CN109355352A - A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification - Google Patents
A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification Download PDFInfo
- Publication number
- CN109355352A CN109355352A CN201811505865.8A CN201811505865A CN109355352A CN 109355352 A CN109355352 A CN 109355352A CN 201811505865 A CN201811505865 A CN 201811505865A CN 109355352 A CN109355352 A CN 109355352A
- Authority
- CN
- China
- Prior art keywords
- beta galactosidase
- fluid nutrient
- nutrient medium
- detection
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/50—Indoles
- C12Q2334/52—5-Bromo-4-chloro-3-indolyl, i.e. BCI
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of fluid nutrient medium for detecting beta galactosidase and its applications in Bacteria Identification.The fluid nutrient medium of present invention detection beta galactosidase; formula are as follows: contain nutriment 5-50g in every 1000ml culture medium; sodium chloride 2-10g; the PBS 50-250ml of 0.2mol/L; 0.5~4g of specific chromogenic substrate; 0.5~4g of enzyme inducer, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.The culture medium can carry out the detection of liquid processes accurate and effective to live bacteria beta galactosidase using hypotoxicity cosolvent and protective agent using the chloro- 3- indoles-β-D- galactoside of the bromo- 4- of 5- as substrate；And the culture medium can apply to commercialized qualification test card, drying substrate in micro hole, which can be realized effectively, to be redissolved and detects to the beta galactosidase of live bacteria, to keep yin and yang attribute result clearly more demarcated, improves and positive bacteria negative to clinical common gram and identify accuracy.
Description
Technical field
The present invention relates to external diagnosis reagent technology field of microbial identification, are specifically related to a kind of detection beta galactose glycosides
The fluid nutrient medium of enzyme and its application in Bacteria Identification.
Background technique
Beta galactosidase, complete entitled β-D- galactoside galactohydrolase (β-D-galactoside galacto-
Hydrolase), referred to as lactase (Lactase), is distributed widely in human body, animal, plant and microorganism, can urge
The galactolipin glycosidic bond changed in beta galactose glycoside compound hydrolyzes, and also has the function of transgalactosidation.Domestic and foreign scholars couple
Beta galactosidase has carried out research extensively and profoundly, wherein to the beta galactosidase in Escherichia coli be study it is most deep,
Most thoroughly, and lactose operon theory is thus proposed.Later, beta galactosidase is widely used in molecular biology, micro-
In biology and clinical examination.Since whether bacterium contains beta galactosidase with inter-species specificity, therefore beta galactose glycosides
Enzyme is used in the differentiation of bacterium kind and Clinical microorganism Bacteria Identification frequently as specific characteristics.
Currently, beta galactosidase is tried frequently as the important composition for the commercialization qualification test card for carrying out Bacteria Identification
It tests, clinical enterobacteriaceae lactobacteriaceae, non-zymocyte, staphylococcus, Streptococcaceae bacterium etc. is identified.As biological Mei Liai is public
The API 20E of department is using ortho-nitrophenyl-β-D- synthesis (ONPG) detection beta galactosidase to enterobacteria and other
Gram-Negative bacillus is identified；API 20NE detects β-gala using p-nitrophenyl-β-D- synthesis (PNPG)
Glycosidase identifies non-fermented bacterium, and VITEK 2GP card is using ONPG detection beta galactosidase to staphylococcus and chain
Coccaceae bacterium is identified.Above-mentioned commercially produced product utilizes bacterium to generate beta galactose glycosides and decomposes substrate PNPG and ONPG,
It generates yellow product to be detected, which is easy the influence by bacterial growth turbidity when positive findings judge.Meanwhile
Herman Tse et al. has found that the nitrophenyl phenolics such as ONPG chromogenic substrate is for example golden yellow in detection staphylococcus bacteria under study for action
It will appear false positive phenomenon when color staphylococcus, staphylococcus epidermis, staphylococcus haemolyticus and Lu Deng staphylococcus, and use 5-
The bromo- chloro- 3- indoles-β-D- galactoside (X-gal) of 4- can accurately then detect the beta galactosidase of staphylococcus.
Using X-gal as detection substrate, bacteriogenic beta galactosidase can decompose it and generate blue
Precipitating, as a result easily determines, therefore obtained extensive research and utilization clearly.CN108285917 discloses a kind of using X-gal
The method of equal chromogenic substrates detection Escherichia coli and salmonella.Using antibacterial substance and chromogenic substrate, pass through plate detection method
Escherichia coli and salmonella are distinguished and identified to observation colony colour with this；CN102827918 also discloses a kind of using X-
Gal and propyleneglycoles observe colony colour by plate detection method and identify salmonella.
Above-mentioned patent is using X-gal as detection substrate, using film solid media to the beta galactosidase of different bacterium
Detection is carried out to tentatively be distinguished to different bacteriums, bacterium cannot precisely be identified, be unable to satisfy clinical demand.
Microorganism identification test card using biochemical identification method is clinical common Bacteria Identification means, is usually to lead to culture medium
It crosses drying to be coated in the micro hole of test card in advance, the physiological saline containing bacterium is made an addition into test hole when clinical detection, general
Culture medium dissolution is to realize test.And X-gal is as a kind of non-water soluble substance, it is difficult to dissolve in aqueous solution, therefore will
It is coated in the micro hole of test card, and detection effect is poor, and as a result interpretation is indefinite and is easy to appear false negative.
Summary of the invention
The purpose of the invention is to overcoming the shortcomings of above-mentioned background technique, a kind of detection beta galactosidase is provided
Fluid nutrient medium and its application in Bacteria Identification.The culture medium is made using the chloro- 3- indoles-β-D- galactoside of the bromo- 4- of 5-
The inspection of liquid processes accurate and effective can be carried out to live bacteria beta galactosidase using hypotoxicity cosolvent and protective agent for substrate
It surveys；And the culture medium can apply to commercialized qualification test card, the drying substrate in micro hole can effectively be realized multiple
It is molten and the beta galactosidase of live bacteria is detected, to keep yin and yang attribute result clearly more demarcated, improve to clinical common
Gram-negative and positive bacteria identify accuracy.
To achieve the purpose of the present invention, the present invention detects the fluid nutrient medium of beta galactosidase, formula are as follows: every 1000ml
Contain nutriment 5-50g, the PBS 50-250ml of sodium chloride 2-10g, 0.2mol/L, specific chromogenic substrate in culture medium
0.5~4g, 0.5~4g of enzyme inducer, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.
Preferably, the specific chromogenic substrate is the chloro- 3- indoles-β-D- galactoside (X-gal) of the bromo- 4- of 5-.
Preferably, the enzyme inducer is isopropyl-β-D-thiogalactoside (IPTG).
Further, the nutriment can be proteose phosphate broth and yeast powder, and it is big to be also possible to tryptose
Beans meat soup, yeast powder and brain heart infusion broth, can also be casein hydrolysate and yeast powder, can also be brain heart infusion broth
And yeast powder.
Preferably, the cosolvent can be dimethyl sulfoxide (DMSO) and polysorbas20, be also possible to dimethylformamide
(DMF) and polysorbas20.
Preferably, the protective agent can be glycerol.
Preferably, the concentration of the dimethyl sulfoxide is 0.01%~0.5%, such as 0.2%.
Preferably, the concentration of the polysorbas20 is 0.01%~0.25%, such as 0.1%.
Preferably, the concentration of the glycerol is 0.1%~0.5%, such as 0.2%.
It is furthermore preferred that the volume ratio of the dimethyl sulfoxide, polysorbas20 and glycerol is (0.5~1.5): 0.5:(0.5~
, such as 1:0.5:1 1.5).
Further, in every 1000ml culture medium contain 5~20g of proteose phosphate broth, yeast powder 2~
8g, the PBS 50-150ml of sodium chloride 3-10g, 0.2mol/L, specific chromogenic 1~3g of substrate, isopropyl-beta D-thio gala
Glucosides (IPTG) 1~4g, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.
Further, in every 1000ml culture medium contain 5~30g of proteose phosphate broth, yeast powder 2~
20g, sodium chloride 5g, 0.2mol/LPBS 50-250ml, specific chromogenic 0.5~3g of substrate, isopropyl-β-D-thiogalactose
Glycosides (IPTG) 0.5~4g, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.
In one embodiment of the invention, it is further improved as above-mentioned chromogenic culture medium, in every 1000ml culture medium
Proteose phosphate broth and yeast powder alternatively at 10~50g of casein hydrolysate, 1~10g of yeast powder.
In one embodiment of the invention, it is further improved as above-mentioned chromogenic culture medium, in every 1000ml culture medium
Proteose phosphate broth and yeast powder alternatively at Tryptose soy 10~50g of meat soup, 1~10g of yeast powder, brain heart infusion
1~10g of meat soup.
In one embodiment of the invention, it is further improved as above-mentioned chromogenic culture medium, in every 1000ml culture medium
Proteose phosphate broth and yeast powder alternatively at 10~50g of brain heart infusion broth, 1~10g of yeast powder.
The present invention also provides application of the fluid nutrient medium of above-mentioned detection beta galactosidase in Bacteria Identification, that is, make
The beta galactosidase detection of clinical viable bacteria is directly used in for fluid nutrient medium.
In addition, the fluid nutrient medium of above-mentioned detection beta galactosidase can also be coated in advance the micro- of test card by dry
In metering-orifice, identified as commercialization detection test card or kit for clinical bacteria.
Further, it is of the present invention commercialization detection test card the preparation method is as follows:
Step (1): preparing fluid nutrient medium, with the nutriment in container precise culture medium prescription, is added later
Purified water mixes dissolution, after nutriment dissolution completely, the desired amount of protective agent is added, and stir and evenly mix, and is placed in high pressure steaming
Vapour autoclave carries out high pressure sterilization, when temperature is down to 50-60 DEG C after sterilizing, chromogenic substrate, enzyme inducer, cosolvent is dissolved mixed
Solution after conjunction after filtration sterilization, which pours into, obtains fluid nutrient medium in container；
Step (2): fluid nutrient medium obtained by step (1) is added to sterility test under clean environment by test card sample-adding
In card；
Step (3): it is dry, sterility test card obtained by step (2) is dried in closed drying equipment, makes test card
The moisture of culture medium gradually scatters and disappears in hole；
Step (4): packaging is sealed packaging to test card obtained by step (3) under clean environment.
Preferably, step (1) the mesohigh sterilizing can be the 15min that sterilizes under the conditions of 121 DEG C.
Compared with prior art, advantages of the present invention is as follows:
(1) X-gal solubility problem and the murder by poisoning to living stems are solved the problems, such as.X-gal poorly water-soluble can only use
Organic solvent is dissolved, and traditional plate detection method is deposited in culture medium since bacterium only contacts with cell culture base
It is smaller on bacterial growth influence in organic solvent, significant impact will not be caused to testing result, therefore X-gal is detected in plate
With relatively universal in method.But in liquid living stems method, since organic solvent has toxic action, limit to bacterial growth
The application of such substrate is made.The present invention is used as hydrotropy using dimethyl sulfoxide and surface active agent tween 20 combination of hypotoxicity
Agent, while not only solving the solubility problem of X-gal chromogenic substrate as protective agent using glycerol, but also reduce culture
Toxic action of the base to bacterium guarantees that bacterial growth is unaffected, can apply to X-gal in liquid living stems.
(2) X-gal substrate color developing effect is enhanced.It is commonly used in detecting the culture medium nutriment of beta galactosidase
Peptone, yeast powder (also known as yeast extract) and beef extract, if CN108285917 culture medium nutriment is peptone, ferment
Female powder and beef extract；CN102827918 culture medium nutriment is peptone and beef extract.Such nutriment is being cultivated
Effect is poor when Streptococcaceae bacterium, and Optimal Medium combination of the present invention meets the culture of the severe bacteria such as streptococcus, promoted simultaneously
Enterobacteriaceae lactobacteriaceae, non-zymocyte and staphylococcic increment add suitable enzyme inducer, improve beta galactose
Glycosides expression of enzymes amount, enhances substrate color developing effect.
(3) present invention is redissolved after solving the problems, such as fluid nutrient medium coating and drying, is conducive to product and is commercialized.The present invention is logical
Addition dimethyl sulfoxide and polysorbas20 are crossed, both ensure that the dissolution of substrate, while making fluid nutrient medium in the drying process firmly
It is adhered to the hole bottom of test card, it is ensured that test card is not in " picking " phenomenon after dry.In addition, the dimethyl retained in hole
Sulfoxide and polysorbas20 can ensure that substrate is redissolved in viable bacteria liquid detecting, to reach optimal color developing effect.Fluid present invention
Culture medium can be dried and apply to be commercialized qualification test card, can effectively realize redissolution and to the beta galactose of live bacteria
Glycosides enzyme is detected, and is improved and positive bacteria negative to clinical common gram and is identified accuracy.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.Additional aspect and advantage of the invention will be set forth in part in the description, partially will be under
Become obvious in the description in face, or practice through the invention is recognized.It is only used to explain this hair it should be appreciated that being described below
It is bright, it is not intended to limit the present invention.
Term "comprising" used herein, " comprising ", " having ", " containing " or its any other deformation, it is intended that covering
Non-exclusionism includes.For example, composition, step, method, product or device comprising listed elements are not necessarily limited to those and want
Element, but may include not expressly listed other elements or such composition, step, method, product or device it is intrinsic
Element.
Conjunction " Consists of " excludes any element that do not point out, step or component.If in claim, this
Phrase will make claim closed, so that it is not included the material in addition to the material of those descriptions, but relative normal
Except rule impurity.When being rather than immediately following after theme in the clause that phrase " Consists of " appears in claim main body,
It is only limited to element described in the clause；Other elements are not excluded except the claim as a whole.
Equivalent, concentration or other values or parameter are excellent with range, preferred scope or a series of upper limit preferred values and lower limit
When the Range Representation that choosing value limits, this should be understood as specifically disclosing by any range limit or preferred value and any range
Any pairing of lower limit or preferred value is formed by all ranges, regardless of whether the range separately discloses.For example, when open
When range " 1 to 5 ", described range should be interpreted as including range " 1 to 4 ", " 1 to 3 ", " 1 to 2 ", " 1 to 2 and 4 to
5 ", " 1 to 3 and 5 " etc..When numberical range is described herein, unless otherwise stated, otherwise the range is intended to include its end
Value and all integers and score in the range.
Singular includes that plural number discusses object, unless the context clearly dictates otherwise." optional " or it is " any
It is a kind of " refer to that the item described thereafter or event may or may not occur, and the description include situation that event occurs and
The situation that event does not occur.
In present specification and claims, range restriction can be combined and/or be exchanged, if without in addition saying
These bright ranges include all subranges contained therebetween.
Indefinite article "an" before element or component of the present invention (goes out the quantitative requirement of element or component with "one"
Occurrence number) unrestriction.Therefore "one" or "an" should be read as including one or at least one, and singular
Element or component also include plural form, unless the quantity obviously only refers to singular.
Moreover, technical characteristic involved in each embodiment of the present invention as long as they do not conflict with each other can
To be combined with each other.
Embodiment 1
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase contains proteose phosphorus in every 1000ml culture medium
The chloro- 3- indoles-β-D- galactoside of hydrochlorate meat soup 25g, yeast powder 5g, the bromo- 4- of sodium chloride 5g, 0.2mol/L PBS 50ml, 5-
1g, isopropyl-β-D-thiogalactoside (IPTG) 1g, dimethyl sulfoxide 2ml, polysorbas20 1ml, glycerol 2ml.
Embodiment 2
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase contains casein water in every 1000ml culture medium
Object 25g, yeast powder 10g, the chloro- 3- indoles-β-D- galactoside 1g of the bromo- 4- of sodium chloride 5g, 0.2mol/L PBS 50ml, 5- are solved,
Isopropyl-β-D-thiogalactoside (IPTG) 1g, dimethyl sulfoxide 2ml, polysorbas20 1ml, glycerol 2ml.
Embodiment 3
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase is big containing tryptose in every 1000ml culture medium
Beans meat soup 20g, yeast powder 5g, brain heart infusion broth 10g, the chloro- 3- Yin of the bromo- 4- of sodium chloride 5g, 0.2mol/L PBS 50ml, 5-
Diindyl-β-D- galactoside 1g, isopropyl-β-D-thiogalactoside (IPTG) 1g, dimethyl sulfoxide 2ml, polysorbas20 1ml are sweet
Oily 2ml.
Embodiment 4
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase contains brain heart infusion in every 1000ml culture medium
Meat soup 35g, yeast powder 5g, the chloro- 3- indoles-β-D- galactoside 1g of the bromo- 4- of sodium chloride 5g, 0.2mol/L PBS 50ml, 5-,
Isopropyl-β-D-thiogalactoside (IPTG) 1g, dimethyl sulfoxide 2ml, polysorbas20 1ml, glycerol 2ml.
Comparative example 1
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase contains proteose phosphorus in every 1000ml culture medium
Hydrochlorate meat soup 25g, yeast powder 5g, sodium chloride 5g, 0.2mol/L PBS 50ml, ortho-nitrophenyl β-D- synthesis 2g are different
Propyl-β-D- thiogalactoside (IPTG) 1g.
Comparative example 2
A kind of liquid chromogenic culture medium of detection bacterium beta galactosidase contains tryptone in every 1000ml culture medium
Take 25g, yeast powder 5g, beef extract 10g, the chloro- 3- indoles-β-D- of the bromo- 4- of sodium chloride 5g, 0.2mol/L PBS50ml, 5- half
Lactoside 1g, isopropyl-β-D-thiogalactoside (IPTG) 1g, dimethyl sulfoxide 2ml, polysorbas20 1ml, glycerol 2ml.
Embodiment 5
Various concentration dimethyl sulfoxide influences bacterial growth and colored intensity.According to 1 Media Components (two of embodiment
Except methyl sulfoxide), using various concentration dimethyl sulfoxide, liquid-liquid culture medium is made, observation various concentration dimethyl is sub-
Sulfone influences substrate colored intensity and bacterial growth.Detection method are as follows: be added final concentration of 10 into detection culture medium5cfu/
The bacterium of ml is placed in 35 DEG C of insulating boxs and is incubated for 18-24h, observes various concentration DMSO culture medium positive for bacteria colored intensity difference
And the difference of bacterial growth and control medium (without chromogenic substrate and cosolvent).The results are shown in Table 1.
1 various concentration DMSO of table influences colored intensity and bacterial growth
Note: colored intensity "+" indicates in upper table: blue；" v " is indicated: light blue；"-" indicates: colourless；
Growth 4+ indicate: bacterial growth with compare unanimously；Growth 3+ is indicated: bacterial growth omits compared with the control
Difference；Growth 1+
Indicate with 2+: bacterial growth is poor compared with the control.
As shown in Table 1, detection beta galactosidase culture medium can be to bacterial growth using various concentration dimethyl sulfoxide
It is impacted with colored intensity (positive phenotypes).It is found that it will not be to bacterial growth using 0.2% dimethyl sulfoxide by test result
Property causes to significantly affect, while substrate color developing effect is preferable, is able to detect that apparent beta galactosidase positive blue phenotype.
Embodiment 6
Adding protective agent glycerol influences colored intensity.Two kinds of fluid nutrient mediums are made referring to embodiment 1, one kind is containing sweet
Oil (consistent with embodiment 1), another kind do not include glycerol (remaining ingredient and embodiment 1 are consistent).Observe two kinds of different culture mediums
Substrate colored intensity difference.Detection method are as follows: be added final concentration of 10 into detection culture medium5The bacterium of cfu/ml, is placed in 35
DEG C insulating box is incubated for 18-24h, observes influence of the glycerol to liquid chromogenic culture medium colored intensity.The results are shown in Table 2.
Influence of 2 glycerol of table to fluid nutrient medium colored intensity
Note: colored intensity "+" indicates in upper table: blue；" v " is indicated: light blue；"-" indicates: colourless；
As shown in Table 2, dimethyl sulfoxide can be reduced to bacterium by qs glycerin (0.2%) being added into fluid nutrient medium
Toxic action makes bacterium normal growth and generates apparent blue positive phenotype using substrate.
Embodiment 7
Specific detection.Fluid nutrient medium is made in embodiment 1, embodiment 2, embodiment 3, embodiment 4 and comparative example 1,
Final concentration of 10 are added into culture medium respectively5The test bacterium of cfu/ml is placed in 35 DEG C of insulating boxs and is incubated for 18-24h.It has been incubated for
Afterwards observe culture medium as a result, blue or yellow then indicate beta galactosidase the positive, be denoted as "+"；It is colourless, indicate beta galactose
Glycosides enzyme is negative, is denoted as "-".The results are shown in Table 3 for observation.
3 different liquids culture medium beta galactosidase testing result of table
As seen from the results in Table 3, using the embodiment 1 of the chloro- 3- indoles-β-D- galactoside of the bromo- 4- of 5-, embodiment 2, implementation
Example 3 and embodiment 4 can accurately detect the beta galactosidase of surveyed reference culture, and use ortho-nitrophenyl β-D- gala pyrans
The comparative example 1 of glucosides, which detects staphylococcus aureus, staphylococcus epidermis and enterococcus faecalis, will appear false positive phenomenon.
Embodiment 8
Different nutrients confrontation beta galactosidase colored intensity influences.By embodiment 1, embodiment 2, embodiment 3, implement
Fluid nutrient medium is made in example 4 and comparative example 2, is added final concentration of 10 into culture medium respectively5The test bacterium of cfu/ml, is placed in
35 DEG C of insulating boxs are incubated for 18-24h.Culture medium result is observed after being incubated for.The results are shown in Table 4 for observation.
4 different nutriments beta galactosidase of table colour developing result
Note: colored intensity "+" indicates in upper table: blue；" v " is indicated: light blue；"-" indicates: colourless；
As shown in Table 4, the beta galactosidase positive findings of embodiment 1, embodiment 2, embodiment 3 and embodiment 4 are obvious,
For blue phenotype.2 staphylococcus xylosus of comparative example and the enterococcus faecium positive are unobvious, are light blue phenotype.It can be seen that the present invention
By Optimal Medium nutriment, beta galactosidase positive phenotypes are enhanced, improve beta galactosidase detection effect.
Embodiment 9
Fluid nutrient medium and after drying microwell plate (hole includes culture medium powder) effect compare.It is formulated according to embodiment 1
Fluid nutrient medium is made, a part is directly used in bacteria beta-galactosidase detection, and another part is made referring to the method for the present invention
Microwell plate.Detection method are as follows: final concentration of 10 are added into fluid nutrient medium and microwell plate5The bacterium of cfu/ml, and make liquid
Liquid volume is consistent after liquid volume is redissolved with microwell plate in culture medium.The 35 DEG C of insulating boxs of being placed on be incubated for 18-24h, observation
Bacteria beta-galactosidase testing result in fluid nutrient medium and microwell plate.The results are shown in Table 5 for observation.
5 fluid nutrient medium of table and the comparison of microwell plate detection effect
Note: "+" indicates in upper table: positive blue phenotype；"-" indicates: negative colourless phenotype；
As shown in Table 5, fluid present invention culture medium and after drying microwell plate energy detection bacterium beta galactosidase, and
Consistent obvious yin and yang attribute result can be obtained.It is aobvious that the present invention not only enables clinical examination person easily configure beta galactosidase
Color culture medium is detected for bacteria beta-galactosidase, and can be made into commercialization beta galactosidase detection test card or reagent
Box has realistic meaning for the working efficiency and Bacteria Identification accuracy that improve clinical examination person.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of fluid nutrient medium for detecting beta galactosidase, which is characterized in that the formula of the culture medium are as follows: every 1000ml
Contain nutriment 5-50g, the PBS 50-250ml of sodium chloride 2-10g, 0.2mol/L, specific chromogenic substrate in culture medium
0.5~4g, 0.5~4g of enzyme inducer, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.
2. the fluid nutrient medium of detection beta galactosidase according to claim 1, which is characterized in that the specificity is aobvious
Color substrate is the chloro- 3- indoles-β-D- galactoside of the bromo- 4- of 5-, and the enzyme inducer is isopropyl-β-D-thiogalactoside.
3. the fluid nutrient medium of detection beta galactosidase according to claim 1, which is characterized in that the nutriment
It is proteose phosphate broth and yeast powder or Tryptose soy meat soup, yeast powder and brain heart infusion broth or casein hydrolysis
Object and yeast powder or brain heart infusion broth and yeast powder.
4. the fluid nutrient medium of detection beta galactosidase according to claim 1, which is characterized in that the cosolvent is
Dimethyl sulfoxide and polysorbas20 or dimethylformamide and polysorbas20.
5. the fluid nutrient medium of detection beta galactosidase according to claim 1, which is characterized in that the protective agent is
Glycerol.
6. the fluid nutrient medium of detection beta galactosidase according to claim 4 or 5, which is characterized in that the diformazan
The concentration of base sulfoxide is 0.01%~0.5%, such as 0.2%；The concentration of the polysorbas20 is 0.01%~0.25%, such as
0.1%；Preferably, the concentration of the glycerol is 0.1%~0.5%, such as 0.2%；It is furthermore preferred that the dimethyl sulfoxide,
The volume ratio of polysorbas20 and glycerol is (0.5~1.5): 0.5:(0.5~1.5), such as 1:0.5:1.
7. the fluid nutrient medium of detection beta galactosidase according to claim 1, which is characterized in that every 1000ml
Contain 5~20g of proteose phosphate broth, 2~8g of yeast powder, the PBS 50- of sodium chloride 3-10g, 0.2mol/L in culture medium
150ml, specific chromogenic 1~3g of substrate, 1~4g of isopropyl-β-D-thiogalactoside, 0.1~10ml of cosolvent, protective agent
Contain 5~30g of proteose phosphate broth, 2~20g of yeast powder, chlorine in 0.1~10ml or every 1000ml culture medium
Change sodium 5g, 0.2mol/L PBS 50-250ml, specific chromogenic 0.5~3g of substrate, isopropyl-β-D-thiogalactoside 0.5
~4g, 0.1~10ml of cosolvent, 0.1~10ml of protective agent.
8. the fluid nutrient medium of detection beta galactosidase according to claim 7, which is characterized in that every 1000ml culture
Proteose phosphate broth and yeast powder in base are alternatively at 10~50g of casein hydrolysate, 1~10g of yeast powder or pancreas egg
White 10~50g of soybean broth, 1~10g of yeast powder, 10~50g of 1~10g of brain heart infusion broth or brain heart infusion broth, yeast
1~10g of powder.
9. fluid nutrient medium the answering in Bacteria Identification of detection beta galactosidase according to claim 1-8
With, which is characterized in that the fluid nutrient medium is directly used in the beta galactosidase detection of clinical viable bacteria, or by dry preparatory
It is coated in the micro hole of test card, is identified as commercialization detection test card or kit for clinical bacteria.
10. application of the fluid nutrient medium of detection beta galactosidase according to claim 9 in Bacteria Identification, special
Sign is, the commercialization detection test card the preparation method is as follows:
Step (1): preparing fluid nutrient medium, and with the nutriment in container precise culture medium prescription, purifying is added later
Water mixes dissolution, after nutriment dissolution completely, the desired amount of protective agent is added, and stir and evenly mix, is placed in high steam and goes out
Bacterium pot carries out high pressure sterilization, when temperature is down to 50-60 DEG C after sterilizing, after chromogenic substrate, enzyme inducer, cosolvent dissolution mixing
Solution after filtration sterilization, which pours into, obtains fluid nutrient medium in container；
Step (2): fluid nutrient medium obtained by step (1) is added to sterility test card under clean environment by test card sample-adding
In；
Step (3): it is dry, sterility test card obtained by step (2) is dried in closed drying equipment, makes to test in card hole
The moisture of culture medium gradually scatters and disappears；
Step (4): packaging is sealed packaging to test card obtained by step (3) under clean environment；
Preferably, step (1) the mesohigh sterilizing can be the 15min that sterilizes under the conditions of 121 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811505865.8A CN109355352A (en) | 2018-12-10 | 2018-12-10 | A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811505865.8A CN109355352A (en) | 2018-12-10 | 2018-12-10 | A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109355352A true CN109355352A (en) | 2019-02-19 |
Family
ID=65331921
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811505865.8A Pending CN109355352A (en) | 2018-12-10 | 2018-12-10 | A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109355352A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929909A (en) * | 2019-03-19 | 2019-06-25 | 广州悦蜂生物防治科技有限公司 | A kind of method, kit and quick measuring card detecting lactase activity |
| CN110904189A (en) * | 2019-12-05 | 2020-03-24 | 湖南长沙天地人生物科技有限公司 | Haemophilus identification drug sensitivity test card and preparation method thereof |
| CN112557381A (en) * | 2021-01-25 | 2021-03-26 | 河南省科学院生物研究所有限责任公司 | Detection test strip for alpha-galactosidase and detection method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038584A1 (en) * | 1995-05-31 | 1996-12-05 | Celsis International Plc | Assay for microorganisms |
| CN101864383A (en) * | 2010-05-11 | 2010-10-20 | 佛山市海天调味食品有限公司 | Coliform bacterium and enzyme increasing culture solution and preparation method thereof |
| CN104388527A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Streptococcus agalactiae selective chromogenic culture medium and detection test paper thereof |
| CN108359707A (en) * | 2018-05-25 | 2018-08-03 | 无锡市赛微生物技术有限公司 | It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof |
-
2018
- 2018-12-10 CN CN201811505865.8A patent/CN109355352A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038584A1 (en) * | 1995-05-31 | 1996-12-05 | Celsis International Plc | Assay for microorganisms |
| CN101864383A (en) * | 2010-05-11 | 2010-10-20 | 佛山市海天调味食品有限公司 | Coliform bacterium and enzyme increasing culture solution and preparation method thereof |
| CN104388527A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Streptococcus agalactiae selective chromogenic culture medium and detection test paper thereof |
| CN108359707A (en) * | 2018-05-25 | 2018-08-03 | 无锡市赛微生物技术有限公司 | It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929909A (en) * | 2019-03-19 | 2019-06-25 | 广州悦蜂生物防治科技有限公司 | A kind of method, kit and quick measuring card detecting lactase activity |
| CN109929909B (en) * | 2019-03-19 | 2022-06-17 | 广州悦蜂生物防治科技有限公司 | Method, kit and rapid test card for detecting lactase activity |
| CN110904189A (en) * | 2019-12-05 | 2020-03-24 | 湖南长沙天地人生物科技有限公司 | Haemophilus identification drug sensitivity test card and preparation method thereof |
| CN110904189B (en) * | 2019-12-05 | 2024-02-27 | 湖南迈瑞医疗科技有限公司 | Haemophilus identification drug sensitivity test card and preparation method thereof |
| CN112557381A (en) * | 2021-01-25 | 2021-03-26 | 河南省科学院生物研究所有限责任公司 | Detection test strip for alpha-galactosidase and detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103282513B (en) | For detecting goods and the method for target microorganism | |
| CN109355352A (en) | A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification | |
| AU2008220705B2 (en) | Bacteria detection and/or identification medium | |
| CN103013822B (en) | Fast drug sensitivity detection kit for aquatic product vibrio | |
| Boemare et al. | Simple bacteriological tests for phenotypic characterization of Xenorhabdus and Photorhabdus phase variants | |
| EP0897428A1 (en) | Test media and quantitative method for identification and differentiation of biological materials in a test sample | |
| CN101395278B (en) | Method for identifying at least two groups of microorganisms | |
| CN101631875B (en) | Medium for detecting and/or identifying bacteria | |
| CN101970682A (en) | Method for detecting and/or identifying clostridium difficile | |
| WO2004063392A2 (en) | Selective culture medium for the isolation and/or detection of species in the streptococcus genus | |
| CN104508140B (en) | The method that detection generates the bacterium of OXA-048 carbapenem enzyme | |
| CN108359707A (en) | It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof | |
| US6130057A (en) | Method for differentiating microorganisms in a sample | |
| PT1300471E (en) | Nutritional mixture and method for early identification and count of gram-negative organisms | |
| CN103820373A (en) | Chromogenic medium for separating and detecting enterobacter cloacae | |
| CN101698826A (en) | Trypticase clostridium botulinum enrichment broth | |
| CN106086159B (en) | A kind of zymolyte culture medium that can detect two kinds of fecal pollution indicator bacterias simultaneously and its application | |
| AU2011298206B2 (en) | Use of a beta-glucosidase activator for detecting and/or identifying C. difficile | |
| US20020086278A1 (en) | Chromogenic media containing blood or hemin | |
| ES2259712T3 (en) | CULTURE MEDIA FOR THE DETECTION AND / OR DISCRIMINATION OF ENTEROCOCO. | |
| RU2264466C2 (en) | Composition and method for detection and early and differential calculation of gram-negative microorganisms | |
| CN105463056A (en) | Culture medium for rapid culture and developing of candida albicans and detection method | |
| CN109706214A (en) | A kind of identification of resistance to quinolone antibiotics Salmonella strains and separation method | |
| CN108660186A (en) | It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof | |
| CN109355226A (en) | Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190219 |
|
| RJ01 | Rejection of invention patent application after publication |