CN109439725A - Chromogenic culture medium and its application for Escherichia coli identification - Google Patents
Chromogenic culture medium and its application for Escherichia coli identification Download PDFInfo
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- CN109439725A CN109439725A CN201811447422.8A CN201811447422A CN109439725A CN 109439725 A CN109439725 A CN 109439725A CN 201811447422 A CN201811447422 A CN 201811447422A CN 109439725 A CN109439725 A CN 109439725A
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- escherichia coli
- culture medium
- chromogenic
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- 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
- C12Q1/10—Enterobacteria
Abstract
The present invention relates to a kind of chromogenic culture mediums for Escherichia coli identification, the chromogenic culture medium includes one of p-nitrophenyl-β-D- glucopyranoside, ortho-nitrophenol β-D glucosiduronate, 4- nitrobenzophenone-β-D- glucopyranoside, 4-methyl umbelliferone acyl-β-D-Glucose thuja acid or the chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide of the bromo- 4- of 5- chromogenic substrate, the glucoside enzyme interacting of the chromogenic substrate and Escherichia coli and discharge colour developing group.Using the chromogenic culture medium identified for Escherichia coli of the invention and its application, component is simple, it is easy to operate, detection sensitivity is high, specificity is good, reduce the inspection positive and false negative, and detection cycle is short, suitable for handling large batch of sample, being easy to industrialization production, can carry out comprehensive, system, accurate detection and preliminary identification to Escherichia Coli in Food.
Description
Technical field
The present invention relates to Testing and appraisal technical field, in particular to Escherichia coli, in particular to one kind are used for Escherichia coli
The chromogenic culture medium of identification.
Background technique
Escherichia coli (Escherichia coli) also known as Escherichia coli, are the normal floras of humans and animals enteron aisle；But
When Abwehrkraft des Koepers decline or its parenteral tissue of intrusion or organ, extra intestinal infection can be caused；Escherichia coli is in the world
Generally acknowledged monitoring of hygiene indicator bacteria, the detection of escherichia coli is this means that direct or indirect recent excrement is dirty in food or water
Dye.
In the standard test methods such as food and commodity inspection, the progress such as eosin methylene blue agar (EMB), maconkey agar is generallyd use
The separation of escherichia coli, it is biochemical anti-in combination with oxidase test, methyl red (MR) test, VP test, urease experiment etc.
Escherichia coli should be identified, it usually needs longer detection cycle.
In recent years, some chromogenic culture mediums are gradually developed, quick separating and identification for escherichia coli,
Received by industries such as food, public health, clinical examinations.Compared with traditional isolation medium, chromogenic culture medium is eliminated pair
Bacterial strain carries out the step of pure culture, directly can make preliminary judgement according to colony colour, has higher sensibility and spy
The opposite sex, it is easy to operate quick, man power and material can be greatlyd save.
Being commonly recognized currently on the market with widely applied escherichia coli chromogenic culture medium is mostly external product, such as method
State's Kerma (unit of kinetic energy) praises the brands such as (CHRO), biology Mei Liai, these stable product qualities, but expensive；Domestic some main trainings
Yang Ji manufacturer starts the independent research for being absorbed in chromogenic culture medium in recent years；Chinese patent CN104278075A is disclosed
A kind of e.coli chromogenic medium, the culture medium add inhibitor and color developing agent on the basis of mTSB broth agar culture medium,
It reduces costs, but color developing agent is not expressed in the invention, and component is more, when operation is relatively cumbersome, uses simultaneously
Basal medium mTSB in ovobiocin to partial target bacterium, there are inhibiting effect, reduce the detection of escherichia coli
Rate, in the detection of high-volume food samples, there may be detection leakage phenomenons, are unfavorable for the detection of food escherichia coli pollution situation.
Summary of the invention
The purpose of the invention is to overcome above-mentioned missing in the prior art, provide a kind of component be simple and convenient to operate,
Detection sensitivity is high, specificity is good, the positive and false negative that reduces inspection and detection cycle is short, be suitable for handling large batch of sample,
It is easy to the chromogenic culture medium identified for Escherichia coli and its application of industrialization production.
To achieve the goals above, one aspect of the present invention provides a kind of chromogenic culture medium for Escherichia coli identification,
Wherein, the chromogenic culture medium includes p-nitrophenyl-β-D- glucopyranoside, ortho-nitrophenol β-D glucosiduronate, 4-
Nitrobenzophenone-β-D- glucopyranoside, 4-methyl umbelliferone acyl-β-D-Glucose thuja acid, the chloro- 3- indoxyl-N- of the bromo- 4- of 5-
One of acetyl-β-D- glucosaminide or a variety of chromogenic substrates, the glucose of the chromogenic substrate and Escherichia coli
Glycosides enzyme interacting and discharge colour developing group.
Preferably, the chromogenic culture medium further includes agar, peptone, lactose, sodium chloride and cholate.
Preferably, the culture medium includes component: 10~15g/L of agar, 5~15g/L of peptone, 5~15g/ of lactose
L, 3~10g/L of sodium chloride, 0.5~5g/L of cholate, 0.05~0.5g/L of chromogenic substrate.
Preferably, the culture medium includes component: agar 15g/L, peptone 5g/L, lactose 11.4g/L, sodium chloride
The chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide 0.15g/L of 3g/L, the bromo- 4- of cholate 2g/L, 5-.
Preferably, the culture medium includes component: agar 12g/L, peptone 15g/L, lactose 5g/L, sodium chloride 5g/
L, cholate 1g/L, 4-methyl umbelliferone acyl-β-D-Glucose thuja acid 0.1g/L.
Preferably, the pH value of the culture medium is 7.2 ± 0.2.
Preferably, the colour developing group is in blue-green or the fluorescence blue under 366nm ultraviolet lamp under natural light.
The present invention also provides chromogenic culture medium the answering in food inspection for Escherichia coli identification described in one kind
With.
It is distinctive using escherichia coli using the chromogenic culture medium identified for Escherichia coli of the invention and its application
Glucuroide adds specific chromogenic substrate, by the interaction release colour developing group of enzyme and substrate, according to colony colour
Identification is made to bacterial strain, the nutrient media components are simple, easy to operate, and detection sensitivity is high, specificity is good, reduce inspection it is positive and
False negative, and detection cycle is short, it, can be uncommon to large intestine angstrom in food suitable for handling large batch of sample, being easy to industrialization production
Bacterium carries out comprehensive, system, accurate detection and preliminary identification, and new way is provided for Escherichia Coli in Food pollution detection
Diameter.
Detailed description of the invention
Fig. 1 is growing state schematic diagram of each bacterium on escherichia coli chromogenic culture medium of the invention.
Fig. 2 is that escherichia coli (CMCC44102) is cultivated 24 hours on ECM, EMB and nutrient agar (NA) of the invention
Growing state schematic diagram.
Specific embodiment
In order to be more clearly understood that technology contents of the invention, specific implementation method of the invention is made into one below
Walk explanation.
Embodiment 1
Escherichia coli chromogenic culture medium component includes: agar 15g/L, peptone 5g/L, lactose 11.4g/L, sodium chloride
3g/L, the bromo- 4- of cholate 2g/L, 5- chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide 0.15g/L, purified water 1000mL.
Escherichia coli chromogenic culture medium is prepared as follows:
(1) each component weighs: weighing agar 15g/L, peptone 5g/L, lactose 11.4g/L, sodium chloride 3g/L, cholate 2g/
The chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide 0.15g/L of the bromo- 4- of L, 5-, is placed in the container of clean dried；
(2) raw material mixes: 1000mL purified water being added in the container in step (1), heating is boiled to being completely dissolved；
(3) adjustment pH is 7.2 ± 0.2；
(4) mixture high pressure sterilization: is subjected to high pressure steam sterilization (115 DEG C, 15 minutes)；
(5) it dispenses: being cooled to 50 DEG C after sterilizing under aseptic condition, topple over sterilized petri dishes；
(6) then 36 ± 1 DEG C of the plate prepared preculture 24 hours is placed at 4 DEG C and is saved to see whether to pollute.
Embodiment 2
Escherichia coli chromogenic culture medium component includes: agar 12g/L, peptone 15g/L, lactose 5/L, sodium chloride 5g/
L, cholate 1g/L, 4-methyl umbelliferone acyl-β-D-Glucose thuja acid 0.1g/L, purified water 1000mL.
Escherichia coli chromogenic culture medium is prepared by method identical in embodiment 1.
Embodiment 3
Escherichia coli chromogenic culture medium component includes: agar 13g/L, peptone 8g/L, lactose 15g/L, sodium chloride
The chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide 0.05g/L of 10g/L, the bromo- 4- of cholate 5g/L, 5-, purified water
1000mL。
Escherichia coli chromogenic culture medium is prepared by method identical in embodiment 1.
Embodiment 4
Escherichia coli chromogenic culture medium component includes: agar 10g/L, peptone 15g/L, lactose 8g/L, sodium chloride 5g/
L, cholate 1g/L, 4- nitrobenzophenone-β-D- glucopyranoside 0.3g/L, purified water 1000mL.
Escherichia coli chromogenic culture medium is prepared by method identical in embodiment 1.
Embodiment 5
1, the growth experiment verifying of chromogenic culture medium
The escherichia coli chromogenic culture medium prepared in Examples 1 to 4 is inoculated with escherichia coli respectively
CMCC44102, Klebsiella Pneumoniae CMCC46117, salmonella typhimurium CMCC50115, staphylococcus aureus
The bacteria suspension 1.0 × 10 of ATCC259238/ mL is diluted by 1:100, is uniformly mixed, is taken the 10 diluted bacteria suspensions of μ L respectively (about
104CFU), the streak inoculation in escherichia coli chromogenic culture medium is cultivated 18~24 hours in 36 ± 1 DEG C of constant incubators, is seen
Bacterial growth situation is examined, growth experiment verifying is carried out.
As shown in figure 1 and table 1, escherichia coli is on ECM for the productive experiment verification result of Examples 1 to 4 culture medium
Apparent blue-green is presented in well-grown；Klebsiella Pneumoniae is grown on ECM, and colourless bacterium colony is presented；Salmonella typhimurium
Growth, bacterium colony is less slightly, is in colourless bacterium colony；Staphylococcus aureus growth is suppressed；Large intestine can be gone out by the differential identification of color
Angstrom uncommon bacterium.
Table 1: each Quality-control strains bacterium colony growing state
| Quality-control strains | Bacterium colony growing state |
| Escherichia coli CMCC 44102 | Growth, blue-green bacterium colony |
| Klebsiella Pneumoniae CMCC 46117 | Growth, colourless bacterium colony |
| Salmonella typhimurium CMCC 50115 | Growth, colourless bacterium colony |
| Staphylococcus aureus ATCC 25923 | It grows suppressed |
2, the growth rate evaluation of chromogenic culture medium
Escherichia coli (CMCC 44102) bacteria suspension after Zengjing Granule is respectively coated on to be made using the embodiment of the present invention 1
It is uncommon that large intestine angstrom is commonly used in standby escherichia coli chromogenic culture medium (ECM), food inspection national standard (GB4789.38-2012)
Bacterium is separately cultured on Yihong methylene blue culture medium (EMB) and nutrient agar (NA), after 36 ± 1 DEG C are cultivated 24 hours, grows
Situation is shown in Fig. 2.
As shown in Figure 2,1~2mm, the blue-green bacterium colony of neat in edge is presented in escherichia coli on ECM.It is on EMB
Existing darkviolet, with black center, have the colonies typical of metallic luster；It is full, smooth wet larger that form is presented on NA
Bacterium colony.The equal well-grown of escherichia coli, clump count no significant difference on three kinds of culture mediums；In terms of bacterium colony size, due to NA
For Nonsele ctive culture media, it being not added with inhibitor, therefore bacterium colony is larger, form is full, and object bacteria bacterium colony is smaller on EMB and ECM, but
Colony edge is neat, moistens, is smooth, and form is also typical, shows that ECM and EMB have certain inhibition to make object bacteria escherichia coli
With, but inhibition is weaker, has no significant effect to colonial morphology and quantity.
Due to the difference of bacterium solution coating, the growth rate of each ware culture medium has certain deviation, takes parallel 10 experiments, meter
Average life rate is calculated, statistical result is shown in Table 2.
Table 2: growth rate of the escherichia coli (CMCC 44102) on ECM and EMB culture medium
From Table 2, it can be seen that the average life rate of ECM is (98.13 ± 3.45) %, show that object bacteria is raw on ECM
Long good, ECM is smaller to the inhibiting effect of object bacteria；The average life rate of EMB be (97.91 ± 3.20) %, compare ECM with
The average life rate of EMB, the two no difference of science of statistics (P=0.88 > 0.05).
3, the Evaluation on specificity of chromogenic culture medium
20 plants of reference culture inoculation escherichia coli chromogenic culture medium ECM, French Kermas (unit of kinetic energy) are praised (CHRO) Escherichia coli and shown
Color culture medium and EMB culture medium, 36 ± 1 DEG C of cultures 18~for 24 hours, growing state of each reference culture on 3 kinds of culture mediums is observed,
As a result 3 be see the table below.
3 culture medium Evaluation on specificity result of table
As can be seen from Table 3, after culture 18~for 24 hours, on 2 kinds of chromogenic culture mediums, escherichia coli is presented 1~
2mm, the neat blue-green bacterium colony in edge, other bacteriums are suppressed in colourless bacterium colony or growth, i.e. the bacterium of escherichia coli Pseudomonas
Strain is positive in detection, other bacterial strains such as staphylococcus aureus, salmonella, Shigella etc. are negative, and shows the present invention
The escherichia coli chromogenic culture medium and same kind of products at abroad of preparation have good specificity, while having price advantage and behaviour
Make simplicity, there is good promotional value.
Simultaneously as can be seen from Table 3, escherichia coli chromogenic culture medium prepared by the present invention and escherichia coli detect
Common EMB culture medium, good (CHRO) e.coli chromogenic medium of French Kerma (unit of kinetic energy) all have good special in food standard
Property, but due to using EMB when carrying out the separation of escherichia coli, increase bacterium processing before need to carrying out, while also needing to combine oxidation
Enzyme test, methyl red (MR) test, VP test, urease experiment etc. biochemical reactions escherichia coli is identified, need compared with
Long detection cycle, and it is external product that French Kerma (unit of kinetic energy), which praises (CHRO) e.coli chromogenic medium, it is expensive, at present there is no
Formula is open, and chromogenic culture medium prepared by the present invention makes Preliminary Identification to bacterial strain by colony colour, shortens detection time,
Cost is reduced simultaneously, breaks technical barrier and monopolization, same technical effect is realized with external chromogenic culture medium, in food inspection
In have a good application prospect.
Embodiment 6
Application of the chromogenic culture medium in food inspection
Food samples are acquired from Putuo District, Shanghai City large supermarket and the market of farm produce, amount to 82 parts.Wherein 12 parts of vegetables,
10 parts of fruit, 16 parts of wheaten food (in bulk, independent packaging, quick-frozen food etc.), egg products 15 parts of (egg, duck's egg, goose egg, quail eggs
Deng), 13 parts of meat products (live fresh pork, beef, mutton, egg, duck and prepared food meat products), drink 11 parts of (dairy produces, beans
Slurry, beverage etc.), 5 parts of aquatic products.
Escherichia coli chromogenic culture medium (ECM), good (CHRO) large intestine of French Kerma (unit of kinetic energy) prepared using the embodiment of the present invention 1
Escherichia coli is commonly used in bacillus chromogenic culture medium and food inspection national standard (GB4789.38-2012) is separately cultured use
Yihong methylene blue culture medium (EMB) detects 82 parts of food samples, and testing result see the table below shown in 4~6.
Table 4: Comparative result of the above-mentioned three kinds of culture mediums in food samples detection
Table 5:ECM and CHRO culture medium count the testing result of food samples
Table 6:ECM and EMB culture medium count the testing result of food samples
From table 4~6 as it can be seen that the positive coincidence rate of ECM and CHRO is 91.89%, overall coincidence rate is 93.90%, kappa
Coefficient=0.85 > 0.75 is that height is consistent, shows that ECM and CHRO have good consistency to the detection of sample；ECM and EMB
Positive coincidence rate be 93.93%, overall coincidence rate be coefficient=0.83 > 0.75 91.46%, kappa, be height it is consistent, table
Bright ECM and EMB has good consistency to the detection of sample.Therefore, the testing result of actual food product sample shows the present invention
It is functional to provide escherichia coli chromogenic culture medium, has a good application prospect in food inspection.
It is distinctive using escherichia coli using the chromogenic culture medium identified for Escherichia coli of the invention and its application
Glucuroide adds specific chromogenic substrate, by the interaction release colour developing group of enzyme and substrate, according to colony colour
Identification is made to bacterial strain, the nutrient media components are simple, easy to operate, and detection sensitivity is high, specificity is good, reduce inspection it is positive and
False negative, and detection cycle is short, it, can be uncommon to large intestine angstrom in food suitable for handling large batch of sample, being easy to industrialization production
Bacterium carries out comprehensive, system, accurate detection and preliminary identification, and new way is provided for Escherichia Coli in Food pollution detection
Diameter.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the invention.Therefore, the description and the appended drawings should be considered as illustrative
And not restrictive.
Claims (8)
1. a kind of chromogenic culture medium for Escherichia coli identification, which is characterized in that the chromogenic culture medium includes to nitro
Phenyl-β-D- glucopyranoside, ortho-nitrophenol β-D glucosiduronate, 4- nitrobenzophenone-β-D- glucopyranoside, 4- first
One of base umbrella shape keto acyl-β-D-Glucose thuja acid or the chloro- 3- indoxyl-N- acetyl-β-D- glucosaminide of the bromo- 4- of 5-
The glucoside enzyme interacting of chromogenic substrate, the chromogenic substrate and Escherichia coli and discharge colour developing group.
2. the chromogenic culture medium according to claim 1 for Escherichia coli identification, which is characterized in that the colour developing training
Feeding base further includes agar, peptone, lactose, sodium chloride and cholate.
3. the chromogenic culture medium according to claim 1 or 2 for Escherichia coli identification, which is characterized in that the training
Feeding base includes component: 10~15g/L of agar, 5~15g/L of peptone, 5~15g/L of lactose, 3~10g/L of sodium chloride, cholate 0.5
~5g/L, 0.05~0.5g/L of chromogenic substrate.
4. the chromogenic culture medium according to claim 1 or 2 for Escherichia coli identification, which is characterized in that the training
Feeding base includes component: agar 15g/L, peptone 5g/L, lactose 11.4g/L, sodium chloride 3g/L, and the bromo- 4- of cholate 2g/L, 5- is chloro-
3- indoxyl-N- acetyl-β-D- glucosaminide 0.15g/L.
5. the chromogenic culture medium according to claim 1 or 2 for Escherichia coli identification, which is characterized in that the training
Feeding base includes component: agar 12g/L, peptone 15g/L, lactose 5g/L, sodium chloride 5g/L, cholate 1g/L, 4-methyl umbelliferone
Acyl-β-D-Glucose thuja acid 0.1g/L.
6. the chromogenic culture medium according to claim 1 or 2 for Escherichia coli identification, which is characterized in that the training
The pH value for supporting base is 7.2 ± 0.2.
7. the chromogenic culture medium according to claim 1 or 2 for Escherichia coli identification, which is characterized in that described is aobvious
Color base group is in blue-green or the fluorescence blue under 366nm ultraviolet lamp under natural light.
8. a kind of application described in claim 1 for the chromogenic culture medium of Escherichia coli identification in food inspection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110564808A (en) * | 2019-08-08 | 2019-12-13 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | Selective chromogenic culture method for gluconacetobacter, acetobacter aceti and gluconobacter in fermented milk and special culture medium thereof |
| CN110819689A (en) * | 2019-12-23 | 2020-02-21 | 中国科学院长春应用化学研究所 | Culture medium and application thereof in detection of escherichia coli |
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Application publication date: 20190308 |