CN109355226A - Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured - Google Patents
Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured Download PDFInfo
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- CN109355226A CN109355226A CN201811377530.2A CN201811377530A CN109355226A CN 109355226 A CN109355226 A CN 109355226A CN 201811377530 A CN201811377530 A CN 201811377530A CN 109355226 A CN109355226 A CN 109355226A
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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
Abstract
The present invention relates to a kind of Zengjing Granules for Legionella and the biphasic culture being separately cultured, including solid slope culture medium and fluid nutrient medium, the component of solid slope culture medium includes: N-2- acetamido -2- aminoethane sulphonic acid, agar, potassium hydroxide, active carbon, yeast extract, α-ketoglutaric acid list first salt, and the component of the fluid nutrient medium includes: N-2- acetamido -2- aminoethane sulphonic acid, 0.1~5g of potassium hydroxide, yeast extract, α-ketoglutaric acid list first salt, L-cysteine, ferric pyrophosphate, poly- anetholesulfonic acid sodium.The invention further relates to a kind of preparation methods of biphasic culture.Using the Zengjing Granule for Legionella of the invention and the biphasic culture and preparation method thereof being separately cultured, it can promote the quick Zengjing Granule of Legionella, being separately cultured for Legionella is realized simultaneously, it is simple and efficient to handle, it is significant to shorten the required incubation time of Legionella detection, increase substantially culture positive rate.
Description
Technical field
The present invention relates to legion's bacterium inspection technology field, in particular to biphasic culture, in particular to one kind is used for legion
The Zengjing Granule of bacterium and the biphasic culture and preparation method thereof being separately cultured.
Background technique
Legionella is the new pathogen confirmed for the first time for 1977, is distributed widely in nature, especially at various water sources and
Air conditioner water, in the artificial water sources such as piped water supply system.Legion Cordycepps (Legionellaceae) only has a category --- Legionella
Belong to (Legionella), there is now 41 kinds, 61 serotypes, most important of which is that legionella pneumophilia, this bacterium is mainly drawn
Play pneumonia, once morbidity or it is popular and without properly treatment in time, respiratory failure can be caused dead, case fatality rate is very high.Therefore, I
State attaches great importance to the preventing and controlling of légionaires' disease.
Legionella is gram-Negative bacillus, facultative aerobic, no gemma, no folder steamed bun, thallus size is 0.3~0.9 × 2~
20 microns, there is one to need cysteine and iron to several poles or secondary pole flagellum, the growth of this bacterium, feed energy using amino acid group
With the source of carbon, i.e. azymic does not also aoxidize carbohydrate, does not restore nitrate, oxidase test feminine gender or weakly positive, catalase test
The positive, urease-negative.The optimal pH of this bacterium growth is 6.9-7.0.2%-5%CO2 can promote the growth of part bacterial strain.Army
Group's bacterium slow growth, is easy to be covered by other bacterium colonies.The colony colour multiplicity of Legionella, usual white, grey, blue or purple
Color can also show dark brown, celadon, peony.
Légionaires' disease is difficult to diagnose by clinical manifestation and various clinical examinations, and clinic mainly passes through laboratory cause of disease
Check that isolated Legionella is made a definite diagnosis.Meanwhile because nutritional condition needed for its growth is harsh, a variety of necessary amino acid and certain are such as needed
A little metal ions, containing 2.5~5.0% CO2 environment in can grow out, affect Legionella detection work it is smooth into
Row.
In recent years, the rapid detection method of domestic common légionaires' disease had: one, immunofluorescence direct method, radiation are exempted from
Epidemic disease measurement, latex agglutination, gene probe, polymerase chain reaction,PCR molecular biology method, these methods because specificity, sensibility,
Price factor etc., and require operator to have certain experience and there are associated assay devices, use scope is by a fixed limit at present
System.Two, culture medium bacterium is separately cultured detection method: cyanobacteria culture medium, ferric nitrate culture medium, BCYE culture medium, GVPC culture medium
Deng, wherein BCYE culture medium be culture the most frequently used culture medium of Legionella.Bacterial cultivation is easy to operate, specificity and sensibility
By force, occupy is that the most reliable method of generally acknowledged this disease of diagnosis and ISO are specified first of each laboratory diagnostic methods of légionaires' disease
The Legionella method of inspection is the goldstandard of légionaires' disease diagnosis.But common culture of the BCYE culture medium as Legionella culture
Base can only be used to the plate isolation of Legionella, can not achieve the Zengjing Granule to sample, cause positive rate relatively low,
With certain limitation.Zhu Qingyi etc. has invented a kind of " legionella transport and enrichment medium " (CN101921719), specifically
It is to disclose a kind of legionella transport and enrichment medium, is used by antibiotic combinations, improves culture medium to non-Legionella
Rejection ability, so that improve Legionella is separately cultured positive rate.But this method is only used for the transport Zengjing Granule of sample,
It can not achieve the dual purpose for increasing bacterium and being separately cultured, it is still necessary to realize separation with the use of BCYE culture medium or GVPC culture medium
Purpose is cultivated, Check-Out Time is longer, complex for operation step.Having not yet to see patent report can be achieved at the same time Zengjing Granule and separation
Cultivate dual purpose legion bacterium culture medium.
Summary of the invention
The purpose of the invention is to overcome above-mentioned missing in the prior art, a kind of increasing bacterium training for Legionella is provided
Feeding and biphasic culture being separately cultured and preparation method thereof.
To achieve the goals above, one aspect of the present invention provides a kind of Zengjing Granule for Legionella and is separately cultured
Biphasic culture, wherein the biphasic culture includes solid slope culture medium and fluid nutrient medium, the solid is oblique
The component of face culture medium include: N-2- acetamido -2- aminoethane sulphonic acid, agar, potassium hydroxide, active carbon, yeast extract,
α-ketoglutaric acid list first salt, the component of the fluid nutrient medium include: N-2- acetamido -2- aminoethane sulphonic acid, hydrogen-oxygen
Change 0.1~5g of potassium, yeast extract, α-ketoglutaric acid list first salt, L-cysteine, ferric pyrophosphate, poly- anetholesulfonic acid sodium.
Preferably, the solid slope culture medium includes the component of following weight proportion: N-2- acetamido -2- amino
1~10g of ethane sulfonic acid, 0.1~5g of potassium hydroxide, 0.1~5g of active carbon, 0.5~5g of yeast extract, agar 5-10g, α -one penta
Diacid list first 0.1~5g of salt, purified water 350mL；
The fluid nutrient medium includes the component of following weight proportion: N-2- acetamido -2- aminoethane sulphonic acid 1~
10g, 0.1~5g of potassium hydroxide, 0.5~5g of yeast extract, α-ketoglutaric acid list first 0.5~5g of salt, L-cysteine hydrochloride
0.2~5g, 0.1~5g of ferric pyrophosphate, poly- anetholesulfonic acid sodium 0.1~10g, purified water 600mL.
Preferably, the solid slope culture medium includes the component of following weight proportion: with the calculating of 350mL volume, N-
2- acetamido -2- aminoethane sulphonic acid 3.5g, potassium hydroxide 0.95g, active carbon 2g, yeast extract 3.5g, agar 7.0g,
α-ketoglutaric acid list first salt 0.5g；
The fluid nutrient medium includes the component of following weight proportion: with the calculating of 600mL volume, N-2- acetamido-
2- aminoethane sulphonic acid 6.0g, potassium hydroxide 1.5g, yeast extract 6.2g, α-ketoglutaric acid list first salt 0.6g, poly- anethole sulphur
Sour sodium 0.18g, L-cysteine 0.4g, ferric pyrophosphate 0.16g.
Another aspect of the present invention provides the Zengjing Granule described in a kind of be used to prepare for Legionella and is separately cultured
Biphasic culture method, the method comprising steps of
(1) raw material needed for weighing solid slope culture medium and fluid nutrient medium respectively；
(2) raw material of solid slope culture medium is mixed, and dispenses the high pressure sterilization into the first culture bottle；
(3) it is formed in the first culture bottle bottom and is frozen into inclined-plane, the solid slope culture medium is made；
(4) raw material of liquid slant medium is mixed, and dispensed into the second culture bottle, the Liquid Culture is made
Base.
Preferably, the step (2) specifically includes: 350mL purified water being added into solid slope culture medium raw material, fills
Point mix and boil dissolution, adjustment pH is 6.9~7.0, packing into the first culture bottle, 121 DEG C high pressure sterilization 15 minutes；
The step (4) specifically includes: ACES being added in the flask equipped with 600mL distilled water, in 50 DEG C of water-baths
Be heated to dissolve, be added potassium hydroxide, yeast extract, SPS and α-ketoglutaric acid list first salt, 121 DEG C high pressure sterilization 15 minutes,
L-cysteine hydrochloride and ferric pyrophosphate after filtration sterilization is added, adjustment pH are 6.9~7.0.
Using the Zengjing Granule for Legionella of the invention and the biphasic culture and preparation method thereof being separately cultured, energy
Enough promote the quick Zengjing Granule of Legionella, while realizing being separately cultured for Legionella, it is simple and efficient to handle, significantly shorten legion
Bacterial examination surveys required incubation time, increases substantially culture positive rate.
Detailed description of the invention
Fig. 1 is the Zengjing Granule and the process for the biphasic culture preparation method being separately cultured for Legionella of the invention
Schematic diagram.
Specific embodiment
In order to be more clearly understood that technology contents of the invention, specific implementation method of the invention is made into one below
Walk explanation.
The present invention provides the embodiments of a kind of two-phase BCYE- α agar and liquid-based culture medium and preparation method thereof, i.e., a kind of
Containing solid and Legionella culture full of nutrition two-phase BCYE- α agar and liquid-based culture medium containing liquid.The culture medium is simultaneously
Containing solid and liquid two parts, sufficient nutriment can be provided for the fast-growth of Legionella, reached and shortened the culture medium time, mention
The purpose of early report testing result.Meanwhile bacterium can form bacterium colony in solid culture primary surface when growing in the culture medium, because
This is made whether the Preliminary Identification for Legionella or non-Legionella to the bacterium of growth, realizes and increase bacterium by observing colony characteristics
With the dual purpose being separately cultured.
Wherein, solid slope culture medium is made of following weight proportion:
It include: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 1~10g, 0.1~5g of potassium hydroxide, active carbon
0.1~5g, 0.5~5g of yeast extract, agar 5-10, α-ketoglutaric acid (monopotassium salt) 0.1~5g, purified water 350mL.
Any of the above-described scheme, it is preferred that N-2- acetamido -2- aminoethane sulphonic acid (ACES) 3.5g, potassium hydroxide
0.95g, active carbon 2g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid (monopotassium salt) 0.5g (with the calculating of 350mL volume).
Fluid nutrient medium part includes:
N-2- acetamido -2- aminoethane sulphonic acid (ACES) 1~10g, 0.1~5g of potassium hydroxide, yeast extract 0.5~
5g, α-ketoglutaric acid (monopotassium salt) 0.5~5g, L-cysteine (hydrochloride) 0.2~5g, 0.1~5g of ferric pyrophosphate, poly- fennel
Brain sodium sulfonate (SPS) 0.1~10g, purified water 600mL.
Any of the above-described scheme, it is preferred that N-2- acetamido -2- aminoethane sulphonic acid (ACES) 6.0g, potassium hydroxide
1.5g, yeast extract 6.2g, α-ketoglutaric acid (monopotassium salt) 0.6g, poly- anetholesulfonic acid sodium (SPS) 0.18g is (with 600mL volume
It calculates).
Any of the above-described scheme, it is preferred that L-cysteine (hydrochloride) 0.4g, ferric pyrophosphate 0.16g, (with 600mL body
Product calculates).
The present invention provides a kind of embodiment of the preparation method of two-phase BCYE- α agar and liquid-based culture medium, including it is as follows
Specific steps:
(1) weighing of solid medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 1~10g, potassium hydroxide
0.1~5g, 0.1~5g of active carbon, 0.5~5g of yeast extract, agar 5-10, α-ketoglutaric acid (monopotassium salt) 0.1~5g are placed
In the container of clean dried.
(2) raw material mixes: 350mL purified water being added in the container in step (1), mixes well and boil dissolution, adjusts
Whole pH is 6.9~7.0.
(3) it dispenses: packing to 100mL Blood culture bottle, every bottle of 30mL, high pressure sterilization (121 DEG C, 15 minutes).
(4) inclined-plane processed: inclined-plane is allowed to be formed and be solidified in bottom of bottle.
(5) weighing of fluid nutrient medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 1~10g, potassium hydroxide
0.1~5g, 0.5~5g of yeast extract, α-ketoglutaric acid (monopotassium salt) 0.5~5g, L-cysteine (hydrochloride) 0.2~5g,
0.1~5g of ferric pyrophosphate, 0.1~10g of poly- anetholesulfonic acid sodium (SPS).
(6) raw material mixes: ACES being added to equipped with 600mL, in the flask of distilled water, it is small in 50 DEG C of water-baths to heat about half
When, or be heated to dissolve.
(7) addition potassium hydroxide, yeast extract, SPS and α-ketoglutaric acid (monopotassium salt), 121 DEG C of high pressure, 15 minutes.
(8) it by L-cysteine and ferric pyrophosphate filtration sterilization, and is added in the mixed liquor after high pressure sterilization.
(9) adjustment pH is 6.9~7.0.
(10) it dispenses: adding the above-mentioned culture medium of 50mL into 100mL Blood culture bottle.
(11) 35 DEG C preculture 24 hours, to see whether to pollute, then place 4 DEG C at.
(12) fluid nutrient medium is added in agar slant with sterile manner before use, in an amount of from about the half for not having inclined-plane,
36 DEG C, 3%CO2,3-7 days are put, and daily that liquid-based covering inclined-plane is primary.
Embodiment 1
A kind of two-phase BCYE- α agar and liquid-based culture medium, solid slope culture medium include: N-2- acetamido -2- amino
Ethane sulfonic acid (ACES) 3.5g, potassium hydroxide 0.95g, active carbon 2g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid are (single
Sylvite) 0.5g (with the calculating of 350mL volume).
Fluid nutrient medium includes: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 6.0g, potassium hydroxide 1.5g, ferment
Female medicinal extract 6.2g, α-ketoglutaric acid (monopotassium salt) 0.6g, poly- anetholesulfonic acid sodium (SPS) 0.18g, L-cysteine (hydrochloride)
0.4g, ferric pyrophosphate 0.16g (with the calculating of 600mL volume).
The preparation method of above-mentioned two-phase BCYE- α agar and liquid-based culture medium, includes the following steps:
(1) weighing of solid medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 3.5g, potassium hydroxide
0.95g, active carbon 2g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid (monopotassium salt) 0.5g are placed in clean dried
In container.
(2) raw material mixes: 350mL purified water being added in the container in step (1), mixes well and boil dissolution, adjusts
Whole pH (should be 6.9-7.0).
(3) it dispenses: packing to 100mL Blood culture bottle, every bottle of 30mL, high pressure sterilization (121 DEG C, 15 minutes).
(4) inclined-plane processed: inclined-plane is allowed to be formed and be solidified in bottom of bottle.
(5) weighing of fluid nutrient medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 6.0g, potassium hydroxide
1.5g, yeast extract 6.2g, α-ketoglutaric acid (monopotassium salt) 0.6g, poly- anetholesulfonic acid sodium (SPS) 0.18g, L-cysteine
(hydrochloride) 0.4g, ferric pyrophosphate 0.16g.
(6) raw material mixes: ACES being added to equipped with 600mL, in the flask of distilled water, it is small in 50 DEG C of water-baths to heat about half
When, or be heated to dissolve.
(7) addition potassium hydroxide, yeast extract, SPS and α-ketoglutaric acid (monopotassium salt), 121 DEG C of high pressure, 15 minutes.
(8) it by L-cysteine and ferric pyrophosphate filtration sterilization, and is added in the mixed liquor after high pressure sterilization.
(9) it adjusts pH: should be 6.9-7.0.
(10) it dispenses: adding the above-mentioned culture medium of 50mL into 100mL Blood culture bottle.
(11) 35 DEG C preculture 24 hours, to see whether to pollute, then place 4 DEG C at.
(12) fluid nutrient medium is added in agar slant with sterile manner before use, in an amount of from about the half for not having inclined-plane,
36 DEG C, 3%CO2,3-7 days are put, and daily that liquid-based covering inclined-plane is primary, whether there is or not bacterial growths for observation.
(13) inoculation Legionella LP1, LP6, BIZ sets 36 DEG C, 3%CO2Culture 3-7 days, and liquid-based is covered into inclined-plane daily
Once, Quality Control bacterium growing state is observed.
(14) in two-phase BCYE- α agar and liquid-based culture medium, Legionella LP1, LP6, BIZ well-grown, liquid-based part
Muddiness, solid slope thallus canescence, smooth wet, edge is complete.
Embodiment 2
A kind of two-phase BCYE- α agar and liquid-based culture medium, solid slope culture medium include: N-2- acetamido -2- amino
Ethane sulfonic acid (ACES) 3g, potassium hydroxide 2.5, active carbon 3g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid (single potassium
Salt) 1g (with the calculating of 350mL volume).
Fluid nutrient medium includes: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 5g, potassium hydroxide 1g, yeast leaching
Cream 5g, α-ketoglutaric acid (monopotassium salt) 0.6g, L-cysteine (hydrochloride) 0.4g, ferric pyrophosphate 1g, poly- anetholesulfonic acid sodium
(SPS) 3g (with the calculating of 600mL volume).
The preparation method of above-mentioned two-phase BCYE- α agar and liquid-based culture medium, includes the following steps:
(1) weighing of solid medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 3g, potassium hydroxide 2.5,
Active carbon 3g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid (monopotassium salt) 1g are placed in the container of clean dried.
(2) raw material mixes: 350mL purified water being added in the container in step (1), mixes well and boil dissolution, adjusts
Whole pH (should be 6.9-7.0).
(3) it dispenses: packing to 100mL Blood culture bottle, every bottle of 30mL, high pressure sterilization (121 DEG C, 15 minutes).
(4) inclined-plane processed: inclined-plane is allowed to be formed and be solidified in bottom of bottle.
(5) weighing of fluid nutrient medium: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 5g, potassium hydroxide 1g, ferment
Female medicinal extract 5g, α-ketoglutaric acid (monopotassium salt) 0.6g, L-cysteine (hydrochloride) 0.4g, ferric pyrophosphate 1g, poly- anethole sulphur
Sour sodium (SPS) 3g.
(6) raw material mixes: ACES being added to equipped with 600mL, in the flask of distilled water, it is small in 50 DEG C of water-baths to heat about half
When, or be heated to dissolve.
(7) addition potassium hydroxide, yeast extract, SPS and α-ketoglutaric acid (monopotassium salt), 121 DEG C of high pressure, 15 minutes.
(8) it by L-cysteine and ferric pyrophosphate filtration sterilization, and is added in the mixed liquor after high pressure sterilization.
(9) it adjusts pH: should be 6.9-7.0.
(10) it dispenses: adding the above-mentioned culture medium of 50mL into 100mL Blood culture bottle.
(11) 35 DEG C preculture 24 hours, to see whether to pollute, then place 4 DEG C at.
(12) fluid nutrient medium is added in agar slant with sterile manner before use, in an amount of from about the half for not having inclined-plane,
36 DEG C, 3%CO2,3-7 days are put, and daily that liquid-based covering inclined-plane is primary.
(13) inoculation Legionella LP7 and LP12 sets 36 DEG C, 3%CO2Culture 3-7 days, and liquid-based is covered into inclined-plane one daily
It is secondary, observe Quality Control bacterium growing state.
(14) in two-phase BCYE- α agar and liquid-based culture medium, Legionella LP7 and LP12 well-grown, liquid-based part is muddy
Turbid, solid slope thallus canescence, smooth wet, edge is complete.
Embodiment 3
A kind of two-phase BCYE- α agar and liquid-based culture medium, solid slope culture medium include: N-2- acetamido -2- amino
Ethane sulfonic acid (ACES) 10g, potassium hydroxide 2g, active carbon 5g, yeast extract 5g, agar 7.0g, α-ketoglutaric acid (monopotassium salt)
2g (with the calculating of 350mL volume).
Fluid nutrient medium includes: N-2- acetamido -2- aminoethane sulphonic acid (ACES) 6.0g, potassium hydroxide 1.47g, ferment
Female medicinal extract 0.5g, α-ketoglutaric acid (monopotassium salt) 5g, L-cysteine (hydrochloride) 4g, ferric pyrophosphate 2g, poly- anetholesulfonic acid
Sodium (SPS) 0.18g (with the calculating of 600mL volume).
Inoculation Legionella LP7 and LP12 set 36 DEG C, 3%CO2Culture 3-7 days, and it is daily that liquid-based covering inclined-plane is primary, it sees
Examine Quality Control bacterium growing state.As the result is shown in two-phase BCYE- α agar and liquid-based culture medium, Legionella LP7 and LP12 growth are good
Good, liquid-based part is muddy, solid slope thallus canescence, and smooth wet, edge is complete.
Biphasic culture provided by the invention can be used for the increasing bacterium of Legionella and be separately cultured, in the mirror for carrying out sample to be tested
Periodically, pass through following step:
(1) prepare sample to be tested: acquire the respiratory tracts powder objects such as the blood, phlegm, tracheae aspirated liquid of examinee and lung tissue,
Water sample sample 1-5mL is inoculated in two-phase BCYE- α agar solid slope and fluid nutrient medium.
(2) above-mentioned culture tube is set 36 DEG C, 3%CO2Culture 3-7 days, it is daily that fluid nutrient medium covering inclined-plane is primary.
(3) observation is as a result, the liquid of culture medium is muddy, bacterium colony canescence occurs in the surface of solids, and smooth wet, edge is complete
Whole bacterium colony, as bacterial growth are positive.
(4) it selects suspicious bacterium colony and makees further identification.
Using the Zengjing Granule for Legionella of the invention and the biphasic culture and preparation method thereof being separately cultured, energy
Enough promote the quick Zengjing Granule of Legionella, while realizing being separately cultured for Legionella, it is simple and efficient to handle, significantly shorten legion
Bacterial examination surveys required incubation time, increases substantially culture positive rate.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the invention.Therefore, the description and the appended drawings should be considered as illustrative
And not restrictive.
Claims (5)
1. a kind of Zengjing Granule for Legionella and the biphasic culture being separately cultured, which is characterized in that the two-phase training
Feeding base includes solid slope culture medium and fluid nutrient medium, and the component of the solid slope culture medium includes: N-2- acetamide
Base -2- aminoethane sulphonic acid, agar, potassium hydroxide, active carbon, yeast extract, α-ketoglutaric acid list first salt, the liquid training
The component for supporting base includes: N-2- acetamido -2- aminoethane sulphonic acid, 0.1~5g of potassium hydroxide, yeast extract, α -one penta 2
Sour list first salt, L-cysteine, ferric pyrophosphate, poly- anetholesulfonic acid sodium.
2. the Zengjing Granule according to claim 1 for Legionella and the biphasic culture being separately cultured, feature exist
In the solid slope culture medium includes the component of following weight proportion: N-2- acetamido -2- aminoethane sulphonic acid 1~
10g, 0.1~5g of potassium hydroxide, 0.1~5g of active carbon, 0.5~5g of yeast extract, agar 5-10g, α-ketoglutaric acid list first salt
0.1~5g, purified water 350mL；
The fluid nutrient medium includes the component of following weight proportion: N-2- acetamido -2- 1~10g of aminoethane sulphonic acid,
0.1~5g of potassium hydroxide, 0.5~5g of yeast extract, α-ketoglutaric acid list first 0.5~5g of salt, L-cysteine hydrochloride 0.2~
5g, 0.1~5g of ferric pyrophosphate, poly- anetholesulfonic acid sodium 0.1~10g, purified water 600mL.
3. the Zengjing Granule according to claim 1 or 2 for Legionella and the biphasic culture being separately cultured, feature
It is, the solid slope culture medium includes the component of following weight proportion: with the calculating of 350mL volume, N-2- acetamido-
2- aminoethane sulphonic acid 3.5g, potassium hydroxide 0.95g, active carbon 2g, yeast extract 3.5g, agar 7.0g, α-ketoglutaric acid list
First salt 0.5g；
The fluid nutrient medium includes the component of following weight proportion: with the calculating of 600mL volume, N-2- acetamido -2- ammonia
Base ethane sulfonic acid 6.0g, potassium hydroxide 1.5g, yeast extract 6.2g, α-ketoglutaric acid list first salt 0.6g, poly- anetholesulfonic acid sodium
0.18g, L-cysteine 0.4g, ferric pyrophosphate 0.16g.
4. a kind of biphasic culture for being used to prepare the Zengjing Granule described in claim 1 for Legionella and being separately cultured
Method, which is characterized in that the method comprising steps of
(1), raw material needed for weighing solid slope culture medium and fluid nutrient medium respectively；
(2), the raw material of solid slope culture medium is mixed, and dispenses the high pressure sterilization into the first culture bottle；
(3), it is formed in the first culture bottle bottom and is frozen into inclined-plane, the solid slope culture medium is made；
(4), the raw material of liquid slant medium is mixed, and dispensed into the second culture bottle, the fluid nutrient medium is made.
5. the two-phase according to claim 4 for being used to prepare the described Zengjing Granule for Legionella and being separately cultured is trained
The method for supporting base, which is characterized in that the step (2) specifically includes: it is pure that 350mL is added into solid slope culture medium raw material
Change water, mix well and boil dissolution, adjustment pH is 6.9~7.0, is dispensed into the first culture bottle, 121 DEG C of high pressure sterilizations 15 divide
Clock；
The step (4) specifically includes: ACES being added in the flask equipped with 600mL distilled water, is heated in 50 DEG C of water-baths
To dissolution, be added potassium hydroxide, yeast extract, SPS and α-ketoglutaric acid list first salt, 121 DEG C high pressure sterilization 15 minutes, be added
L-cysteine hydrochloride and ferric pyrophosphate after filtration sterilization, adjustment pH are 6.9~7.0.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811377530.2A CN109355226A (en) | 2018-11-19 | 2018-11-19 | Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured |
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| CN201811377530.2A CN109355226A (en) | 2018-11-19 | 2018-11-19 | Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured |
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| CN201811377530.2A Pending CN109355226A (en) | 2018-11-19 | 2018-11-19 | Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured |
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