CN108060109A - People's mycoplasma pneumoniae isolation medium and cultural method - Google Patents
People's mycoplasma pneumoniae isolation medium and cultural method Download PDFInfo
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- 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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- 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 mycoplasmas to be separately cultured field, and in particular to the cultural method of a kind of people's mycoplasma pneumoniae isolation medium SLB and this culture medium.It is respectively including basal medium, horse serum, antibiotic and other adding ingredients, component：Basal medium 560ml, horse serum：50 200ml, antibiotic 4ml, 386 236ml of other adding ingredients, the horse serum of addition and other adding ingredient total volumes are 436ml；It is 7.4 7.6 to adjust final ph with 1M NaOH.Antibiotic is benzyl penicillin, thaliium acetate, polymyxin B and anphotericin.Other adding ingredients are 1066 tissue cultures complementary elements of CMRL, glucose, yeast cold soaking powder containing glutamine.The culture based selective of the present invention is good, and high specificity, the speed of growth is fast, and separation rate is high.To fluid nutrient medium after culture substantially by red change Huang and the sample of as clear as crystal no precipitation, ware is applied in substantially visible " fried egg " sample colony growth on solid medium, PCR results are the positive.
Description
Technical field
The present invention relates to mycoplasmas to be separately cultured field, and in particular to a kind of people's mycoplasma pneumoniae isolation medium SLB, with
And the cultural method of this culture medium.
Background technology
People's mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is the common community acquired pneumonia of children
One of the main pathogens of (community acquired pneumonia CAP), it can not only be drawn by invading respiratory tract
Laryngitis, pharyngitis, bronchitis and atypical pneumonia are played, can also induce and aggravate children and pant.After people's mycoplasma pneumoniae infection
Also can extrapulmonary complication be formed by complicated immune response mechanism.The resistance to macrolides people mycoplasma pneumoniae sense of clinical research confirmation
Dye can form fatal pneumonia (Fatal pneumonia FP), long with the clinical symptoms duration, clinical symptoms weight and lung
The features such as outer complication is more.
People's mycoplasma pneumoniae nutritional requirement is higher, is once known as " fastidious bacteria ".People's mycoplasma pneumoniae is binary division, during multiplication
Between it is small more than 6 when, the so long doubling time makes one mycoplasma pneumoniae culture as a very long process, is far longer than thin
Bacterium.In addition, people's mycoplasma pneumoniae genome is smaller, the gene of Amino acid synthesis itself is not found, while it is multiple also to lack synthesis
The lots of genes of heteroproteose cell wall construction.It is related to the gene dosage of DNA reparations, DNA restructuring, cell division and protein secretion also than general
Logical bacterium is few.Smaller genome and limited biosynthesis ability limit the biosynthesis of people's mycoplasma pneumoniae and metabolic energy
Power, resulting in the need for extremely complex culture medium adding ingredient could be separately cultured in vitro.
People's mycoplasma pneumoniae is separately cultured that difficulty is larger, and preparing for isolation medium is particularly important.At present, people's pneumonia branch
There are mainly two types of substance basal mediums：PPLO and SP-4 basal mediums, it is generally understood that SP-4 culture mediums are used to separate MP
Shi Xiaoguo is more preferable, and serum provides important nutriment, wherein most commonly used for calf serum and horse serum, horse serum due to
Its cholesterol level is higher and becomes preferred, but this laboratory was once cultivated with the horse serum of Gibco, and discovery is ineffective,
Flavescence sample is few and incubation time is longer.
It is separately cultured for people's mycoplasma pneumoniae of clinical sample, centainly also to add in suitable antibiotic and inhibit miscellaneous bacteria
Growth, what is usually added is benzyl penicillin and thaliium acetate.In addition, it adds in liquid medium phenol red as indicator, mycoplasma
Metabolizable glucose production acid, when culture medium color is by red change Huang, you can judge the growth of MP.But the actually separation training of mycoplasma
Difficulty is supported, cultivation cycle is long.After fluid nutrient medium turns yellow substantially, visible " fried egg " the shape mycoplasma colonies typical of ware is applied.
There are poor specificity, miscellaneous bacteria easy to pollute, false positive rate height etc. mostly to ask for isolation medium of the country in relation to MP at present
Topic, most of clinical samples muddiness after culture have precipitation, thus it cannot be determined whether infection people's mycoplasma pneumoniae, domestic each reality
The mycoplasma isolation medium that room uses import mostly is tested, it is expensive.Because a kind of people's mycoplasma pneumoniae growth of the invention is more fast
Speed and the higher clinical-use human mycoplasma pneumoniae isolation medium of specificity, have for the research and diagnosis of people's mycoplasma pneumoniae
Significant application value.
The content of the invention
The object of the present invention is to provide a kind of high specificity, people's mycoplasma pneumoniae separation that the speed of growth is fast, separation rate is high
Culture medium.
It is a further object of the present invention to provide the cultural methods of this culture medium.
People's mycoplasma pneumoniae isolation medium name of the present invention is people mycoplasma pneumoniae isolation medium SLB, is wrapped
Including basal medium, horse serum, antibiotic and other adding ingredients, component is respectively：
First, basal medium 560ml：Mycoplasma broth bouillon 2.5-5g, bacto peptone 4.5-6g；Bacterium pancreas egg
White peptone 8-12g, yeast extract 4-6g, 0.2% phenol red 10ml, add in pure water and are settled to 560ml, and pH value is adjusted with 1M NaOH
115 DEG C of high pressure sterilizations 30 minutes after to 7.5-7.7；
2nd, horse serum：50-200ml, 56 DEG C of inactivation 30min；
3rd, antibiotic 4ml：Adding in 1ml benzyl penicillins makes its final concentration of 250-600units/ml, and 1ml thaliium acetates make it
Final concentration of 0.1mg/ml, 1ml polymyxin B make its final concentration of 2-3.5units/ml, and 1ml anphotericins make its final concentration
For 2-3.5 μ g/ml；
4th, other adding ingredient 386-236ml：1066 tissue cultures complementary elements 3 of CMRL containing glutamine~
5.5g, 1~20g of glucose, yeast cold soaking 1.1~2.5g of powder, it is 386-236ml to add in pure water constant volume, is filtered with 0.22 μm of aperture
Membrane filtration degerming；The horse serum of addition and other adding ingredient total volumes are 436ml；
5th, it is 7.4-7.6 to adjust final ph with 1M NaOH.
The horse serum can be existing horse serum or self-control horse serum.
The self-control horse serum preparation method is as follows：
First, the selection of healthy horses：Selection 5-8 Sui age, without wound, do not injected antibiotic health male horses；
2nd, take a blood sample：Progress horses neck surface disinfection → shaving → sterilizes → removing vein blood vessel → and disappears again in order
Heparin tube is connect blood into 500ml blood collecting bottles by malicious inserting needle → sterile working along bottle wall, per bottled 400ml；
3rd, serum separates：Blood collecting bottle is put into 37 DEG C of insulating box 1.5-2.5h → 4 DEG C and places 2-4h → 3000rpm
Once → 4 DEG C overnight → sterile working absorption supernatant yellow liquid → -20 DEG C freeze for 40min centrifugations；
4th, the filtration sterilization of serum：→ 0.22 μm of aperture filter column filtering of defrosting serum → 0.45 μm aperture filter column filtering →
0.1 μm of aperture filter column filters → -20 DEG C and freezes.
The isolated culture method of people's mycoplasma pneumoniae isolation medium SLB of the present invention comprises the following steps：
First, clinical sample is inoculated in the fluid nutrient medium and cultivated, condition of culture is 37 DEG C, 5%CO2Culture
Case；
2nd, step 1 sample is continued in 10% ratio blind passage of percent by volume to the culture medium after cultivating 6-24h；
3rd, the culture of step 2 sample is observed after 3-10 days, with continuing after 0.45 μm of aperture membrane filtration if having precipitation
Observe in 10% ratio blind passage of percent by volume to the culture medium and frequently its color change；Culture medium face is observed if without precipitation
Color；Obtain preliminary judgement people's mycoplasma pneumoniae clinical separation strain result；
4th, continuation blind passage, the culture of inoculation solid medium 3-7 days, observation training are carried out to step 3 initial gross separation strain sample
It supports base color change, colonial morphology and PCR and identifies 3 kinds of processing, obtain and finally judge separating resulting.
The standard of preliminary judgement people's mycoplasma pneumoniae clinical separation strain result is：If fluid nutrient medium is substantially by red
Turn yellow and as clear as crystal without precipitation and floating material, can tentatively judge to be separated to people's mycoplasma pneumoniae clinical separation strain；Described
Finally the standard of judgement separating resulting is：If after blind passage fluid nutrient medium again substantially it is yellow by red change and it is as clear as crystal without precipitation and
Floating material, it is positive to have " fried egg " sample colony growth and PCR on solid medium, is finally judged to being successfully separated people's lung
Scorching Mycoplasma Isolated From Clinical.
Compared with existing domestic people's mycoplasma pneumoniae isolation medium, the present invention has the following advantages and beneficial effects：
Culture medium of the invention uses self-control horse serum, the main cholesterol and long-chain that provides mycoplasma and itself cannot synthesize
Aliphatic acid and haemocyanin, nutritional ingredient are more more rich than commercial horse serum, and for cultivating people's mycoplasma pneumoniae, the speed of growth is bright
It is aobvious to accelerate, be conducive to being separately cultured for people's mycoplasma pneumoniae.The self-control horse serum that the culture medium of the present invention uses, cholesterol contain
The more general calf serum of amount is high, can effectively facilitate the synthesis of bovis cells growth and breeding and cell membrane.
The culture medium addition glucose of the present invention, can effectively facilitate bovis cells growth and breeding, especially work as glucose
When content reaches 1%, the mycoplasma speed of growth is substantially accelerated, and the time that turns yellow shortens.
The culture medium of the present invention adds 4 kinds of antibiotic, can effectively inhibit the growth of other miscellaneous bacterias and to people's mycoplasma pneumoniae
Growth effect is smaller.In order to avoid germ contamination in isolate, benzyl penicillin and thaliium acetate are added in culture medium, the former inhibits to remove from office
Lan Shi positive bacterias, the latter inhibit Gram-negative bacteria.In addition gram-negative can effectively be inhibited by adding polymyxin B and anphotericin
Property bacterium and fungi growth so that pollution rate is substantially reduced, and so as to effectively reduce false positive rate, specificity is high.
The culture based selective of the present invention is good, and high specificity, the speed of growth is fast, and separation rate is high.To Liquid Culture after culture
Base substantially by red change Huang and the sample of as clear as crystal no precipitation, applies ware in substantially visible " fried egg " sample bacterium colony on solid medium
Growth, PCR results are the positive.
SLB culture medium prescriptions of the present invention, suitable for being separately cultured for people's mycoplasma pneumoniae clinical strain, are separated to
Bacterial strain can stablize passage.
Description of the drawings
The microscope figure (× 100) of Fig. 1 behaviour mycoplasma pneumoniae reference culture ATCC29342 bacterium colonies.
The microscope figure (× 100) of Fig. 2 people's mycoplasma pneumoniae clinical separation strain bacterium colony.
Fig. 3 people's mycoplasma pneumoniae clinical separation strain PCR electrophoretograms.
Description of the drawings：It is that PCR is carried out using people's mycoplasma pneumoniae custom primer to 12 plants of separating samples in Fig. 3, as a result
To the band of 144bp sizes, isolated 12 plants of sample PCR qualification result behaviour mycoplasma pneumoniaes.1-12 in figure：People's pneumonia
Mycoplasma Clinical isolation；13：ATCC 15531；14：ATCC 29342；15：Negative control, M：DNA Marker
DL5000。
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1：
The preparation of people's mycoplasma pneumoniae isolation medium SLB：
Basal medium：Mycoplasma broth bouillon 3.5g, bacto peptone 5.3g；Bacto-tryptone 10.0g, ferment
Female extract 5g, 0.2% phenol red 10ml add in pure water and are settled to 560ml, pH to 7.6,115 DEG C of sterilizings are adjusted after mixing
30min, it is spare after cooling.
Horse serum：Defrosting 200ml horse serums, 56 DEG C of inactivation 30min.
The preparation method of horse serum is as follows：
1. the selection of healthy horses：Selection 5-8 Sui age, without wound, do not injected antibiotic health male horses.
2. it takes a blood sample：Horses neck surface disinfection → shaving → is carried out in order sterilizes → remove vein blood vessel → disinfection again
Heparin tube is connect blood into 500ml blood collecting bottles by inserting needle → sterile working along bottle wall, 400ml/ bottles.Note：Blood collecting bottle is first with a small amount of raw
Brine wetting is managed, blood collection procedure cannot impact bubble, handle with care, and otherwise erythroclasis, which generates haemolysis, influences the matter of serum
Amount.
3. serum separates：Blood collecting bottle is put into 37 DEG C of insulating box 2h → 4 DEG C and places 2-4h → 3000rpm40min centrifugations one
Secondary → 4 DEG C of overnight → sterile workings are drawn supernatant yellow liquid → -20 DEG C and are frozen.
4. the filtration sterilization of serum：→ 0.22 μm of aperture filter column filtering of defrosting serum → 0.45 μm aperture filter column filtering →
0.1 μm of aperture filter column filters → -20 DEG C of packing and freezes.
Antibiotic：500000units/ml benzyl penicillins, 10% thaliium acetate, 2500units/ml polymyxins are prepared in advance
4 kinds of antibiotic of B, 2.5mg/ml anphotericin, each antibiotic are divided after preparing with after 0.22 μm of aperture membrane filtration degerming respectively
Dress preserves for use；Add above-mentioned 1ml benzyl penicillins during use respectively in 1L culture mediums, 1ml thaliium acetates, 1ml polymyxin Bs,
1ml anphotericins.
Other adding ingredients：The CMRL 1066 tissue cultures complementary element 5g of connected valleys glutamine, glucose 10g, yeast are cold
Powder 2g is soaked, pure water is added in and is settled to 236ml, it is now with the current with 0.22 μm of aperture membrane filtration degerming after mixing.
It is 7.5 to adjust final ph with 1M NaOH.
After mentioned component mixing, dispensed by 2ml/ pipes for use to sterile cillin bottle.
Using the prepared people's mycoplasma pneumoniae isolation medium SLB of the present embodiment, Yunnan Province's Yuxi are separately cultured
Virgin hospital and the throat swab sample of Kunming Traditional Chinese Medicine Hospital Hospitalized Children, specific cultural method are as follows：
First, throat swab is inoculated in the prepared people's mycoplasma pneumoniae isolation medium SLB of the present embodiment and cultivated, cultivated
Condition is 37 DEG C, 5%CO2 incubators.
2nd, step 1 sample is continued in 10% ratio blind passage to the present embodiment of percent by volume to be matched somebody with somebody after cultivating 6-24h
People's mycoplasma pneumoniae isolation medium SLB of system.
3rd, the culture of step 2 sample is observed after 3-10 days, with continuing after 0.45 μm of aperture membrane filtration if having precipitation
It is observed in 10% ratio blind passage of percent by volume to the prepared people's mycoplasma pneumoniae isolation medium SLB of the implementation case and frequently
Its color change；Culture medium color is observed if without precipitation, obtains preliminary judgement people's mycoplasma pneumoniae clinical separation strain result.
4th, continuation blind passage, inoculation solid are carried out to step 3 preliminary judgement behaviour mycoplasma pneumoniae positive separation strains sample
Medium culture 3-7 days, observation culture medium color change, colonial morphology and PCR identify 3 kinds of processing, obtain and finally judge separation knot
Fruit.
5th, the standard of preliminary judgement people's mycoplasma pneumoniae clinical separation strain result is：If fluid nutrient medium is apparent
It is yellow by red change and as clear as crystal without precipitation and floating material, it can tentatively judge to be separated to people's mycoplasma pneumoniae clinical separation strain；Institute
That states finally judges that the standard of separating resulting is：If fluid nutrient medium again sink by substantially yellow by red change and as clear as crystal nothing after blind passage
It forms sediment and floating material, it is positive to have " fried egg " sample colony growth and PCR on solid medium, is finally judged to being successfully separated
Everybody mycoplasma pneumoniae clinical separation strain.Wherein PCR uses people's mycoplasma pneumoniae custom primer：
Preceding primer 5'-GAAGCTTATGGTACAGGTTGG-3 '；
Primer 5'-ATTACCATCCTTGTTGTAAGG-3 ' afterwards；
Amplified fragments size is 144bp.The experimental result is shown in Table 1, Fig. 2, Fig. 3.
1 people's mycoplasma pneumoniae Clinical isolation --- throat swab sample results of table
The result shows that：It is separately cultured 2017 9 using 1 prepared people's mycoplasma pneumoniae isolation medium SLB of embodiment
Month Yuxi children's hospital of Yunnan Province and 196 parts of oropharyngeal swab specimens of Kunming Traditional Chinese Medicine Hospital Hospitalized Children are cultivated big after 6-24h
Part turns yellow, and is suspected to be varied bacteria growing, and it is substantially yellow and limpid by red change that 4 plants of fluid nutrient mediums are obtained after continuing blind passage culture 3-10 days
It is transparent without precipitation sample, by these samples apply ware after solid medium culture 3 days, it is seen that have " fried egg " sample colony growth,
The all positives of PCR the results shows, sequencing result show that separation strains are all people's M. hyopneunzoniae strain.Show the people's pneumonia branch
Substance isolation medium SLB inhibition miscellaneous bacteria effect is good, high specificity, and separation rate is high.But people mycoplasma pneumoniae clinical separation strain bacterium
It is substantially smaller than type strain to fall form, it may be possible to also completely not suitable due to separating and being transferred in a new culture medium from human body
Answer new growing environment.
Using the prepared people's mycoplasma pneumoniae isolation medium SLB of the present embodiment, Yunnan Province's Yuxi are separately cultured
Virgin hospital and the sputum sample sheet of Kunming Traditional Chinese Medicine Hospital Hospitalized Children, specific cultural method are as follows：
First, by sputum sample, this is former with 0.5ml is taken to be inoculated in the prepared people's pneumonia branch of the implementation case after phlegm diluted
It is cultivated in body isolation medium SLB, condition of culture is 37 DEG C, 5%CO2 incubators.
2nd, step 1 sample is continued in 10% ratio blind passage to the prepared people's lung of the implementation case after cultivating 18-24h
Scorching mycoplasma isolation medium SLB, the percentage are percent by volume.
3rd, the culture of step 2 sample is observed after 3-10 days, with continuing after 0.45 μm of aperture membrane filtration if having precipitation
Observe in 10% ratio blind passage to the prepared people's mycoplasma pneumoniae isolation medium SLB of the implementation case and frequently the change of its color
Change, the percentage is percent by volume；Culture medium color is observed if without precipitation, preliminary judgement people's mycoplasma pneumoniae is obtained and faces
Bed separation strains result.
4th, continuation blind passage, inoculation solid are carried out to step 3 preliminary judgement behaviour mycoplasma pneumoniae positive separation strains sample
Medium culture 3-7 days, observation culture medium color change, colonial morphology and PCR identify 3 kinds of processing, obtain and finally judge separation knot
Fruit.
5th, the standard of preliminary judgement people's mycoplasma pneumoniae clinical separation strain result is：If fluid nutrient medium is apparent
It is yellow by red change and as clear as crystal without precipitation and floating material, it can tentatively judge to be separated to people's mycoplasma pneumoniae clinical separation strain；Institute
That states finally judges that the standard of separating resulting is：If fluid nutrient medium again sink by substantially yellow by red change and as clear as crystal nothing after blind passage
It forms sediment and floating material, it is positive to have " fried egg " sample colony growth and PCR on solid medium, is finally judged to being successfully separated
People's mycoplasma pneumoniae clinical separation strain.Wherein PCR uses people's mycoplasma pneumoniae custom primer：
Preceding primer 5'-GAAGCTTATGGTACAGGTTGG-3 '；
Primer 5'-ATTACCATCCTTGTTGTAAGG-3 ' afterwards；
Amplified fragments size is 144bp.The experimental result is shown in Table 2, Fig. 2, Fig. 3.
2 people's mycoplasma pneumoniae Clinical isolation --- phlegm sample results of table
The result shows that：It is separately cultured 2017 9 using 1 prepared people's mycoplasma pneumoniae isolation medium SLB of embodiment
Month Yuxi children's hospital of Yunnan Province and 233 parts of sputum sample sheets of Kunming Traditional Chinese Medicine Hospital Hospitalized Children, culture obtain 8 after 3-10 days
Strain fluid nutrient medium is substantially yellow by red change and as clear as crystal without precipitation sample, these samples are applied ware in solid medium culture 3-
7 days, it is seen that have " fried egg " sample colony growth, all positives of PCR the results shows, sequencing result shows that separation strains are all people
M. hyopneunzoniae strain.Show that the people's mycoplasma pneumoniae isolation medium SLB inhibition miscellaneous bacteria effect is good, high specificity, separation rate
It is high.Find that this separation rate of sputum sample is higher simultaneously, it may be possible to people's mycoplasma pneumoniae content is high compared with throat swab in sputum sample or sampling amount compared with
Greatly, but people's mycoplasma pneumoniae clinical separation strain colonial morphology is substantially smaller than type strain, it may be possible to due to separating and shifting from human body
In the culture medium new to one, new growing environment is not adapted to completely also.
Ware solid medium culture 72h is applied to separating sample and observes " fried egg " sample bacterium colony, as a result institute as shown in Figure 1, Figure 2
Show.
Claims (3)
1. a kind of people's mycoplasma pneumoniae isolation medium, it is characterised in that name as people mycoplasma pneumoniae isolation medium SLB, bag
Including basal medium, horse serum, antibiotic and other adding ingredients, component is respectively：
First, basal medium 560ml：Mycoplasma broth bouillon 2.5-5g, bacto peptone 4.5-6g；Bacto-tryptone
8-12g, yeast extract 4-6g, 0.2% phenol red 10ml, add in pure water be settled to 560ml, with 1M NaOH adjust pH value to
115 DEG C of high pressure sterilizations 30 minutes after 7.5-7.7；
2nd, horse serum：50-200ml, 56 DEG C of inactivation 30min；
3rd, antibiotic 4ml：Adding in 1ml benzyl penicillins makes its final concentration of 250-600units/ml, and 1ml thaliium acetates make it dense eventually
It spends for 0.1mg/ml, 1ml polymyxin Bs make its final concentration of 2-3.5units/ml, and 1ml anphotericins make its final concentration of 2-
3.5μg/ml；
4th, other adding ingredient 386-236ml：1066 tissue cultures 3~5.5g of complementary element of CMRL containing glutamine, Portugal
Grape 1~20g of sugar, yeast cold soaking 1.1~2.5g of powder, it is 386-236ml to add in pure water constant volume, is removed with 0.22 μm of aperture membrane filtration
Bacterium；The horse serum of addition and other adding ingredient total volumes are 436ml；
5th, it is 7.4-7.6 to adjust final ph with 1M NaOH.
2. people's mycoplasma pneumoniae isolation medium as described in claim 1, it is characterised in that the horse serum is self-control horse
Serum, preparation method are as follows：
First, the selection of healthy horses：Selection 5-8 Sui age, without wound, do not injected antibiotic health male horses；
2nd, take a blood sample：In order carry out horses neck surface disinfection → shaving → sterilize again → removing vein blood vessel → sterilize into
Heparin tube is connect blood into 500ml blood collecting bottles by pin → sterile working along bottle wall, per bottled 400ml；
3rd, serum separates：By blood collecting bottle be put into 37 DEG C of insulating box 1.5-2.5h → 4 DEG C place 2-4h → 3000rpm 40min from
Once → 4 DEG C overnight → sterile working absorption supernatant yellow liquid → -20 DEG C freeze the heart；
4th, the filtration sterilization of serum：Defrosting serum → 0.45 μm aperture filter column filters → 0.22 μm of aperture filter column and filters → 0.1 μm
Aperture filter column filters → -20 DEG C and freezes.
3. people's mycoplasma pneumoniae isolation medium as described in claim 1, it is characterised in that people's mycoplasma pneumoniae point
Isolated culture method from culture medium SLB comprises the following steps：
First, clinical sample is inoculated in the fluid nutrient medium and cultivated, condition of culture is 37 DEG C, 5%CO2Incubator；
2nd, step 1 sample is continued in 10% ratio blind passage of percent by volume to the culture medium after cultivating 6-24h；
3rd, the culture of step 2 sample is observed after 3-10 days, with continuing after 0.45 μm of aperture membrane filtration by body if having precipitation
Product 10% ratio blind passage of percentage observes to the culture medium and frequently its color change；Culture medium color is observed if without precipitation；
Preliminary judgement people's mycoplasma pneumoniae clinical separation strain result；
4th, continuation blind passage, the culture of inoculation solid medium 3-7 days is carried out to step 3 initial gross separation strain sample, observes culture medium
Color change, colonial morphology and PCR identify 3 kinds of processing, obtain and finally judge separating resulting.
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| CN111424100A (en) * | 2020-02-25 | 2020-07-17 | 宁波明舟生物科技有限公司 | Detection method of legionella pneumoniae, neisseria meningitidis and mycoplasma pneumoniae |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424100A (en) * | 2020-02-25 | 2020-07-17 | 宁波明舟生物科技有限公司 | Detection method of legionella pneumoniae, neisseria meningitidis and mycoplasma pneumoniae |
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