CN106119164B - A method of avain tuberculosis mycobacteria is separately cultured with synthetic media - Google Patents
A method of avain tuberculosis mycobacteria is separately cultured with synthetic media Download PDFInfo
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- CN106119164B CN106119164B CN201610517944.5A CN201610517944A CN106119164B CN 106119164 B CN106119164 B CN 106119164B CN 201610517944 A CN201610517944 A CN 201610517944A CN 106119164 B CN106119164 B CN 106119164B
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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
- C12N1/20—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
Abstract
The present invention relates to a kind of methods that synthetic media is separately cultured avain tuberculosis mycobacteria.What the culture of avain tuberculosis mycobacteria generallyd use is Pei Shi (Petragnani) solid medium, the quality of the fresh skim milk of culture medium main component and egg is influenced by the factors such as milk cow and egg source, freshness, holding time, causes quality stability between batch poor.In addition production itself is cumbersome, and the laboratory of fresh milk source of drawing material difficulty is difficult to carry out.And synthetic media provided by the present invention and preparation method thereof, overcome more than defect, the culture medium ratio Pei Shi culture medium being prepared into substantially reduces avain tuberculosis mycobacteria incubation time, and is clinically separated effect and is substantially better than Pei Shi culture medium.Have the advantages that easy to operate, quality is stable, be suitble to be clinically separated culture avain tuberculosis mycobacteria, provides sound assurance for fowl tuberculosis diagnosis.
Description
Technical field belongs to the present invention relates to a kind of method for being separately cultured avain tuberculosis mycobacteria with synthetic media
Veterinary microbiology field.
Background technique
Fowl tuberculosis is that (also known as mycobacterium avium is shown in China Veterinery Drug Inspection Office, Chinese beast by avain tuberculosis mycobacteria
Doctor Microbiological Culture Collection administrative center writes, and Chinese animal doctor's strain catalogue second edition 2002, Scientia Agricultura Sinica technology is published
Society, 2002, p83) cause a kind of infectious disease of poultry and bird.It is characterized in that the histoorgans such as liver, spleen and the enteron aisle of disease fowl form meat
Bud swells and cheesy calcium scoring.This disease can infringer and many animals.World Organization for Animal Health is classified as B class animal epidemic disease
Disease, and China is set to three classes animal epidemic.The disease feature be in chronic process, gradual syntexis, anaemia, cockscomb atrophy, limping with
And lay eggs reduction or stopping.The course of disease continues 2~3 months, sometimes up to 1 year.Sick fowl because failure or because liver be denaturalized rupture due to it is unexpected
It is dead.Once incoming poultry-farm, then long-term existence, is difficult to eradicate.There is different degrees of fowl tuberculosis when national in recent years,
Disease incidence is high, and harm is serious, and economic loss is huge, and a kind of easy isolated culture method is established in urgent need, so as to clinical beast
Doctor and researcher carry out diagnosis in time, accurately, to carry out rationally effectively disposition to epidemic situation.
It is diagnosis fowl tuberculosis " goldstandard " that avain tuberculosis mycobacteria is successfully separated out in suspected lesion tissue.Fowl knot
Core mycobacterium generally includes four subspecies, and the first subspecies are avain tuberculosis mycobacterium serotype 1,2,3 and genotype IS901+
And IS1245+；Second subspecies have avain tuberculosis mycobacterium people/pig subspecies (hominissuis) serotype 4,5,6,8,9,10,11
With 21 and genotype IS901-And IS1245+；Third subspecies are avain tuberculosis mycobacterium perituberculosis subspecies；4th subspecies are fowl knot
Core mycobacterium silvaticum (translate, and OIE terrestrial animal is examined by Ministry of Agriculture's veterinary Bureau/China Animal Health and Epidemiology Center
Disconnected test and vaccine handbook, 2010).
Currently, isolated in China avain tuberculosis mycobacteria mainly uses Pei Shi (Petragnani) solid medium (Chinese beast
Cure Culture Collection, China Veterinery Drug Inspection Office compiles, Chinese veterinary microorganism strain catalogue, Chinese agriculture publication
Society, hereinafter referred to as " strain catalogue " in 2002；The Ministry of Agriculture of the People's Republic of China, MOA, People's Republic of China's veterinary biologics
Regulation, 2000 editions, Chinese agriculture publishing house, hereinafter referred to as " regulation " in 2001).The culture medium main component is fresh degreasing
Milk and egg, quality are influenced by the factors such as milk cow and egg source, freshness, holding time, cause culture medium batch
Quality stability is poor between secondary.In addition Pei Shi is cultivated in matrix manufacturing, need to be acquired fresh milk and be carried out degreasing, and need to prepare potato
Immersion liquid simultaneously separates yolk, and complex manufacturing technology is cumbersome.In addition, can not for the laboratory of fresh milk source of drawing material difficulty at all
Realize culture matrix manufacturing.Thus seriously restrict and affect nationwide timely diagnosis and prevention and control to fowl tuberculosis.
Summary of the invention
The purpose of the present invention
Technical solution of the present invention
1. a kind of method for being separately cultured avain tuberculosis mycobacteria with synthetic media, it is characterized in that fowl type tuberculosis will be produced
The appropriate normal saline dilution of avain tuberculosis mycobacteria reference culture C68201, C68202 and C68203 freeze-drying lactobacillus of rhzomorph
Afterwards, using skimmed milk power, egg yolk lecithin as in the solid medium of synthetic media primary raw material, 37 DEG C are cultivated 7, are made for inoculation
To prepare fowl type tuberculin first order seed.
2. the isolated culture method of a kind of avain tuberculosis mycobacteria described in accordance with the claim 1, it is characterized in that wherein institute
It is using the formula (W/V) of synthetic media: skimmed milk power 20g, PPLO agar powder 20g, yolk lecithin 10g, asparagine
2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, vitamin K 0.1g, malachite green 0.6g, dehydrated alcohol 20ml, deionization
Water 1000ml.Its preparation method are as follows: go to heat after completely dissolution from water with 500ml by PPLO agar powder and malachite green, 116 DEG C
Sterilize 15min, is cooled to 60 DEG C and is used as A liquid；Skimmed milk power, asparagine, peptone, yeast powder, glycerol, vitamin K are used
500ml deionized water, after 50 DEG C of heating water bath dissolutions, 0.22 μm of filtration sterilization is as B liquid；By yolk lecithin with sterile working
Mode use the dehydrated alcohol of 20ml after completely dissolution as C liquid in sterile vessel, then by A liquid and B liquid and C liquid, (V/V is
After 500:500:20) being sufficiently mixed, culture medium slant is made with 8ml/ pipe and is used.
Detailed description of the invention
1. strain is used in the production of avain tuberculosis rhzomorph
(1) source C68201/C68202/C68203 plants of avain tuberculosis mycobacterias, identified by China Veterinery Drug Inspection Office,
Keeping and supply.
(2) cultural character and virulence
By C68201/C68202/C68203 plants avain tuberculosis mycobacteria freeze-drying lactobacillus each 1,1ml physiological saline is used respectively
After dissolved dilution, respectively it is inoculated with 0.2ml/ branch with skimmed milk power, PPLO agar powder and egg yolk lecithin as main synthetic media
The culture medium slant of raw material, 37 DEG C are cultivated 7, and media surface grows up to yellowish white butyrous, the sticky allusion quotation of bacterium colony
Type bacterium colony scrapes the intramuscular injection of 10mg viable bacteria from inclined-plane or viable bacteria 1mg is taken to be injected intravenously each 5 of 8 week old SPF chicken, clinical observation 4
Week, equal 5/5 morbidity or dead are used suitable for breeding fowl type tuberculin production seed.
2 culture mediums synthetic media designed by the invention
(1) formula (W/V) is as follows:
Skimmed milk power 20g, PPLO agar powder 20g, yolk lecithin 10g, asparagine 2.6g, peptone 2g, yeast powder
2g, glycerol 36ml, malachite green 0.6g, micro-element K 0.1g, dehydrated alcohol 20ml, deionized water to 1000ml.
(2) synthetic media preparation method
PPLO agar powder and malachite green are gone to heat after completely dissolution from water with 500ml, 116 DEG C of sterilizing 15min, cooling
A liquid is used as to 60 DEG C；By skimmed milk power, asparagine, peptone, yeast powder, glycerol, vitamin K 500ml deionized water,
After 50 DEG C of heating water bath dissolutions, 0.22 μm of filtration sterilization is as B liquid；By yolk lecithin in sterile device in a manner of sterile working
It uses the dehydrated alcohol of 20ml after completely dissolution as C liquid in ware, then fills A liquid and B liquid and C liquid (V/V 500:500:20)
After dividing mixing, it is spare that culture medium slant is made with 8ml/ pipe.
(3) synthetic media is examined
1) character is in faint yellow solid.
2) steriling test presses Republic of China Veterinary Pharmacopoeia (the People's Republic of China (PRC), Chinese veterinary pharmacopoeia committee veterinary drug
2005 editions three Chinese agriculture publishing houses of allusion quotation, 2006, hereinafter referred to as " Chinese veterinary pharmacopoeia ") as defined in method carry out, answer sterile
Growth.
3) growth test
By avain tuberculosis mycobacteria (C68201/C68202/C68203 plants, China Veterinery Drug Inspection Office provides) freeze-dried vaccine
Kind with after 1ml normal saline dilution, is inoculated with synthetic media solid slope respectively with 0.2ml/ branch, 37 DEG C are cultivated 7, and bacterium colony is
Yellowish white butyrous, bacterium colony is sticky, and picking colony can be in wire drawing shape.First order seed is used as after pure passed examination.2~
8 DEG C of preservations should be no more than (" regulation ") on the 45th.
The present invention relates to microbial resources information
Microbial resources involved in the present invention are C68201 plants, C68202 plants and C68203 plants of avain tuberculosis mycobacteria,
By China Veterinery Drug Inspection Office's identification, keeping and supply (see China Veterinery Drug Inspection Office, Chinese veterinary microorganism strain
Preservation administrative center is write, Chinese animal doctor's strain catalogue second edition 2002, Scientia Agricultura Sinica technology publishing house, 2002, p83).
The positive effect of the present invention
Bacterium separation is carried out from doubtful fowl tuberculosis tissue, is diagnosis fowl tuberculosis " goldstandard ".Isolated in China training
What feeding avain tuberculosis mycobacteria generallyd use is Pei Shi (Petragnani) solid medium, and the culture medium main component is fresh de-
The quality of rouge milk and egg is influenced by the factors such as milk cow and egg source, freshness, holding time, causes batch interstitial
It is poor to measure stability.In addition production itself is cumbersome, and the laboratory of fresh milk source of drawing material difficulty is difficult to carry out, thus
Influence avain tuberculosis mycobacteria is separately cultured and the diagnosis of clinical fowl tuberculosis.And synthetic media provided by the present invention and its
Preparation method, overcome more than defect, the culture medium ratio Pei Shi culture medium being prepared into substantially reduce avain tuberculosis mycobacteria training
It supports time (can reduce by 3 days or more incubation times), and is clinically separated effect and is substantially better than Pei Shi culture medium.It efficiently solves new
The problem that fresh milk source of drawing material is difficult, technique production is complicated.Have the advantages that easy to operate, quality is stable, is suitble to be clinically separated
Avain tuberculosis mycobacteria is cultivated, provides sound assurance for fowl tuberculosis diagnosis.
Embodiment
Embodiment 1
--- the research of avain tuberculosis mycobacteria synthetic media
From 1%~3% skimmed milk power, 0.5%~1.5% yolk lecithin, 1%~2%PPLO agar powder is combined into
In formula, the avain tuberculosis mycobacteria (C68201/C68202/C68203 plants) being lyophilized by inoculated and cultured filters out 2% degreasing
The synthetic media formula of+1% yolk lecithin+2%PPLO agar powder of milk powder.The formula is applied as a result, 37 DEG C of stationary cultures 7
Day, avain tuberculosis mycobacteria (C68201/C68202/C68203 plants) media surface grows up to yellowish white butyrous, bacterium colony
Sticky colonies typical.From the scraping 10mg viable bacteria intramuscular injection of culture inclined-plane or viable bacteria 1mg is taken to be injected intravenously 8 week old SPF chickens each 5
Only, clinical observation 4 weeks, equal 5/5 morbidity or dead was almost the same with the bacterium result of the same race of Pei Shi solid medium culture.Meet
The requirement of " fowl type tuberculin manufacture strain " in " regulation ".
1 material
1.1 culture medium raw material
(lot number 232100 is purchased from BD Difco to skim milkTMCompany)；(lot number 214010, is purchased from yolk lecithin
OXOID company)；PPLO agar powder (lot number: 214230, it is purchased from BD DifcoTMCompany)；Pancreas casein peptone (lot number VM732731-
644, it is purchased from MERCK company)；Yeast extract (lot number 911948 is purchased from OXOID company)；Asparagine, glycerol, malachite green,
Dehydrated alcohol, glucose, sucrose, thio ` sodium sulphate, hemin, vitamin K etc. are conventional chemical reagent, purchased from north
Capital chemical reagents corporation.
1.2 fresh milks (same day picks up from Beijing cattle farm), egg, potato, potato starch are super purchased from Beijing
City, and physiological saline (lot number: 0619,4.5ml/ branch, 100ml/ bottles), A type test tube uses specification 25ml, Chinese veterinary medicament supervision
Institute's culture medium group provides.
1.3 strain
Produce the strain of fowl type tuberculin: fowl type mycobacterium tuberculosis (C68201/C68202/C68203 plants)
(2006.8.29 freeze-drying, 0.3ml/ branch), by China Veterinery Drug Inspection Office's identification, keeping and supply.
2 methods and result
The preparation of 2.1 avain tuberculosis mycobacteria seed liquors
It is carried out referring to " regulation ", takes each 1 of avain tuberculosis mycobacteria C68201, C68202 and C68203 freeze-drying lactobacillus, point
Yong not be after 1ml normal saline dilution, with the switching of 0.2ml/ branch by the Pei Shi culture medium of 2.2 methods preparation, 37 DEG C are cultivated 20,
As first order seed, lower surface lawn then is washed with appropriate physiology salt respectively, is placed in containing in sterilizing glass bulb, uses physiology
Salt-water Oscillator mixing is diluted to Maxwell than turbid concentration about 106The bacterium solution of CFU/ml, uses as seed liquor.
2.2 Pei Shi (Petragnani) solid medium
2.2.1 ingredient: fresh skim milk 450ml, potato starch 18g, asparagine 2.6g (or peptone 3g) are gone
Skin potato 225g, 15, egg (remove 3 egg white)
2.2.2 preparation method is as follows referring to " regulation ": 1) wiping peeling potatoes at silk, potato starch, Tianmen is added
Winter element (or peptone), skim milk are set water-bath in beaker and are boiled 40~60 minutes, and stir evenly frequently, and paste is made into.To
The egg (eggshell is first cleaned with 75% alcohol disinfecting) smashed is added when being cooled to 50 DEG C, with four layers of gauze filtration slagging-off after mixing.
It is eventually adding glycerol and malachite green solution stirs evenly, be sub-packed in the test tube of sterilizing.2) packing is had to the test tube of culture medium
It sets in flowing steam pot and puts into inclined-plane, through flowing steam discontinuous sterilization 3 times, one time a day.65 DEG C of sterilizing 30min on the 1st, the 2nd, 3
Day, 75~80 DEG C of sterilizing 30min.
2.3 synthetic media
2.3.1 basic components screen
It is prepared by following ingredient: skimmed milk power 10g or 20g or 30g, yolk lecithin 5g or 10g or 15g, PPLO
Agar powder 10g or 20g, asparagine 2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, malachite green 0.6g, dehydrated alcohol
20ml, deionized water 1000ml.Then by skimmed milk power, yolk lecithin and PPLO agar powder, table 1 is designed to by different proportion
Shown 18 formulas labeled as 1~18 (specific ratio and number are shown in Table 1), while setting up the control of Pei Shi culture medium.
2.3.2 preparing
PPLO agar powder and malachite green are gone to heat after completely dissolution from water with 500ml in ratio shown in table 1 and amount,
116 DEG C of sterilizing 15min are cooled to 60 DEG C and are used as A liquid；By skimmed milk power, asparagine, peptone, yeast powder, glycerol, dimension life
Plain K 500ml deionized water, after 50 DEG C of heating water bath dissolutions, 0.22um filtration sterilization is as B liquid；By yolk lecithin with nothing
The mode of bacterium operation uses the dehydrated alcohol of 20ml after completely dissolution as C liquid in sterile vessel, then by A liquid and B liquid and C liquid
After (V/V 500:500:20) is sufficiently mixed, it is spare that culture medium slant is made with 8ml/ pipe.
The inoculation of 2.4 seed liquors
The seed liquor that 2.1 are prepared, after making 10000 times of dilutions with physiological saline, by 0.2ml/ branch, inoculation is made respectively
Synthetic media and Pei Shi medium slant, 37 DEG C cultivate 20 days.Observe each culture inclined-plane bacterium colony growth result.
The bacterium colony count results (unit: CFU) of 1 synthetic media basic components of table screening
Note: seed liquor about 106CFU/ml。
Find out from 1 result of table, its ability for cultivating 3 kinds of bacterium of formula 14 is best, and is apparently higher than control Pei Shi culture medium.
The screening of 2.5 culture medium growth factors
It chooses the higher formula 14 of breeding bacteria and is used as synthetic media basic ingredient, wherein A liquid and C liquid press 2.3
Method carries out.Glucose, sucrose, sodium thiosulfate, the chlorine of various dose is added in ratio shown in table 2 and amount respectively in B liquid
Change the growth factors such as ferroheme, vitamin K.Then 500ml deionized water can be used together 50 with basis B liquid ingredient in formula 14
After the dissolution of DEG C heating water bath, 0.22um filtration sterilization is used as B liquid, then is made into solid medium by 2.3 methods.
The bacterium colony count results (unit: CFU) of 2 culture medium growth factor of table screening
Note: access viable bacteria about 106The seed liquor of CFU/ml, it is 151103 that Pei Shi, which compares lot number,.
Find out that vitamin K is obvious to culture medium growth promoting function from 2 result of table, and other growth factor growth promoting functions
It is unobvious.
2.6 growth factor different amounts comparative tests
By the growth factor filtered out (vitamin K), by final concentration 0.005%, 0.01%, 0.02%, 0.05%,
0.1% is added in synthetic media, then accesses seed liquor by 2.4 methods, and 37 DEG C are cultivated 20 respectively, and observation the results are shown in Table
3。
3 different amounts growth factor comparative test bacterium colony count results (unit: CFU) of table
Note: access viable bacteria about 106The seed liquor of CFU/ml
Find out from 3 result of table, synthetic media bacterium number highest of the vitamin K at 0.01%.
2.7 synthetic medias and the comparative test of Pei Shi culture medium
By formula 14 vitamin Ks of optimal dose (addition), prepare 3 batches of synthetic medias (lot number is respectively 201501,
201502 and 201503), together with 3 batches of Pei Shi culture mediums (lot number is respectively 201511,201512 and 201513), respectively it is sub-packed in A
Type test tube (8ml/ pipe).Dilution strain is accessed by 0.2ml/ branch, 37 DEG C are cultivated 20 respectively.Respectively at culture 7 days, 10 days, 14
Day and 20 days, result is observed respectively.
4 culture medium comparative test result (unit: CFU) of table
Note: 3 kinds of bacterium strains: 1 it is C68201,2 is C68202,3 is C68203, seed liquor is each about 106CFU/ml；
"/" represent bacterium colony it is invisible or naked eyes can not count
Find out from 4 result of table, under same culture conditions, the breeding bacteria of 3 batches of synthetic medias is cultivated compared with 3 crowdes of Pei Shi
The breeding bacteria of base is high, and incubation time shortens, in addition the Pei Shi training between batch than being prepared with fresh skim milk and yolk
It is preferable to support base homogeneity.
The virulence test of 2.4 culture thallus
The lawn of 37 DEG C of inclined-plane base cultures 7 days of synthesis culture and 37 DEG C of Pei Shi medium slant are cultivated to lawn on the 10th,
After respectively being scraped with sterile soupspoon, lawn weight is weighed, then with after appropriate normal saline dilution and the mixing that suspends.Muscle note respectively
8 week old SPF chickens 5 are penetrated, 1ml/ only (every 1ml viable bacteria containing 10mg), clinical observation 4 weeks, records survival condition；It is injected intravenously respectively
8 week old SPF chickens 5,0.5ml/ only (every 0.5ml viable bacteria containing 1mg), clinical observation 4 weeks, record survival condition.It the results are shown in Table 5.
Table 5 cultivates thallus virulence determination result
Find out from 5 result of table, the thallus and both thallus of Pei Shi culture medium culture 14 days of synthetic media culture 7 days are malicious
Power measurement result is almost the same, meets and produces avain tuberculosis rhzomorph strain virulence standard in " regulation ".
4 conclusions
4.1 are above Pei Shi training with the synthetic media breeding bacteria of culture medium prescription 14 plus 0.01% vitamin K composition
Base is supported, and strain growth speed is better than Pei Shi culture medium on synthetic media, incubation time ratio Pei Shi culture medium is short, right
SPF chicken toxicity test result and Pei Shi culture medium are almost the same, meet in " regulation " " production avain tuberculosis rhzomorph strain virulence mark
The requirement of standard ".
Embodiment 2
--- synthetic media is compared with Pei Shi culture medium is to the growth and breeding of other avain tuberculosis mycobacteria reference cultures
As a result
The synthetic media (201501 batches) and Pei Shi culture medium (201512 batches) of optional 1 batch of preparation, purchased from national beast
22 plants of other each 1 of avain tuberculosis mycobacteria reference culture freeze-drying lactobacillus that Culture Collection saves are cured, then respectively
After the dilution of 1ml physiological saline solution, above two medium slant is inoculated with 0.2ml/ branch respectively, 37 DEG C are cultivated 14, the phase
Between 7,10 and 14 days observation observe growing state respectively.It the results are shown in Table 6.
6 different strain growing state of table
Note: "+" growth；"-" is without growth；
Find out from 6 result of table, synthetic media is cultivated 10 at 37 DEG C, can successfully turn out 22 plants of strains, and bacterium is raw
Long speed is substantially better than Pei Shi culture medium.
Embodiment 3
--- synthetic media is with Pei Shi culture medium to the separation comparison result of clinical pathological material of disease
Optional 1 batch of synthetic media (201501 batches) and Pei Shi culture medium (201512 batches), to 5 parts of doubtful avain tuberculosis of clinic
Pathological material of disease is separated.It, will with sterile scissors sterile working picking pathological material of disease interior tissue about 0.1g under Biohazard Safety Equipment environment
It is put into the 1.5ml eppdorf pipe of the sterilizing containing stainless steel ball, is then respectively adding 200ul sterile saline,
It is homogenized with tissue refiner's (parameter setting are as follows: 100r/min vibrates 1min, interval 1min, total 3min).Finally by tissue
Homogenate respectively with after the dilution of 200 μ l sterile salines, is transferred synthetic media and Pei Shi culture medium respectively with 200 μ l/ pipes again
Each 1 pipe, 37 DEG C are cultivated 14~20, and inclined-plane culture result is observed.It the results are shown in Table 7.
7 different culture medium of table is clinically separated bacterium situation and compares
Note: "+" bacterium colony is grown and PCR is accredited as the positive；"-" is without growth；
Find out from 7 result of table, synthetic media is to clinical doubtful sample separation rate up to 100%, and Pei Shi culture medium is only
80%.
Claims (2)
1. a kind of method for being separately cultured avain tuberculosis mycobacteria with synthetic media, it is characterized in that fowl type tuberculin will be produced
C68201 plants, C68202 plants and C68203 plants appropriate normal saline dilutions of freeze-drying lactobacillus of avain tuberculosis mycobacteria reference culture
Afterwards, using skimmed milk power, egg yolk lecithin as in the solid medium of synthetic media primary raw material, 37 DEG C are cultivated 7, are made for inoculation
To prepare fowl type tuberculin first order seed；
The synthetic media formula that the culture uses is based on W/V: skimmed milk power 20g, PPLO agar powder 20g, yolk ovum
Phosphatidase 1 0g, asparagine 2.6g, peptone 2g, yeast powder 2g, glycerol 36ml, vitamin K 0.1g, malachite green 0.6g, nothing
Water-ethanol 20ml, deionized water 1000ml.
2. the isolated culture method of a kind of avain tuberculosis mycobacteria described in accordance with the claim 1, it is characterized in that used in it
The preparation method of synthetic media are as follows: PPLO agar powder and malachite green 500ml deionized water are heated after completely dissolution,
116 DEG C of sterilizing 15min are cooled to 60 DEG C and are used as A liquid；By skimmed milk power, asparagine, peptone, yeast powder, glycerol, dimension life
Plain K 500ml deionized water, after 50 DEG C of heating water bath dissolutions, 0.22 μm of filtration sterilization is as B liquid；By yolk lecithin with nothing
The mode of bacterium operation uses the dehydrated alcohol of 20ml after completely dissolution as C liquid in sterile vessel, is then 500:500 by V/V:
After 20 A liquid and B liquid and C liquid is sufficiently mixed, culture medium slant is made with 8ml/ pipe and is used.
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| CN102449137A (en) * | 2008-12-05 | 2012-05-09 | 地中海(埃克斯-马赛第二)大学 | Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex |
| CN103993065A (en) * | 2014-05-07 | 2014-08-20 | 济宁医学院 | Diphasic quick differential medium of mycobacterium tuberculosis and application of medium |
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| CN102449137A (en) * | 2008-12-05 | 2012-05-09 | 地中海(埃克斯-马赛第二)大学 | Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex |
| CN103993065A (en) * | 2014-05-07 | 2014-08-20 | 济宁医学院 | Diphasic quick differential medium of mycobacterium tuberculosis and application of medium |
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