CN109810931A - A kind of culture medium and its application method for cultivating thermophilic mucin Ackermam Salmonella - Google Patents
A kind of culture medium and its application method for cultivating thermophilic mucin Ackermam Salmonella Download PDFInfo
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Abstract
The present invention provides a kind of culture mediums and its application method for cultivating thermophilic mucin Ackermam Salmonella, belong to microbiological culture media technical field.The component of the culture medium includes soy peptone, threonine, glucose, N-acetyl-glucosamine, sodium chloride and disodium hydrogen phosphate.Bacterium amount needed for obtaining thermophilic mucin Ackermam Salmonella functional study can be cultivated using culture medium of the invention, potential bio-hazard is not present in the Component Source safety of culture medium.
Description
Technical field
The invention belongs to microbiological culture media technical field more particularly to a kind of trainings for cultivating thermophilic mucin Ackermam Salmonella
Support base and its application method.
Background technique
Fat and its relevant metabolic disease such as diabetes and cardiovascular disease cause great prestige to public health
The side of body.The situation in China is especially severe, existing more than 100,000,000 diabetics, while having nearly 500,000,000 crowd to be in prediabetes.Therefore,
The measure that research prevents and treats this kind of disease is particularly important.
Intestinal flora is closely related with human health, and fat and insulin resistance is that two of metabolic disease early stage are important
Feature, intestinal flora play an important role in the generation and progress of metabolic disease.The change of intestinal flora, inflammation and enteron aisle screen
It is the fat important feature with diabetes B that barrier function, which is destroyed,.It is existing to study the category being concentrated mainly on taxology, section even
Door is horizontal, and the research for being accurate to kind of level and strain level is less.Study various bacterial strains and metabolic disease in human body intestinal canal
Relationship, it can be found that there is the biomarker of prediction and diagnostic value, and provide novel targets to carry out effective prevention.It is existing
Studies have shown that commensal gut bacterium Akkermansia muciniphila may be exactly a kind of such function bacterium.
Thermophilic mucin Ackermam Salmonella Akkermansia muciniphila is that the first in current wart germ door is separated
Intestinal Anaerobic Bacteria, separated successfully in 2004.Current research discovery, the Akkermansia that Akkermansia belongs to
Muciniphila (hereinafter referred to as Akk) may be one of the important symbol microorganism measuring intestinal microecology and whether balancing.Akk
To be widely present in Healthy People enteron aisle with the function bacterium for adjusting the characteristics such as glycaemic homeostasis.It has the spy of degradation mucin
Property, it is to live in one of host's intestinal mucosa mucus surface important microbe.
Because of the effect of these metabolism improvings possessed by Akk, many scholars are referred to as next-generation probiotics.It considers
Akk as a kind of candidate new probiotics, with its microorganism formulation as main component must have vast development prospect and
Market is promoted, or even the external experimental study for having had progress Akk human experiment at present.But it is applied in the market for realizing Akk
Before, it needs to solve there are many more problem.
The optimization of Akk synthetic media is exactly such a required and vital content.At present both at home and abroad for
The culture of Akk all relies on greatly the culture medium being formulated by bovine brain heart infusion agar dry powder and pig stomach mucin.Although culture
Effect it is fine, but higher cost, and brain heart infusion agar dry powder and mucin both are from animal, may be carried not
The virus known and other hazard components, the safety of the medium component in this source are unable to get abundant guarantee.
There is document report (Plovier H, Everard A, Druart C, et al.A purified membrane
protein from Akkermansia muciniphila or the pasteurized bacterium improves
Metabolism in obese and diabetic mice. [J] .Nature Medicine, 2016,23 (1): 107.)
Akk synthetic media ingredient includes soy peptone, threonine, glucose and N-acetyl-glucosamine.But it is reported with the document
Culture medium carry out Akk amplification cultivation, do not succeed, can not cultivate to obtain Akk bacterium.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of culture medium for cultivating thermophilic mucin Ackermam Salmonella and its making
With method, can cultivate to obtain thermophilic mucin Ackermam Salmonella, and the bacterium that culture obtains using culture medium provided by the invention
Amount can satisfy the required of functional study.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a kind of culture medium for cultivating thermophilic mucin Ackermam Salmonella, the component of the culture medium includes big
Legumin peptone, threonine, glucose, N-acetyl-glucosamine, sodium chloride and disodium hydrogen phosphate.
Preferably, the culture medium takes water as a solvent, and every liter includes: 20~45mmol glucose, 12~32g soybean protein
Peptone, 3~8g threonine, 20~35mmolN- acetylglucosamine, 4~6g sodium chloride and 2~3.5g disodium hydrogen phosphate.
Preferably, the culture medium takes water as a solvent, and every liter includes: 25~40mmol glucose, 16~28g soybean protein
Peptone, 4~7g threonine, 25~30mmol N-acetyl-glucosamine, 4.5~5.5g sodium chloride and 2.5~3g disodium hydrogen phosphate.
Preferably, the culture medium takes water as a solvent, and every liter includes: 30~35mmol glucose, 20~24g soybean protein
Peptone, 5~6g threonine, 35mmol N-acetyl-glucosamine, 5g sodium chloride and 2.6~2.8g disodium hydrogen phosphate.
The present invention also provides the application methods of the culture medium described in above-mentioned technical proposal, comprising: by the thermophilic mucin
The bacterium solution of Ackermam Salmonella is inoculated in culture medium described in above-mentioned technical proposal, carries out Anaerobic culturel.
Preferably, the instrument that the Anaerobic culturel uses includes anaerobic box, includes 85%CO in the anaerobic box2, 10%N2
And 5%H2。
Preferably, the content of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in the bacterium solution8cfu/mL。
Preferably, the inoculum concentration of the bacterium solution is 100 μ L/200 μ L.
Preferably, the temperature of the Anaerobic culturel is 35~40 DEG C.
Preferably, the time of the Anaerobic culturel is 3~4d.
The present invention provides a kind of culture medium for cultivating thermophilic mucin Ackermam Salmonella, the component of the culture medium includes big
Legumin peptone, threonine, glucose, N-acetyl-glucosamine, sodium chloride and disodium hydrogen phosphate.Contain in culture medium in the prior art
There are soy peptone, threonine, glucose and N-acetyl-glucosamine, can not cultivate to obtain thermophilic mucin Ackermam Salmonella, this
Invention is added to sodium chloride and disodium hydrogen phosphate on the basis of existing, solves and lacks buffer substance in existing culture medium and cause
The drawbacks of thermophilic mucin Ackermam Salmonella can not grow can make thermophilic mucin Acker after adding sodium chloride and disodium hydrogen phosphate
Mans bacterium normal growth, can obtain carrying out bacterium amount required when the functional study of the bacterium.
Detailed description of the invention
Fig. 1 is form result under the mirror of thermophilic mucin Ackermam Salmonella；
Fig. 2 is the colonial morphology and purity result of thermophilic mucin Ackermam Salmonella；
Fig. 3 is imaging results of the amplified production of thermophilic mucin Ackermam Salmonella after agarose gel electrophoresis；
Fig. 4 be isometric mucin culture medium and culture medium provided by the invention after being inoculated with equivalent Akk in identical ring
It after cultivating same time in border, takes out and is centrifuged obtained bacterial precipitation figure, wherein the centrifuge tube on the left side is mucin culture medium
Cultivate obtaining as a result, the centrifuge tube on the right is the result that culture medium culture of the invention obtains；
Fig. 5 be isometric mucin culture medium and culture medium provided by the invention after being inoculated with equivalent Akk in identical ring
It after cultivating same time in border, is resuspended, viable bacteria dyeing is carried out to bacterium solution and counted using isometric sterile PBS after being centrifuged
Figure, wherein upper left be the culture medium of the present invention without inoculation be used as negative control, upper right for Akk in the medium of the present invention
The viable bacteria dyeing carried out after culture, lower-left are the mucin culture medium without inoculation as negative control, and bottom right is Akk in glutinous egg
The viable bacteria dyeing carried out after being cultivated in white culture medium.
Specific embodiment
The present invention provides a kind of culture medium for cultivating thermophilic mucin Ackermam Salmonella, the component of the culture medium includes big
Legumin peptone, threonine, glucose, N-acetyl-glucosamine, sodium chloride and disodium hydrogen phosphate.In the present invention, the culture medium
Take water as a solvent, every liter preferably includes: 20~45mmol glucose, 12~32g soy peptone, 3~8g threonine, 20~
35mmolN- acetylglucosamine, 4~6g sodium chloride and 2~3.5g disodium hydrogen phosphate；It include: optionally 25~40mmol grape
Sugar, 16~28g soy peptone, 4~7g threonine, 25~30mmol N-acetyl-glucosamine, 4.5~5.5g sodium chloride and 2.5
~3g disodium hydrogen phosphate；Optionally include: 30~35mmol glucose, 20~24g soy peptone, 5~6g threonine,
35mmolN- acetylglucosamine, 5g sodium chloride and 2.6~2.8g disodium hydrogen phosphate.In the present invention, the sodium chloride and phosphoric acid
Buffer substance of the disodium hydrogen as culture medium solves and is containing soy peptone, threonine, glucose and N-acetyl-glucosamine
Culture medium in the drawbacks of cannot accessing thermophilic mucin Ackermam Salmonella, the bacterium can be obtained.In the present invention, when described every
When rising the concentration of the sodium chloride in culture medium higher than 6g, it is unfavorable for the growth of thermophilic mucin Ackermam Salmonella.
The present invention also provides the application methods of the culture medium described in above-mentioned technical proposal, comprising: by the thermophilic mucin
The bacterium solution of Ackermam Salmonella is inoculated in culture medium described in above-mentioned technical proposal, carries out Anaerobic culturel.
In the present invention, the Anaerobic culturel is preferably in 85%CO2, 10%N2And 5%H2Lower progress, the Anaerobic culturel
The instrument used is preferably anaerobic box.In the present invention, the temperature of the Anaerobic culturel is preferably 35~40 DEG C, the anaerobism training
The feeding time is preferably 3~4d.
In the present invention, the inoculum concentration of the bacterium solution of the thermophilic mucin Ackermam Salmonella is preferably 100 μ L/200 μ L, described
The content of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8cfu/mL.The present invention is to the thermophilic mucin Ackermam Salmonella
The source of bacterium solution be not particularly limited, be prepared using customary preparation methods.Method of the present invention to the inoculation
It is not particularly limited with the tool used, using routine.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.72g glucose, 2.4g soy peptone,
0.6g threonine, 0.88gN- acetylglucosamine, 0.9g sodium chloride and 0.4g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0183。
It after bacterium solution is inoculated in mucin fluid nutrient medium, cultivates 2~3 days, takes out centrifugation, precipitating is carried out to remove from office blue dye
Color, microscopy observe conservation bacterium mirror under form (result such as Fig. 1).Meanwhile viable bacteria precipitating sectional streak is inoculated in mucin and is put down
Plate, culture are taken out after 2 days, observe the colonial morphology and purity (result such as Fig. 2) of conservation bacterium.Finally, being used using bacterium solution as template
Thermophilic mucin Ackermam Salmonella primer carries out PCR amplification, carries out agarose gel electrophoresis to amplified production, passes through full automatic gel
Imaging system judges the molecular weight (result such as Fig. 3) of primary template DNA.
As can be drawn from Figure 1, the bacterium in bacterium solution is Grain-negative rod-short, meets the mirror of thermophilic mucin Ackermam Salmonella
Lower Morphological Features, and under mirror and have no other obvious miscellaneous bacterias, it was demonstrated that the bacterium in bacterium solution is pure bacterium.As can be drawn from Figure 2, bacterium
Bacterium colony in liquid is tiny, is white clear shape, meets the colonial morphology feature of thermophilic mucin Ackermam Salmonella, and on plate simultaneously
Without other varied bacteria growings, it was demonstrated that the bacterium in bacterium solution is pure bacterium.As can be drawn from Figure 3, bacterium solution carries out PCR amplification as template
Afterwards, DNA molecular amount size is between 250~500bp, position partially under, slightly close to 250bp line, meet passing report,
I.e. thermophilic mucin Ackermam Salmonella DNA molecular amount after same primer amplification is 327bp.To sum up, it can be derived that in bacterium solution
Bacterium is pure thermophilic mucin Ackermam Salmonella.
Cysteine is a kind of reducing agent, can consume the oxygen in culture medium, is conducive to thermophilic mucin Ackermam Salmonella
Anaerobic growth.In following embodiment and comparative example, the effect of cysteine is same as Example 1.
Embodiment 2
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.72g glucose, 3.2g soy peptone,
0.8g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0343。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 3
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 0.72g glucose, 4g soy peptone, 1g
Threonine, 1.328gN- acetylglucosamine, 1.1g sodium chloride and 0.6g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0423。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 4
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.72g glucose, 4.8g soy peptone,
1.2g threonine, 1.548g N-acetyl-glucosamine, 1.2g sodium chloride and 0.7g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.063。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 5
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.9g glucose, 2.4g soy peptone,
0.6g threonine, 1.056g N-acetyl-glucosamine, 1.1g sodium chloride and 0.7g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0143。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 6
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.9g glucose, 3.2g soy peptone,
0.8g threonine, 0.884g N-acetyl-glucosamine, 1.2g sodium chloride and 0.6g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0253。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 7
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 0.9g glucose, 4g soy peptone, 1g
Threonine, 1.548gN- acetylglucosamine, 0.9g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is 0.027.
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 8
Take the conical flask of 250ml specification, after 200mL pure water is added, add 0.9g glucose, 4.8g soy peptone,
1.2g threonine, 1.328gN- acetylglucosamine, 1g sodium chloride and 0.4g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0513。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 9
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 2.4g soy peptone,
0.6g threonine, 1.328g N-acetyl-glucosamine, 1.2g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0087。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 10
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 3.2g soy peptone,
0.8g threonine, 0.884g N-acetyl-glucosamine, 1.1g sodium chloride and 0.4g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.035。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 11
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 4g soy peptone,
1.25g threonine, 1.548g N-acetyl-glucosamine, 1g sodium chloride and 0.7g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0687。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 12
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 4.8g soy peptone,
1.2g threonine, 1.056g N-acetyl-glucosamine, 0.9g sodium chloride and 0.6g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.087。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 13
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 2.4g soy peptone,
0.6g threonine, 1.548gN- acetylglucosamine, 1g sodium chloride and 0.6g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0093。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 14
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 3.2g soy peptone,
0.8g threonine, 1.328g N-acetyl-glucosamine, 0.9g sodium chloride and 0.7g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0187。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 15
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 1.26g glucose, 4g soy peptone, 1g
Threonine, 1.056gN- acetylglucosamine, 1.2g sodium chloride and 0.4g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.0043。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 16
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 4.8g soy peptone,
1.2g threonine, 0.884g N-acetyl-glucosamine, 1.1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.097。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 17
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 1.08g glucose, 4g soy peptone, 1g
Threonine, 1.056gN- acetylglucosamine, 0.8g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1083。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 18
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 4.8g soy peptone,
1.2g threonine, 1.056g N-acetyl-glucosamine, 0.9g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1193。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 19
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 5.6g soy peptone,
1.4g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.137。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 20
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.08g glucose, 6.4g soy peptone,
1.6g threonine, 1.056g N-acetyl-glucosamine, 1.1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1427。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 21
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 1.26g glucose, 4g soy peptone, 1g
Threonine, 1.056gN- acetylglucosamine, 0.9g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 4d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1050。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 22
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 4.8g soy peptone,
1.2g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1120。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 23
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 5.6g soy peptone,
1.4g threonine, 1.056g N-acetyl-glucosamine, 1.1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1423。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 24
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.26g glucose, 6.4g soy peptone,
1.6g threonine, 1.056g N-acetyl-glucosamine, 0.8g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1367。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 25
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 1.44g glucose, 4g soy peptone, 1g
Threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium into
Horizontal high voltage sterilizing, after culture medium is cooling, to the bacterium solution (in bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, and observation is heavy
Shallow lake bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1063。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 26
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 4.8g soy peptone,
1.2g threonine, 1.056g N-acetyl-glucosamine, 1.1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1337。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 27
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 5.6g soy peptone,
1.4g threonine, 1.056g N-acetyl-glucosamine, 0.8g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1407。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 28
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 6.4g soy peptone,
1.6g threonine, 1.056g N-acetyl-glucosamine, 0.9g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (temperature of culture be 37 DEG C) 3d, 3000rpm centrifugation
10min, observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value,
OD600Value is 0.1400.
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 29
The conical flask of 250ml specification is taken, after 200mL pure water is added, adds 1.44g glucose, 4g soy peptone, 1g
Threonine, 1.056gN- acetylglucosamine, 1.1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture medium
High pressure sterilization is carried out, after culture medium is cooling, to the bacterium solution (bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
In thermophilic mucin Ackermam Salmonella bacteria containing amount be 2.6 × 108Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobic box
(85%CO2, 10%N2And 5%H2) in take out after culture (temperature of culture be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1143。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 30
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 4.8g soy peptone,
1.2g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (temperature of culture be 37 DEG C) 3d, 3000rpm is centrifuged 10min, sees
Precipitating bacterium amount is examined, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1043。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 31
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 5.6g soy peptone,
1.4g threonine, 1.056g N-acetyl-glucosamine, 0.9g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to training
It supports base and carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
(bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in bacterium solution8Cfu/mL it) is placed on 200 μ L cysteines, inoculation
Anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min,
Observation precipitating bacterium amount, then with after the sterile PBS resuspension precipitating of 5mL, use the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1470。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 32
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 6.4g soy peptone,
1.6g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
Bacterium amount is precipitated, then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value, OD600Value is
0.1470。
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
Embodiment 33
Take the conical flask of 250ml specification, after 200mL pure water is added, add 1.44g glucose, 6.4g soy peptone,
1.6g threonine, 1.056gN- acetylglucosamine, 1g sodium chloride and 0.5g disodium hydrogen phosphate, after mixing is sufficiently stirred, to culture
Base carries out high pressure sterilization, after culture medium is cooling, to the bacterium solution (bacterium of the thermophilic mucin Ackermam Salmonella of 100 μ L of inoculation of medium
The bacteria containing amount of thermophilic mucin Ackermam Salmonella is 2.6 × 10 in liquid8Cfu/mL) and 200 μ L cysteines, inoculation are placed on anaerobism
Case (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d, 3000rpm is centrifuged 10min, observation
It precipitates bacterium amount (result such as Fig. 4), then with after the sterile PBS resuspension precipitating of 5mL, uses the OD of NanoDrop measurement bacterium solution600Value.
Three parallel laboratory tests, OD600Value is respectively 1.494,1.395 and 1.412.
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
The bacterium amount in bacterium solution is counted using clinical laboratory's Urine Analyzer, thermophilic mucin Acker in the bacterium solution of 200 μ L
The bacteria containing amount of Mans bacterium is 8.8 × 109cfu。
Fluorescent staining is carried out to thermophilic mucin Ackermam Salmonella using fluorescent dye, concrete operations are as follows: take kit (BacLightTMBacterialViability Kits) in A, each 5 μ L of B dyestuff be protected from light after mixing to
With after Akk culture bacterium solution is done 500 times of dilutions using sterile PBS, the Akk for taking the 3 μ L of dyestuff after mixing to be added to 1mL dilutes
In bacterium solution, mixing fullys shake, is protected from light in room temperature and is incubated for 15min, then takes 5 μ L dyeing viable bacteria suspension on glass slide, covers
In fluorescence microscopy microscopic observation after slide.As a result such as Fig. 5.As can be drawn from Figure 5, the Akk of equivalent is provided in the present invention respectively
Culture medium in cultivate after, more viable counts when than cultivating in mucin culture medium can be obtained, this shows that the present invention mentions
The culture medium of confession is fully able to realize the amplification in vitro culture of Akk, and can be realized subsequent functional study and industrialization
Development.
Comparative example 1
The conical flask of 250ml specification is taken, after 200mL pure water is added, 0.4g mucin is added and 7.4g brain heart infusion is dry
Powder after mixing is sufficiently stirred, carries out high pressure sterilization to culture medium, thermophilic glutinous to 100 μ L of inoculation of medium after culture medium is cooling
(content of thermophilic mucin Ackermam Salmonella is 2.6 × 10 to the bacterium solution of albumen Ackermam Salmonella in bacterium solution8) and 200 μ L half cfu/mL
Cystine, inoculation are placed on anaerobic box (85%CO2, 10%N2And 5%H2) in take out after culture (cultivation temperature be 37 DEG C) 3d,
3000rpm is centrifuged 10min, observation precipitating bacterium amount (result such as Fig. 4), then with after the sterile PBS resuspension precipitating of 5mL, uses
The OD of NanoDrop measurement bacterium solution600Value.Three parallel laboratory tests, OD600Value is respectively 0.915,0.945 and 0.976.
Whether bacterium in bacterium solution is the detection of thermophilic mucin Ackermam Salmonella and the purity of the bacterium with embodiment 1.
The bacterium amount in bacterium solution is counted using clinical laboratory's Urine Analyzer, thermophilic mucin Acker in the bacterium solution of 200 μ L
The bacteria containing amount of Mans bacterium is 4.2 × 109cfu。
Fluorescent staining is carried out to thermophilic mucin Ackermam Salmonella using fluorescent dye, concrete operations are as follows: take kit (BacLightTMBacterial Viability Kits) in A, each 5 μ L of B dyestuff be protected from light after mixing to
With after Akk culture bacterium solution is done 500 times of dilutions using sterile PBS, the Akk for taking the 3 μ L of dyestuff after mixing to be added to 1mL dilutes
In bacterium solution, mixing fullys shake, is protected from light in room temperature and is incubated for 15min, then takes 5 μ L dyeing viable bacteria suspension on glass slide, covers
In fluorescence microscopy microscopic observation after slide.As a result such as Fig. 5.
As can be drawn from Figure 5, after the Akk of equivalent is cultivated in culture medium provided by the invention respectively, can be compared
More viable counts when cultivating in mucin culture medium, this shows that culture medium provided by the invention is fully able to realize Akk's
Amplification in vitro culture, and can be realized subsequent functional study and industrialized development.
By above embodiments and comparative example it can be concluded that, using culture medium of the invention can cultivate to obtain thermophilic mucin Ah
Gram Mans bacterium, the component safety in culture medium, there is no potential risks, and can be realized subsequent functional study and
Industrialized development.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of culture medium for cultivating thermophilic mucin Ackermam Salmonella, the component of the culture medium includes soy peptone, Soviet Union's ammonia
Acid, glucose and N-acetyl-glucosamine, which is characterized in that further include sodium chloride and disodium hydrogen phosphate.
2. culture medium according to claim 1, which is characterized in that the culture medium takes water as a solvent, every liter include: 20~
45mmol glucose, 12~32g soy peptone, 3~8g threonine, 20~35mmol N-acetyl-glucosamine, 4~6g chlorination
Sodium and 2~3.5g disodium hydrogen phosphate.
3. culture medium according to claim 1 or 2, which is characterized in that the culture medium takes water as a solvent, and every liter includes:
25~40mmol glucose, 16~28g soy peptone, 4~7g threonine, 25~30mmol N-acetyl-glucosamine, 4.5~
5.5g sodium chloride and 2.5~3g disodium hydrogen phosphate.
4. culture medium according to claim 1 or 2, which is characterized in that the culture medium takes water as a solvent, and every liter includes:
30~35mmol glucose, 20~24g soy peptone, 5~6g threonine, 35mmol N-acetyl-glucosamine, 5g sodium chloride and
2.6~2.8g disodium hydrogen phosphate.
5. the application method of the described in any item culture mediums of Claims 1 to 4 characterized by comprising by the thermophilic mucin
The bacterium solution of Ackermam Salmonella is inoculated in the described in any item culture mediums of Claims 1 to 4, carries out Anaerobic culturel.
6. application method according to claim 5, which is characterized in that the instrument that the Anaerobic culturel uses includes anaerobism
Case includes 85%CO in the anaerobic box2, 10%N2And 5%H2。
7. application method according to claim 5, which is characterized in that thermophilic mucin Ackermam Salmonella contains in the bacterium solution
Amount is 2.6 × 108cfu/mL。
8. application method according to claim 5, which is characterized in that the inoculum concentration of the bacterium solution is 100 μ L/200 μ L.
9. application method according to claim 5, which is characterized in that the temperature of the Anaerobic culturel is 35~40 DEG C.
10. application method according to claim 5, which is characterized in that the time of the Anaerobic culturel is 3~4d.
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