CN107384830A - The cultural method and its culture medium of a kind of riemerella anatipestifer - Google Patents
The cultural method and its culture medium of a kind of riemerella anatipestifer Download PDFInfo
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- CN107384830A CN107384830A CN201710738815.3A CN201710738815A CN107384830A CN 107384830 A CN107384830 A CN 107384830A CN 201710738815 A CN201710738815 A CN 201710738815A CN 107384830 A CN107384830 A CN 107384830A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention discloses a kind of cultural method of riemerella anatipestifer, comprise the following steps：10.0g tryptones, 5.0g soy peptones, 5.0g yeast extracts, 5.0g sodium chloride and 12.0g agar powders are dissolved in 1000ml deionized water, pH value is adjusted to 7.1 7.2, after autoclaving, when being cooled to 65 DEG C 70 DEG C, sterile de- fiber duck blood 100ml is added by sterile manner, shake up rear pour plate, after steriling test, obtain duck blood pancreas egg peptone Soy Agar plates culture medium；It is sterile to take the streak inoculation of duck brain tissue to be placed in CO on duck blood pancreas egg peptone Soy Agar plates from morbidity duck and dead duck separation riemerella anatipestifer bacillus2Cultivated 24 48 hours for 37 DEG C in candle cylinder, produce riemerella anatipestifer bacterium colony.The present invention can be suitably used for the flat board culture needs of routine experimentation, and riemerella anatipestifer can be made to grow preferable bacterium colony in 24h.
Description
Technical field
The present invention relates to biological field, and in particular to the cultural method and its culture medium of a kind of riemerella anatipestifer.
Background technology
China is a duck culturing big country, and duck culturing industry is seized of consequence, and people in the agricultural economy in China
One of important sources of meat egg needed for life.As the industry is obvious to scale, the development in intensive direction, its economic benefit
Improve, but some important epidemic diseases make it form serious prestige by very big economic loss and to its further development
The side of body.
Riemerella anatipestifer disease is one of the infectious disease the most serious that damaged at present to countries in the world duck culturing industry.Duck
Mo Shi bacillosises are that main infringement is young caused by riemerella anatipestifer (Riemerella anatipestifer, RA) in epidemic disease
The acute or chronic contagious disease of a variety of birds such as duck, poult, also known as " new duck disease ", " pest of duck syndrome ", " duck
Septicemia ", " duck infectious serositis ", " Pasteurella anatipestipestifer infection " etc..The disease is with fibrinous pericarditis, perihepatitis, gas
Capsulitis, meningitis and caseous salpingitis are characterized.Its incidence of disease in 5%~90%, case fatality rate typically 1%~80%,
Up to 90% or even 100% during peak, the resistance to more growth retardations of duck of crossing nearly all have stream into stiff duck, the disease in each duck culturing area in the world
OK, huge economic loss is caused to world's duck culturing industry.
The bacterium by Hendrickso and Hilbert, in 1932, identified first, and be named as the striking Buddhist Salmonella of pest of duck by separation
(Pfeifferella, Anatipestifer).Bruner and Fabricant in 1954 according to identical characteristic by this bacterium row people
Morakot Bordetella, referred to as pest of duck Morakot Salmonella (Moraxellaantipestifer).Hereafter it is again special with dyeing by its form
The factors such as sign are put into Pasteurella, are named as Pasteurella anatipestipestifer (Pastenrella anatipestifer).Thereafter
Bangun etc. ((1981,1987) by this bacterium and Morakot Salmonella and Pasteurella biochemical reaction, growth temperature, to physics and chemistry because
The resistance of element, DNA base composition, the polyacrylamine gel electrophoresis pattern etc. of esterase characteristic, mycoprotein are compared
Compared with, and by its homology of DNA-rRNA hybridization analysis, concluded with gas chromatography analysis cell fatty acid image,
The bacterium should mark off from Morakot Bordetella or Pasteurella to come.Chinese scholar Guo Yu uncut jades (1986) are by this bacterium and kill bar more
The ultra microstructure of family name bacillus is compared, it was demonstrated that the two is different on morphosis, at the 9th edition《Primary Jie Shi Bacteria Identifications handbook》
It is middle to be classified as the uncertain kind in position.Piechulla etc. (1986) combines according to NDA, menaquinone and branched fatty acid
Produce, it is proposed that RA is included in Flavobacterium/bite in CELLULOLYTIC BACTERIUM, Rosson etc. (1991) also for confirm the bacterium and Flavobacterium/
Biting the member of CELLULOLYTIC BACTERIUM has substantial amounts of similitude.Segers etc. (1993) is according to DNA-RNA hybridization analysis, protein, fat
The phenotypic characteristic of fat acid composition, it is proposed that be divided into independent one and belong to, riemerella anatipestifer is renamed as by Pasteurella anatipestipestifer.
This name at present obtains the accreditation of domestic and foreign scholars, and is adopted and quotes on internal authority magazine.China original one
Directly suggest preferably using riemerella anatipestifer with this disease of Pasteurella anatipestipestifer disease and duck infectious serositis appellation, Guo Yupu (1999)
Disease, because E. coli isolated from ducks, salmonella etc. can cause scrositis.But traditionally also continue to use " duck infectious serositis " extremely
It is modern.
Riemerella anatipestifer is Gram-negative dialister bacterium, no brood cell, it is impossible to move.Pure culture smear, thalline remove
Outside shaft-like, some is ellipse, most single presence, a small number of into way double-line.Size is 0.7~6.5 μm of 0.3~0.5 μ m,
Accidental indivedual thalline are thread in length, up to 11~24 μm.Seeing through prepared Chinese ink negative staining has folder film, and smear is through Wright's staining visible part bacterium
The dense dye in body the two poles of the earth.The periphery pod membrane of bacterium is an electron lucent layer under Electronic Speculum, and bacterial cell wall is 1 electron-dense layers, cell membrane
Have three layers structure, and outer layer and internal layer are in electron-dense layers (Guo Yupu, 1996).Between having one between cell membrane and cellular layer significantly
Gap, there is circular or oval empty balloon-shaped structure on thalline periphery, positioned at thalline one end, part RA tops or side also have bud shape to go to live in the household of one's in-laws on getting married
Biology.
Cultured Property about Riemerella anatipestifer, the growth of riemerella anatipestifer require harshness, it is necessary to CO2It can grow,
Can be in chocolate agar, tryptic soy agar, LB culture mediums, tryptose meat soup, tryptic soy broth and improvement martin's bouillon
Deng being grown on culture medium；It can not be grown on plain agar and maconkey agar, this can be as the foundation of antidiastole.Early stage is just
There is scholar to find that thiamine (1000mL contain thiamine 0.01g) can make RA growths preferable, and the pyridine amine of low concentration element with
Amprolium has inhibitory action to RA growths.The bacterium colony table generated on tryptic soy agar (TSA) or chocolate agar plate
Face is smooth, micro-protuberance, and circle is in butyrous, and colony diameter about 1~1.5mm, if continuing to cultivate, bacterium colony is slightly larger, reachable 2.0mm,
The equal green-emitting of Fluirescence observation (Harry EG, 1969).But cultivated in normal atmospheric conditions, bacterium colony is smaller, and reveal pearl.
Bacterium colony on blood agar plate culture tomb is anhemolytic dewdrop shape petite, if on this culture medium after subculture, bacterium colony becomes
Greatly, lawn is slightly sticky thick, can obtain optimum growh, and bacterium colony is rounded, slightly projection, surface is smooth, transparent, 1~2mm of diameter.According to report
Road (magnifies third, 1999), and the RA speeds of growth of different serotypes difference, 10 type RA growths are most fast, and culture 7h can grow up to,
2 types are most slow.In the meat soup containing serum or pancreatin yeast broth, 48h is cultivated, it is seen that slight haze unanimous between the higher and lower levels, pipe is low to be had
A small amount of precipitation.Subculture can also grow on the cool tire culture medium of serum (horse serum), and bacterium colony is translucent, but can not be in common fine jade
Fat on wheat health culture medium with growing.The blood agar slant culture of this bacterium, preserved in 4 DEG C of refrigerators it is easily dead, typically through 4~
5d answers subculture once, but virulence gradually reduces.
The growth of riemerella anatipestifer requires harsh, and culture in vitro needs higher nutritional condition, accordingly, phase at present
Close scholar and inquired into the various culture mediums suitable for the growth of inner Mo Shi bacillus such as soybean pancreatin, serum agar, blood agar, but do not have
The cultural method of unified standard, systematic research is not carried out for the growth characteristics on various culture mediums.
The content of the invention
To solve the above problems, the invention provides a kind of cultural method of riemerella anatipestifer,.
To achieve the above object, the technical scheme taken of the present invention is：
A kind of cultural method of riemerella anatipestifer, comprises the following steps：
S1, prepare duck blood pancreas egg peptone Soy Agar plates culture medium
10.0g tryptones, 5.0g soy peptones, 5.0g yeast extracts, 5.0g sodium chloride and 12.0g agar powders are dissolved in
In 1000ml deionized water, stir, pH value is adjusted to 7.1-7.2 with sodium hydroxide, after 121 DEG C of autoclaving 15min,
When being cooled to 65 DEG C -70 DEG C, sterile de- fiber duck blood 100ml is added by sterile manner, shakes up rear pour plate, it is sterile
After inspection, duck blood pancreas egg peptone Soy Agar plates culture medium is obtained, 4 DEG C of refrigerators save backup；
S2, from morbidity duck and dead duck separation riemerella anatipestifer bacillus, it is sterile to take the streak inoculation of duck brain tissue in duck blood pancreas
On egg peptone Soy Agar plates, CO is placed in237 DEG C of culture 24-48 hours in candle cylinder, produce and grow circular micro- protuberance, surface light
Cunning, butyrous, diameter 1-2mm non-pigment bacterium colonies, not haemolysis on blood plate.
Present invention also offers a kind of riemerella anatipestifer culture medium, it is prepared by the raw material of following components：
It is tryptone 10.0g, soy peptone 5.0g, yeast extract 5.0g, sodium chloride 5.0g, agar powder 12.0g, sterile
De- fiber duck blood 100ml, deionized water 1000ml.
The invention has the advantages that：
The cultural method of the present invention is easy to operate, can be suitably used for the flat board culture needs of routine experimentation, can make to write from memory in pest of duck
Family name bacillus grows preferable bacterium colony in 24h；And culture medium low manufacture cost, culture effect is good, can high-volume, the increasing of high quality
Bacterium is cultivated, and to being diagnosed the illness in clinical practice with directive significance, is laid a good foundation for riemerella anatipestifer production of vaccine.
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
In following examples, the separation of riemerella anatipestifer is completed by following steps：
It is easily separated to riemerella anatipestifer from morbidity duck and dead duck, it is sterile to take pathological tissues, it is big in trypsase
Rule on beans agar or chocolate agar, put in candle cylinder 37 DEG C and cultivate 24 hours, bacterial growth is 0.5~1mm sizes, surface
It is smooth, slightly raised, in the circular colonies of cheese shape.
The identification of riemerella anatipestifer is completed by following steps：
Riemerella anatipestifer cultivated on tryptic soy agar 24h formed circular protrusions, it is transparent, in dewdrop sample, diameter
For 0.5~1.5mm bacterium colony, glow green is observed with skew ray, E. coli isolated from ducks bacterium colony is 2~5mm, Salmonella anatis bacterium colony
For 2~3mm, and both rear cultures on TSA are canescence；Further it is inoculated with Mai Kangkai flat boards, it is seen then that write from memory in pest of duck
Family name bacillus does not grow, and E. coli isolated from ducks takes on a red color bacterium colony, the white bacterium colony of Salmonella anatis.
Embodiment
1st, the preparation of duck blood pancreas egg peptone Soy Agar plates culture medium：
10.0g tryptones, 5.0g soy peptones, 5.0g yeast extracts, 5.0g sodium chloride and 12.0g agar powders are dissolved in
In 1000ml deionized water, stir, regulation pH value after 121 DEG C of autoclaving 15min, is cooled to 65 to 7.1-7.2
At DEG C -70 DEG C, sterile de- fiber duck blood 100ml is added by sterile manner, shakes up rear pour plate, after steriling test, is obtained
Duck blood pancreas egg peptone Soy Agar plates culture medium, 4 DEG C of refrigerators save backup；
2nd, exemplified by the type YNRA-1 of one plant of serum 13 that identification is separated from this laboratory, by YNRA-1 inoculation duck blood pancreases
On egg peptone Soy Agar plates, 5%~10%CO is put2Incubator, 24h is cultivated under the conditions of 37 DEG C, can obtain circular micro- protuberance, surface
Smooth, butyrous, diameter 1~2mm non-pigment bacterium colonies, not haemolysis on blood plate.
3rd, duck blood pancreas egg peptone Soy Agar plates culture medium riemerella anatipestifer growing state
By taking the type YNRA-1 of serum 13 as an example, bacterial strain is smeared on inoculation duck blood pancreas egg peptone Soy Agar plates (diameter 90mm),
In 12h, 24h, lower lawn is washed with 10ml sterile salines, OD is determined with 721 micro-spectrophotometers420It is worth (OD420It is worth conduct
The reference index of bacterium total bacteria count, bacterium total bacteria count Y=2.85X1.24, X OD420Value).Measurement result 12h OD420Value 0.23,
Bacterium total bacteria count 4.6x109CFU/ml, 24h OD420Value 0.52, bacterium total bacteria count 1.27x1010CFU/ml。
Cultural character is observed
Bacterium growing state on different culture media is observed, 24- is cultivated on duck blood pancreas egg peptone Soy Agar plates culture medium
The colonial morphology of 48 hours for it is circular, translucent, protuberance, table and it is smooth, rich in gloss, butyrous, diameter about 1-2mm bacterium
Fall, do not grown on maconkey agar.CO2Under the conditions of anaerobism candle cylinder, grown on duck blood pancreas egg peptone Soy Agar plates culture medium
Well, the undergrowth and on plain agar flat board；Thalline Gram-negative, form is small coccobacillus under microscope, single
Individual thalline is in the majority, the slightly dense dye in the two poles of the earth.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (2)
1. a kind of cultural method of riemerella anatipestifer, it is characterised in that comprise the following steps：
S1, prepare duck blood pancreas egg peptone Soy Agar plates culture medium
10.0g tryptones, 5.0g soy peptones, 5.0g yeast extracts, 5.0g sodium chloride and 12.0g agar powders are dissolved in
In 1000ml deionized water, stir, pH value is adjusted to 7.1-7.2 with sodium hydroxide, after 121 DEG C of autoclaving 15min,
When being cooled to 65 DEG C -70 DEG C, sterile de- fiber duck blood 100ml is added by sterile manner, shakes up rear pour plate, it is sterile
After inspection, duck blood pancreas egg peptone Soy Agar plates culture medium is obtained, 4 DEG C of refrigerators save backup；
S2, from morbidity duck and dead duck separation riemerella anatipestifer bacillus, it is sterile to take the streak inoculation of duck brain tissue in duck blood pancreas egg peptone
On Soy Agar plates, CO is placed in237 DEG C of culture 24-48 hours in candle cylinder, produce grow circular micro- protuberance, surface it is smooth,
Butyrous, diameter 1-2mm non-pigment bacterium colonies, not haemolysis on blood plate.
2. a kind of riemerella anatipestifer culture medium, it is characterised in that be prepared by the raw material of following components：
Tryptone 10.0g, soy peptone 5.0g, yeast extract 5.0g, sodium chloride 5.0g, agar powder 12.0g, sterile de- fibre
Tie up duck blood 100ml, deionized water 1000ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949619A (en) * | 2018-07-02 | 2018-12-07 | 广东省农业科学院动物卫生研究所 | A kind of zymotechnique of riemerella anatipestifer |
| CN116790451A (en) * | 2023-08-23 | 2023-09-22 | 云南省畜牧兽医科学院 | Antigen and kit for detecting duck-origin salmonella enteritidis antibody and preparation method thereof |
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| CN101948781A (en) * | 2010-09-01 | 2011-01-19 | 四川农业大学实验动物工程技术中心 | Culture medium for production of riemerella anatipestifer vaccine and preparation method thereof |
| CN102250792A (en) * | 2011-06-17 | 2011-11-23 | 四川农业大学 | Culture medium for riemerella anatipestifer |
| CN103007261A (en) * | 2012-12-26 | 2013-04-03 | 青岛康地恩药业股份有限公司 | Riemerella anatipestifer (RA) and application thereof |
| CN105695375A (en) * | 2016-04-25 | 2016-06-22 | 四川农业大学 | Novel riemerella anatipestifer culture medium and preparation method thereof |
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2017
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101089190A (en) * | 2006-06-16 | 2007-12-19 | 阚方琦 | Nutrition blood culture medium |
| CN101948781A (en) * | 2010-09-01 | 2011-01-19 | 四川农业大学实验动物工程技术中心 | Culture medium for production of riemerella anatipestifer vaccine and preparation method thereof |
| CN102250792A (en) * | 2011-06-17 | 2011-11-23 | 四川农业大学 | Culture medium for riemerella anatipestifer |
| CN103007261A (en) * | 2012-12-26 | 2013-04-03 | 青岛康地恩药业股份有限公司 | Riemerella anatipestifer (RA) and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108949619A (en) * | 2018-07-02 | 2018-12-07 | 广东省农业科学院动物卫生研究所 | A kind of zymotechnique of riemerella anatipestifer |
| CN116790451A (en) * | 2023-08-23 | 2023-09-22 | 云南省畜牧兽医科学院 | Antigen and kit for detecting duck-origin salmonella enteritidis antibody and preparation method thereof |
| CN116790451B (en) * | 2023-08-23 | 2023-11-24 | 云南省畜牧兽医科学院 | Antigen and kit for detecting duck-origin salmonella enteritidis antibody and preparation method thereof |
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