CN106244667B - For quickly identifying culture medium and its application of eggs crack detection pathogenicity - Google Patents
For quickly identifying culture medium and its application of eggs crack detection pathogenicity Download PDFInfo
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- CN106244667B CN106244667B CN201610852455.5A CN201610852455A CN106244667B CN 106244667 B CN106244667 B CN 106244667B CN 201610852455 A CN201610852455 A CN 201610852455A CN 106244667 B CN106244667 B CN 106244667B
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Abstract
The invention discloses a kind of for quickly identifying the culture medium of eggs crack detection pathogenicity, and identifying the method for eggs crack detection pathogenicity using the culture medium, the culture medium includes being dehydrated small bovine brain leaching powder, tryptone, glycerol, sodium citrate, manganese sulfate, magnesium sulfate, zinc sulfate, dipotassium hydrogen phosphate, carragheen, lactoalbumin hydrolysate, glucose, L-sodium, vitamin B6, vitamin B12, horse serum, chicken red blood cell lysate, purified agar powder and deionized water.The method can intuitively carry out eggs crack detection bacterial screening, have many advantages, such as simply, easily and fast.Compared with traditional animal lethal test, reduces the dosage of experimental animal, improve animal welfare, shorten experimental period.
Description
Technical field
The invention belongs to veterinary microorganism fields, and in particular to one kind is caused a disease for quickly identifying eggs crack detection
The culture medium of power, and the method using culture medium identification eggs crack detection pathogenicity.
Background technique
Avian cholera is a kind of poultry bacterial sexually transmitted disease as caused by fowl pasteurella multocida.The disease is widely current in the world
Various birds are encroached in various regions.For avian cholera characterized by hueppe's disease, the morbidity fowl death rate is up to 60%-100%, provisions
Fowl industry causes biggish harm, is poultry main bacteria sexually transmitted disease for many years always to be of interest both at home and abroad.It is domestic at present logical
Mitigate the harm of epidemic disease frequently with the method for drug control, but prolonged and repeated medication not only rises feeding cost, but also causes
A series of problems, such as bacterial drug resistance enhancing and medicament residue, facts proved that relying only on drug control is not prevention and treatment avian cholera
On select countermeasure.In comparison, using vaccine immunity, it neither will cause bacterial drug resistance enhancing, medicament residue will not be generated, because
And it is easier to be received by numerous culturists and consumer.
Vaccine for preventing avian cholera includes virulent inactivated vaccine and attenuated live vaccines, wherein virulent inactivated vaccine is immune
Protection can only be provided to the strong virus attack of phase homologous serotype afterwards, and the Cross immunogenicity to different serotypes bacterial strain cannot be obtained
Power.And after attenuated live vaccines inoculation poultry, the antigen with cross-protection is generated when can be proliferated in vivo --- intersect and protects
It protects the factor (cross-protective factor, CPF), there is wider immune spectrum using attenuated live vaccines, can be difference
The virulent attack of serotype provides immunoprotection, thus is concerned.
The key for developing avian cholera live vaccine is to carry out the screening of less-virulent strain, i.e., judgement bacterial strain is pathogenic.At present really
The pathogenicity for determining eggs crack detection separation strains mainly passes through the test of animal lethal and is determined that this method not only disappears
A large amount of experimental animal, and time-consuming and laborious, increase experimental cost, violation animal welfare are consumed, to avian cholera attenuated live vaccines
Development brings biggish difficulty.
Summary of the invention
The purpose of the present invention be intended to provide it is a kind of for quickly identifying the culture medium of eggs crack detection pathogenicity, with
And identify the method for eggs crack detection pathogenicity using the culture medium.Its principle is to be inoculated with eggs crack detection
In solid medium provided by the invention, pass through micro- sem observation under the conditions of adding oblique ray outside in 40 DEG C of culture 12-15h
The difference (fluorescent orange is presented in virulent strain, and sky blue fluorescence is presented in less-virulent strain) of bacterium colony fluorescent color, and then to bacterial strain
It is pathogenic quickly to be identified.
The present invention provides a kind of for identifying the pathogenic culture medium of eggs crack detection, component are as follows: dehydration
Small bovine brain soaks powder 15-20g/L, tryptone 10-15g/L, glycerol 5.0-6.0ml/L, sodium citrate 0.8-1.0g/L, manganese sulfate
0.3-0.4g/L, magnesium sulfate 0.15-0.2g/L, zinc sulfate 0.15-0.2g/L, dipotassium hydrogen phosphate 2.5-3.0g/L, carragheen
0.2-0.3g/L, lactoalbumin hydrolysate 4.0-5.0g/L, glucose 10-12g/L, L-sodium 1.4-1.5g/L, vitamin
B60.2-0.3g/L, vitamin B12 0.15-0.2g/L, horse serum 50-60ml/L, chicken red blood cell lysate 4.0-5.0ml/L,
Purified agar powder 15g/L, remaining is deionized water.
When preparing above-described culture medium, need first be dehydrated small bovine brain leaching powder, tryptone, glycerol, sodium citrate, sulphur
Sour manganese, magnesium sulfate, zinc sulfate, dipotassium hydrogen phosphate, carragheen, lactoalbumin hydrolysate, glucose, L-sodium, purified agar powder
Etc. components deionized water is added, adjust PH to 7.4,121 DEG C of sterilizing 15min through sodium hydroxide solution after heating for dissolving.It is to be cooled
To after 50 DEG C, the vitamin B6 and vitamin B12 solution of horse serum, chicken red blood cell lysate and filtration sterilization is added, it is sufficiently mixed
Solid medium is made in injection plate after even.
The beneficial effects of the present invention are: with eggs crack detection culture medium provided by the invention can intuitively into
Row eggs crack detection bacterial screening has many advantages, such as simply, easily and fast.It is compared with the traditional method, reduces experiment
The dosage of animal, while animal welfare is improved, shorten experimental period.Studies have shown that culture medium provided by the invention can be with
Make eggs crack detection bacterium colony that there is typical pathogenicity identification feature, even if repeatedly can still stablize holding bacterium colony after passage
Original fluorescence identification feature.
Specific embodiment
The following examples are a further detailed description of the invention.
Embodiment 1
Take the small bovine brain leaching powder 20g, tryptone 10g, glycerol 5.0ml, sodium citrate 0.8g, manganese sulfate 0.3g, sulphur of dehydration
Sour magnesium 0.2g, zinc sulfate 0.2g, dipotassium hydrogen phosphate 3.0g, carragheen 0.2g, lactoalbumin hydrolysate 5.0g, glucose 12g, L- paddy ammonia
Sour sodium 1.5g and purified agar powder 15g is added in 900ml deionized water, is heated to boiling, and is stirred continuously until mixed liquor is complete
Fully dissolved adjusts pH value to 7.4,121 DEG C of sterilizing 15min with sodium hydroxide solution.It is cooled to 50 DEG C after sterilizing, is separately added into
Horse serum 50ml, chicken red blood cell lysate 5.0ml, filtration sterilization 1% vitamin B6 solution 20ml (contain vitamin B6 0.2g)
With 1% vitamin B12 solution 20ml (contain vitamin B6 0.2g), sterilizing flat board is injected after mixing well, every pair plate about 15ml,
Eggs crack detection culture medium is obtained after solidification.
Embodiment 2
Take the small bovine brain leaching powder 15g, tryptone 15g, glycerol 6.0ml, sodium citrate 1.0g, manganese sulfate 0.4g, sulphur of dehydration
Sour magnesium 0.15g, zinc sulfate 0.15g, dipotassium hydrogen phosphate 2.5g, carragheen 0.3g, lactoalbumin hydrolysate 4.0g, glucose 10g, L- paddy
Propylhomoserin sodium 1.4g and purified agar powder 15g is added in 900ml deionized water, is heated to boiling, and is stirred continuously until mixed liquor
It is completely dissolved, adjusts pH value to 7.4,121 DEG C of sterilizing 15min with sodium hydroxide solution.50 DEG C are cooled to after sterilizing, respectively plus
Enter horse serum 60ml, chicken red blood cell lysate 4.0ml, filtration sterilization 2% vitamin B6 solution 15ml (containing vitamin
B60.3g) and 1% vitamin B12 solution 15ml (contain vitamin B6 0.15g), sterilizing flat board is injected after mixing well, it is every secondary flat
Plate about 15ml, obtains eggs crack detection culture medium after solidification.
Embodiment 3
The eggs crack detection virulent strain of pathogenicity known to 6 plants or less-virulent strain are inoculated in by 1 institute of embodiment
Eggs crack detection solid medium made of method is stated, is taken out after 40 DEG C of culture 12h, is adding 45 ° of skew ray lines outside
Under part, the fluorescent color of bacterium colony is observed by disecting microscope, as the result is shown 3 plants of fowl for being known as virulent strain more killing property Pasteur
Bacillus (C48-1, X73 and P1059) is presented fluorescent orange, and 3 plants of eggs crack detections for being known as less-virulent strain
Sky blue fluorescence (table 1) is presented in (CU, P7810 and 1010), is consistent with discrimination method of the invention.The result shows that using this
The invention culture medium can intuitively be identified virulent strain and less-virulent strain by the difference of bacterium colony fluorescent color.
The eggs crack detection bacterium colony fluorescent color of the different pathogenicities of table 1
| Strain number | Source | Separate host | Pathogenicity | Bacterium colony fluorescent color |
| C48-1 | Chinese Industrial Standards (CIS) strain | Chicken | Virulent strain | It is orange |
| X73 | International standard strain | Chicken | Virulent strain | It is orange |
| P1059 | International standard strain | Turkey | Virulent strain | It is orange |
| CU | U.S.'s vaccine strain | Turkey | Less-virulent strain | Sky blue |
| P7810 | China Vaccine strain | Duck | Less-virulent strain | Sky blue |
| 1010 | China Vaccine strain | Chicken | Less-virulent strain | Sky blue |
Embodiment 4
Using method of the present invention to 234 plants of eggs crack detection separation strains (all fowl more killing property Pasteur's bar
Bacterium separation strains are saved by Jiangsu Inst. of Fowls Science and are provided, wherein 98 plants of chicken source separation strains, 64 plants of duck source separation strains, goose
61 plants of source separation strains, 11 plants of turkey source separation strains) carry out pathogenicity identification.234 plants of separation strains are inoculated with 1 the method for embodiment
Manufactured eggs crack detection solid medium takes out after 40 DEG C of culture 15h, outside plus under the conditions of 45 ° of oblique rays, leads to
The fluorescent color for crossing disecting microscope observation bacterium colony, the results are shown in Table 2.By table as it can be seen that 234 plants of eggs crack detection separation strains
Middle bacterium colony fluorescent color is that orange bacterial strain accounts for 96.2% (225/234), and fluorescent color is that sapphire bacterial strain only accounts for 3.8%
(9/234), determine that virulent strain there are 225 plants in accordance with the method for the present invention, less-virulent strain there are 9 plants.
2 234 plants of eggs crack detection separation strains bacterium colony partings of table
In order to further verify the accuracy of the method for the present invention, 22 plants (including 13 plants are chosen from 234 separation strains
Velogen strain and all 9 plants of low virulent strains).Separation strains are after brain heart infusion broth increases bacterium, with 1 × 103Colony Forming Unit
The dosage of (colonyforming units, CFU) carries out neck subcutaneous injection to 3 monthly ages nonimmune chicken and attacks malicious (each separation strains
Attack malicious 5 nonimmune chickens), it is observed continuously 14 days, the separation strains that the death rate reaches 80% or more are velogen strain, and the death rate is lower than
20% separation strains are low virulent strain.It the results are shown in Table 3, be sapphire fowl more killing property Pasteur by the visible 9 plants of bacterium colony fluorescent colors of table
After poison is attacked in the injection of bacillus separation strains, all test chickens are survived, and show that above-mentioned bacterial strains are less-virulent strain.And 13 plants of bacterium colony fluorescence
Color is after poison is attacked in orange eggs crack detection separation strains injection, except D002 separation strains animal survival rate is 20% (1/
5), the lethality of remaining 12 plants of separation strains is 100% (5/5), shows that this 13 plants of bacterial strains are virulent strain.The above results table
It is bright, use eggs crack detection pathogenicity discrimination method of the present invention and animal lethal test result complete one
It causes.
The test of 3 eggs crack detection separation strains pathogenicity of table
Embodiment 5
The eggs crack detection for choosing pathogenicity known to 10 plants, respectively in sheep blood agar medium, brain heart infusion
The continuous passage of indiscriminate ground 30 times on agar medium, improvement Martin's agar medium and culture medium provided by the invention, often
In 10 generations, observed and recorded bacterium colony fluorescent color, the results are shown in Table 4.The result shows that either virulent strain or less-virulent strain, in this hair
Its bacterium colony fluorescent color still has typical identification feature after passing on 30 times on the culture medium of bright offer.And it is trained in sheep blood agar
Base, brain heart infusion agar culture medium, improvement 3 kinds of eggs crack detections of Martin's agar medium are supported often with the bacterium on culture medium
Fall in the culture medium that unstressed configuration, fluorescent brightness and fluorescent color etc. are significantly not so good as offer of the invention, and continuous biography
In generation, causes bacterium colony fluorescent color to occur to connect on above-mentioned 3 kinds of culture mediums compared with Big mutation rate, either virulent strain or less-virulent strain
Its bacterium colony does not observe fluorescence completely after resuming 30 generations of generation.The above results show can using culture medium provided by the invention
So that eggs crack detection bacterium colony has typical pathogenicity identification feature, bacterium is kept even if repeatedly can still stablize after passage
Fall original fluorescence identification feature.
Fluorescent characteristic of 4 eggs crack detection of table after different culture medium passage
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of for quickly identifying the culture medium of eggs crack detection pathogenicity, it is characterised in that: the culture medium contains
Have dehydration small bovine brain leaching powder 15-20g/L, tryptone 10-15g/L, glycerol 5.0-6.0ml/L, sodium citrate 0.8-1.0g/L,
Manganese sulfate 0.3-0.4g/L, magnesium sulfate 0.15-0.2g/L, zinc sulfate 0.15-0.2g/L, dipotassium hydrogen phosphate 2.5-3.0g/L, card
Draw glue 0.2-0.3g/L, lactoalbumin hydrolysate 4.0-5.0g/L, glucose 10-12g/L, L-sodium 1.4-1.5g/L, dimension life
Plain B60.2-0.3g/L, vitamin B12 0.15-0.2g/L, horse serum 50-60ml/L, chicken red blood cell lysate 4.0-5.0ml/
L, purified agar powder 10-20g/L, remaining is deionized water.
2. culture medium described in claim 1 is carrying out the application in the identification of eggs crack detection pathogenicity, described is answered
With being non-diagnostic purpose.
3. carrying out eggs crack detection pathogenicity mirror method for distinguishing, the side using culture medium described in claim 1
Method is non-diagnostic purpose.
4. discrimination method according to claim 3, which is characterized in that by eggs crack detection inoculation medium, training
After supporting under the conditions of adding oblique ray outside, by the difference of micro- sem observation bacterium colony fluorescent color, fluorescent orange is presented and is determined as
Virulent strain is presented sky blue fluorescence and is determined as less-virulent strain.
5. discrimination method according to claim 4, which is characterized in that the condition of culture is 40 DEG C of culture 12-15h.
6. discrimination method according to claim 4, which is characterized in that the microscope is disecting microscope.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0694560A2 (en) * | 1994-07-29 | 1996-01-31 | American Cyanamid Company | Production of antigens of Pasteurella |
| CN103966142A (en) * | 2014-05-21 | 2014-08-06 | 福建省农业科学院 | Culture medium for decreasing production cost of pasteurella multocida capsule and preparation method thereof |
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2016
- 2016-09-26 CN CN201610852455.5A patent/CN106244667B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0694560A2 (en) * | 1994-07-29 | 1996-01-31 | American Cyanamid Company | Production of antigens of Pasteurella |
| CN103966142A (en) * | 2014-05-21 | 2014-08-06 | 福建省农业科学院 | Culture medium for decreasing production cost of pasteurella multocida capsule and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Transcriptional Response of Pasteurella multocida to Nutrient Limitation;PAUSTIAN,M.L.等;《JOURNAL OF BACTERIOLOGY》;20020731;第184卷(第13期);3734-3739 |
| 用一种选择指示培养基快速鉴别多杀巴氏杆菌;苏雄;《华中农业大学学报》;19881231;第7卷(第2期);163-165 |
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