＞ 1.2, then process.Draw aseptic Martin's meat
Soup 1.5mL is in glass cuvette, then the sample 1.5mL after absorption process is in same glass cuvette
Mixing, i.e. 2 times dilutions, put into sample position, record OD600Value is 1.016.
5, calculate: first press formula (4129.4 × OD600+509.31)×106CFU/mL calculates, and obtains
4.7×109CFU/mL, then the Pasteurella multocida content being multiplied by extension rate 2, i.e. this suspension is
9.4×109CFU/mL。
Embodiment 3:
Pasteurella multocida CVCC44801 strain (purchased from National Veterinary Culture Collection),
Streak inoculation, in Martin's agar plate, cultivates 20h for 37 DEG C, and the single colony inoculation of picking is in 20mL Martin's meat
Tang Zhong, adds 50% glucose injection 10mL, cultivates 6h for 37 DEG C.Measure the tool containing bacterium number in this culture fluid
Body operational approach is as follows:
1, sampling: take 4mL culture fluid respectively and add in two 5mL EP pipes.
2, sample treatment: open TGL-15B type centrifuge (Anting Scientific Instrument Factory, Shanghai), by two EP
Pipe symmetry is put into, and 5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.5000g is centrifuged 5min, sucking-off supernatant, discards.Add the aseptic martin's bouillon of 4mL,
Blow and beat mixing gently.
3, detection: open UV1600 type ultraviolet spectrophotometer (Beijing Rayleigh Analytical Instrument Co., Ltd), regulation
Wavelength value is 600nm.Draw aseptic martin's bouillon 3mL in glass cuvette, put into reference position, draw
Sample 3mL after process, in glass cuvette, puts into sample position, records OD600Value is 0.103.
4, sample reprocessing and detection: because OD600Value ＜ 0.4, then process.Take 4mL respectively to cultivate
Liquid adds in 8 5mL EP pipes, and symmetry is put in centrifuge, and 5000g is centrifuged 5min, sucking-off supernatant,
Discard.Each addition aseptic martin's bouillon of 4mL, blows and beats mixing gently, and 5000g is centrifuged 5min, in sucking-off
Clearly, discard.By the bacterium mud 3.2mL aseptic martin's bouillon Eddy diffusion of 8 pipes, the sample after absorption process
Product 3mL, in glass cuvette, puts into sample position, records OD600Value is 0.983.
5, calculate: first press formula (4129.4 × OD600+509.31)×106CFU/mL calculates, and obtains
4.6×109CFU/mL, then the Pasteurella multocida content divided by cycles of concentration 10, i.e. this suspension
It is 4.6 × 108CFU/mL。
Claims (1)
1. the method for total number of bacteria in a quick mensuration Pasteurella multocida culture, it is characterised in that it comprises the following steps:
1. cleaning mixture preparation: preparation is suitable for the fluid medium of Pasteurella multocida growth as cleaning mixture, room temperature preservation；
Described fluid medium is martin's bouillon；
2. sampling: take the testing sample of certain volume；
3. sample treatment: sample 5000g is centrifuged 5min, sucking-off supernatant, adds and the step isopyknic cleaning mixture of taken testing sample, blowing and beating mixing gently, 5000g is centrifuged 5min, sucking-off supernatant again, add the isopyknic cleaning mixture of testing sample taken with step, blow and beat mixing gently；
4. detection: utilizing ultraviolet spectrophotometer, the OD value when 600nm wavelength of the sample after mensuration process, with step 1. middle cleaning mixture as reference；
5. calculate: when OD value is in the range of 0.4-1.2, sample is (4129.4 × OD containing bacterium number600+509.31)×106CFU/mL, as OD value ＜ 0.4, measures after suitably being concentrated by sample, as OD value ＞ 1.2, measures after suitably being diluted by sample, and calculates containing bacterium sum.
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|---|---|---|---|---|
| CN101936890A (en) * | 2010-08-19 | 2011-01-05 | 四川农业大学实验动物工程技术中心 | Method for quickly determining bacterial number in Riemerella anatipestifer culture |
| CN102854165A (en) * | 2012-09-03 | 2013-01-02 | 重庆市畜牧科学院 | Method for rapidly determining number of bacterial in lactic acid bacillus subtilis production liquid culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936890A (en) * | 2010-08-19 | 2011-01-05 | 四川农业大学实验动物工程技术中心 | Method for quickly determining bacterial number in Riemerella anatipestifer culture |
| CN102854165A (en) * | 2012-09-03 | 2013-01-02 | 重庆市畜牧科学院 | Method for rapidly determining number of bacterial in lactic acid bacillus subtilis production liquid culture |
Non-Patent Citations (3)
| Title |
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| 分枝杆菌计数方法的比较研究;罗泰来等;《第四军医大学学报》;20071231;第28卷(第04期);373-375页 * |
| 应用紫外分光光度法进行禽多杀性巴氏杆菌(C_(48-1))细菌计数的研究;陈士运等;《山东畜牧兽医》;20090531;第30卷(第05期);12-13页 * |
| 运用OD值法进行细菌计数的研究;乔军等;《中国家禽》;19960430(第04期);摘要，第一、三节，表4 * |
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