CN106498023A - A kind of Listeria monocytogenes chromogenic culture medium and test piece - Google Patents
A kind of Listeria monocytogenes chromogenic culture medium and test piece Download PDFInfo
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Abstract
The invention discloses a kind of Listeria monocytogenes chromogenic culture medium and test piece, culture medium is made up of peptone, yeast extract powder, sodium chloride, Sodium Pyruvate, lithium chloride, magnesium glycerophosphate, enzyme-specific chromogenic substrate and miscellaneous bacteria inhibitor.The chromogenic culture medium of the present invention, the usage amount of enzyme-specific chromogenic substrate are few, and the cost of culture medium is greatly reduced.The chromogenic culture medium of the present invention, preparation method are simple, and sterilization steps are simple, simplify the requirement to sterilising conditions, are very beneficial for the use that unit detects in basic unit.The test piece of the present invention is easy to use, can complete the detection of Listeria monocytogenes in 37 DEG C of incubated 24～36 h, can save substantial amounts of manpower and materials after sample-adding.
Description
Technical field
The invention belongs to microbiological Test field, more particularly to a kind of colour developing culture for detecting Listeria monocytogenes
Base and the test piece made of chromogenic culture medium.
Background technology
Listeria monocytogenes (Listeria monocytogenes) are widely present in nature, and it is dynamic to be that one kind can cause
Thing and the Main Pathogenic Bacteria of mankind's listeriosis.Pregnant woman, infant, immune tolerance patient infectious age up to 20%～
30%, far above other common foodborne bacterial pathogenses.The bacterium is four one of big food origin disease pathogenic bacteria nineties in 20th century,
Through being classified as one of food-borne germ of emphasis detection by WHO.
The national standard method (GB/T 4789.30-2010) of Listeria monocytogenes inspection, from sampling, increases bacterium, then with difference
Culture medium isolated and purified, then carry out Physiology and biochemistry identification need 7 days, prepare and round-off work heavier, operating process
Loaded down with trivial details, more manpower and materials are needed, is unfavorable for the timely inspection and supervision of Listeria monocytogenes, the reality for limiting the method should
With.The detection that microorganism is carried out using quick, accurate, convenient method is a kind of discovery trend.
The specific enzyme-to-substrate contact produced by observing or detecting pathogenic microorganism produces color change, and develops
The enzyme-specific developing technology that comes, with required time is short, sensitivity is high, the high advantage of high specificity and accuracy rate.
CN104388522A discloses a kind of Listeria monocytogenes selective coloration culture medium, wherein contains per 1000mL culture mediums as follows
The component of weight：The heart-brain extract 5-20g, agar 2-6g, peptone 2-5g, enzyme-specific chromogenic substrate 0.1-1g, dimethyl
Formamide 0.05-20g, magnesium sulfate 0.2-0.5g, lithium chloride 0.2-0.5g, oxygen scavenger 0.3-0.6g, 3- morpholino propane -1- sulphurs
Sour 0.2-1.0g, miscellaneous bacteria inhibitor 6-17g, balance of water；The enzyme-specific chromogenic substrate is the chloro- 3- indoles-fleshes of the bromo- 4- of 5-
Alcohol -1- phosphate.Chromogenic culture medium in the program needs to carry out 120 DEG C of sterilizing 15min of high steam, and the consumption of developer
Larger, production cost is of a relatively high.
CN105349611A discloses Listeria monocytogenes quick detection cold water gel test paper in a kind of food and Dining tool
Piece and preparation method and application, belongs to microbial safety inspection technical field.The present invention utilizes cold water gel to chromatograph
Filter paper is carrier, medium component, developer and cold water gel is mixed, is equably loaded in chromatography filter paper, and sample can be equal
Even be distributed in cold water gel, form the colony characteristicses of distinctness on its surface, realize Listeria monocytogenes quick and precisely qualitative, fixed
Amount detection.And the present invention is with new compound (2R, 3S, 4R, 5R, 6R) -1,2,3,4,5- pentahydroxy- butylcyclohexyls-(2- nitro -4-
Fluorophenyl) phosphate is developer, compared with traditional Listeria monocytogenes chromogenic culture medium, its sensitivity that reacts and special
Property is significantly better than conventional medium, and resistance to irradiation, and chemical stability is higher.In addition, test-paper obtained in the present invention is with low cost,
Method of operating is easy, completes, and detection time only needs 24～36h by being simplified to one-step method.The program depends critically upon new
Developer, synthesis procedure are complex, and testing cost is relatively high.
As developer is typically in occupation of most of cost of chromogenic culture medium, develops a kind of enzyme-specific chromogenic substrate and use
The low chromogenic culture medium of amount, reduces the cost of chromogenic culture medium and test piece, it may have very actual meaning.
In addition, existing chromogenic culture medium generally needs to use autoclaving equipment, and a lot of basic unit's detection units do not have
Standby this sterilising conditions, develop one kind and can heat the i.e. spendable culture medium of boiling sterilization, Ke Yi great under normal conditions
The disinfecting action of big simplified culture base, beneficial to promoting the use of.
Content of the invention
It is an object of the invention to provide a kind of Listeria monocytogenes chromogenic culture medium and being prepared using the culture medium
Fast test strip.
The technical solution used in the present invention is：
A kind of Listeria monocytogenes chromogenic culture medium, contains peptone 7-25g, yeast extract powder in every 1000mL culture mediums
4-10g, sodium chloride 4-15g, Sodium Pyruvate 0.5-3g, lithium chloride 0.5-5g, magnesium glycerophosphate 0.5-3g, enzyme-specific colour developing bottom
Thing 0.02-0.15g, miscellaneous bacteria inhibitor 20-200mg.
As the further improvement of above-mentioned Listeria monocytogenes chromogenic culture medium, in every 1000mL culture mediums, contain peptone
8-18g, dusty yeast 5-8g, sodium chloride 5-9g, Sodium Pyruvate 1-3g, lithium chloride 0.5-3g, magnesium glycerophosphate 1-3g, enzyme-specific
Chromogenic substrate 0.04-0.12g, miscellaneous bacteria inhibitors 4 0-150mg.
Used as the further improvement of above-mentioned Listeria monocytogenes chromogenic culture medium, enzyme-specific is inositol monophosphate phosphatidase.
Enzyme-specific chromogenic substrate is the bromo- 4- chloro-3-hydroxyls indoles inositolophosphates of 5-.
Used as the further improvement of above-mentioned Listeria monocytogenes chromogenic culture medium, miscellaneous bacteria inhibitor is by cefotaxime, sulfuric acid
Polymyxin, nalidixic acid, anphotericin composition.Cefotaxime, sulfuric acid polymyxin, nalidixic acid, anphotericin mass ratio
For 1：(1-4)：(2-6)：(1-3).
As the further improvement of above-mentioned Listeria monocytogenes chromogenic culture medium, in every 1000mL culture mediums, fine jade is also added with
Fat 12-18g.
A kind of Listeria monocytogenes test piece, are followed successively by base plate from down to up, water absorbent gel layer, filter paper layer and upper epiphragma,
Filter paper layer is adsorbed with above-mentioned Listeria monocytogenes chromogenic culture medium.
A kind of Listeria monocytogenes detection method, shows including sample drop is added to the above-mentioned Listeria monocytogenes containing agar
In color culture medium, or on Listeria monocytogenes test piece, 37 DEG C incubated 24～36 hours, and chromogenic culture medium tests piece
Upper there is glaucous bacterium point as Listeria monocytogenes positive bacterium colony.
The invention has the beneficial effects as follows：
1) chromogenic culture medium of the invention, the usage amount of enzyme-specific chromogenic substrate are few, and the cost of culture medium is greatly reduced.
2) chromogenic culture medium of the invention, preparation method are simple, and sterilization steps are simple, simplify and sterilising conditions are wanted
Ask, be very beneficial for the use that unit detects in basic unit.
3) chromogenic culture medium of the invention is easy to use, can complete single increasing in 37 DEG C of incubated 24～36h after sample-adding
Listerial detection, can save substantial amounts of manpower and materials.
Description of the drawings
Fig. 1 Listeria monocytogenes chromogenic culture medium is inoculated with Listeria monocytogenes design sketch；
Fig. 2 Listeria monocytogenes test piece inoculation Listeria monocytogenes design sketch；
Fig. 3 Listeria monocytogenes chromogenic culture medium and test piece shelf-life test chart.
Specific embodiment
The culture medium of the present invention and test piece can be prepared according to a conventional method, for the sake of being easy to compare, the training of the present invention
Foster base and test piece are prepared as follows obtaining.
The preparation method of culture medium is as follows：
1) developer dimethylformamide dissolves, and makes mother liquor and adds mixing in culture medium；
2) by the composition in addition to bacteriostatic agent, the stirring that adds water is boiled to boiling, is kept for boiling 3-5 minutes after all dissolution of raw material
Sterilized；
3) when culture medium temperature is down to 40～50 DEG C, add miscellaneous bacteria inhibitor composition and mix, you can be down flat
Plate, is prepared into corresponding chromogenic culture medium flat board.
The preparation method of test piece is as follows：
1) according to water absorbent gel each component proportions water absorbent gel, which is uniformly coated on base plate, 25 DEG C of dehumidifiers are gone
Water, makes the base plate for scribbling water absorbent gel；
2) developer dimethylformamide dissolves, and makes mother liquor and is added in the culture medium without agar composition, mixes；
3) filter paper is added in culture medium absorption culture medium, on the base plate for being then affixed to scribble water absorbent gel, 25 DEG C are taken out
Wet remove water；
4) epiphragma is sticked, and co-60 radiation sterilizes, that is, make test piece.
Embodiment 1
Contain peptone 7g, yeast extract powder 10g, sodium chloride 4g, Sodium Pyruvate 3g, lithium chloride in per 1000mL culture mediums
0.5g, magnesium glycerophosphate 3g, enzyme-specific chromogenic substrate 0.02g, miscellaneous bacteria inhibitor 200mg.Wherein enzyme-specific chromogenic substrate
For the bromo- 4- chloro-3-hydroxyls indoles inositol monophosphate sodium of 5-.Mentioned component is dissolved in the water of 1000mL, agar 12g, heating is added
Boil twice, treat that culture medium is cooled to 40-50 DEG C, it is 1 to add miscellaneous bacteria/inhibitor mixture mass ratio：4：6：3 cefotaximes, sulphur
Sour polymyxin, nalidixic acid, anphotericin, mix and can use.
Embodiment 2
Contain peptone 25g, yeast extract powder 4g, sodium chloride 15g, Sodium Pyruvate 0.5g, chlorine in per 1000mL culture mediums
Change lithium 5g, magnesium glycerophosphate 0.5g, enzyme-specific chromogenic substrate 0.07g, miscellaneous bacteria inhibitor 20mg.Wherein enzyme-specific colour developing bottom
Thing is the bromo- 4- chloro-3-hydroxyls indoles inositol monophosphate potassium of 5-.Mentioned component is dissolved in the water of 1000mL, agar 15g is added, plus
Heat is boiled twice, treats that culture medium is cooled to 40-50 DEG C, and it is 1 to add miscellaneous bacteria/inhibitor mixture mass ratio：1：2：1 cefotaxime,
Sulfuric acid polymyxin, nalidixic acid, anphotericin, mix and can use.
Embodiment 3
Contain peptone 16g, yeast extract powder 6g, sodium chloride 5g, Sodium Pyruvate 2.5g, chlorination in per 1000mL culture mediums
Lithium 1.5g, magnesium glycerophosphate 1.5g, enzyme-specific chromogenic substrate 0.15g, miscellaneous bacteria inhibitor 120mg.Wherein enzyme-specific colour developing
Substrate is the bromo- 4- chloro-3-hydroxyls indoles inositol monophosphate ammoniums of 5-.Mentioned component is dissolved in the water of 1000mL, agar 18g is added,
Heating is boiled twice, treats that culture medium is cooled to 40-50 DEG C, and it is 1 to add miscellaneous bacteria/inhibitor mixture mass ratio：3：4：2 cephalos he
Pyridine, sulfuric acid polymyxin, nalidixic acid, anphotericin, mix and can use.
Embodiment 4
Listeria monocytogenes test piece, and including base plate, water absorbent gel layer, filter paper layer and upper epiphragma, base plate are waterproof, no
Antibacterial synthetic material composition, water absorbent gel layer is 1 by mass ratio：1：1：20 trehalose：Sodium Polyacrylate：Guar gum：Carboxylic first
Base sodium cellulosate constitute, filter paper material be non-woven fabrics, be adsorbed with 1 Listeria monocytogenes chromogenic culture medium of embodiment be not added with agar into
It is grouped into.Upper epiphragma is transparent, permeable watertight, is printed on the grid of square 1cm × 1cm.
Embodiment 5
Listeria monocytogenes test piece, and including base plate, water absorbent gel layer, filter paper layer and upper epiphragma, base plate are waterproof, no
Antibacterial synthetic material composition, water absorbent gel layer is 1 by mass ratio：4：5：60 trehalose：Sodium Polyacrylate：Guar gum：Carboxylic first
Base sodium cellulosate constitute, filter paper material be non-woven fabrics, be adsorbed with 2 Listeria monocytogenes chromogenic culture medium of embodiment be not added with agar into
It is grouped into.Upper epiphragma is transparent, permeable watertight, is printed on the grid of square 1cm × 1cm.
Embodiment 6
Listeria monocytogenes test piece, and including base plate, water absorbent gel layer, filter paper layer and upper epiphragma, base plate are waterproof, no
Antibacterial synthetic material composition, water absorbent gel layer is 1 by mass ratio：2：2：40 trehalose：Sodium Polyacrylate：Guar gum：Carboxylic first
Base sodium cellulosate constitute, filter paper material be non-woven fabrics, be adsorbed with 3 Listeria monocytogenes chromogenic culture medium of embodiment be not added with agar into
It is grouped into.Upper epiphragma is transparent, permeable watertight, is printed on the grid of square 1cm × 1cm.
1～3 Listeria monocytogenes chromogenic culture medium of embodiment makes flat board, after sample-adding 37 DEG C incubated 24～36 hours
Observation result；4～6 Listeria monocytogenes of embodiment test piece, 37 DEG C of incubated 24～36 hours observation results after sample-adding.Aobvious
There is glaucous bacterium point on color culture medium or test piece and be Listeria monocytogenes positive bacterium colony (as depicted in figs. 1 and 2).
Specificity experiments
By Listeria monocytogenes ATCC19115, Ying Nuoke Listeria ATCC33090, EHEC ATCC25922,
Not citric acid bacillus ATCC43864, pseudomonas aeruginosa CMCC (B) 10104, staphylococcus aureus ATCC6538, dysentery will
Congratulate bacterium CMCC (B) 51252, Candida albicans ATCC10231, Enterobacter sakazakii ATCC29544, secondary arc hemolytic bacterium
ATCC17802, enteritis door sand bacterium CMCC (B) 50335, Bacillus cereus CMCC (B) 63303, streptococcus fecalis ATCC29212.
By all of reference culture rejuvenation after purification, 10 are made with SPSS2～103The bacteria suspension of cfu/mL concentration, is connect respectively
Plant Listeria monocytogenes chromogenic culture medium (embodiment 1～3) and the Listeria monocytogenes test piece (embodiment 4 in the present invention
～6), and bacteria suspension concentration (0.6% ferment of Listeria monocytogenes and Ying Nuoke Listerias is detected with plate count agar PCA
The trypticase soy agar TSA-YE plate counts of female medicinal extract), two repetitions of each setting.36 DEG C ± 1 DEG C be inverted culture 24～
Observation log (being shown in Table 1) after 36h.
Table 1, specific test result
Note："+" represents positive findings, has blue-green bacterium point to occur；"-" represents negative findings, and aseptic point grows or nothing
Blue-green point occurs.
As can be seen from Table 1, only Listeria monocytogenes can be grown on chromogenic culture medium and test piece, other 12 kinds
Quality-control strains do not grow system or colour developing on chromogenic culture medium and test piece for other colors, are negative findings, and instruction sheet increases
The specificity of Listeria chromogenic culture medium and test piece is good.
Minimum detectability is tested
After by purified for Listeria monocytogenes reference culture rejuvenation, 10 times are prepared into SPSS and are serially diluted mark
Quasi- bacteria suspension, to 10-8The later standard bacteria of dilution factor carries out sesquialter dilution, then by each dilution factor bacteria suspension respectively with this
Bright middle Listeria monocytogenes chromogenic culture medium and test piece are detected, while being contrasted with national standard method, each setting two
Individual repetition, observes after 36 DEG C ± 1 DEG C inversion 24～36h of culture and records experimental result (being shown in Table 2).
Table 2, minimum detectability result of the test
As can be seen from Table 2, Listeria monocytogenes chromogenic culture medium and test piece all have very high sensitivity, single increasing Lee
This special bacterium chromogenic culture medium LDL can reach 1CFU/mL, and Listeria monocytogenes test piece minimum detectability can reach
2CFU/mL is arrived, the National Standard Method culture medium detection bottom line of control is 3CFU/mL (being shown in Table 2).
Artificial contaminated bacteria samples are tested
Will be through GB/T 4789.30 2010《National food safety standard, food microbiological examination, single increasing Liszt
Bacterium is checked》Be detected as the negative sample of Listeria monocytogenes, sterile working weigh 25g (mL) sample be added to equipped with 225mL without
In the aseptic homogenizing bag of bacterium physiological saline, 2min is smashed with homogenizer.By Listeria monocytogenes, Ying Nuoke Listerias, large intestine
Escherichia, staphylococcus aureus Quality-control strains are in nutrient broth (trypticase of the Listeria monocytogenes with 0.6% yeast extract
Soybean broth TSB-YE) in respectively after incubated overnight activation, make plastc ring according to random ratio, draw 1mL
Plastc ring is added in sample liquid, makes artificial contamination's sample, after placing 4～5h, selects suitable three gradients difference
Carried out with Listeria monocytogenes chromogenic culture medium, Listeria monocytogenes test piece, commercialization OXOID Listerias chromogenic culture medium
Test, two repetitions of each setting.
Table 3, artificial contaminated bacteria samples sampling test
As can be seen from Table 3, Listeria monocytogenes chromogenic culture medium of the invention and OXOID Listeria chromogenic culture mediums
Coincidence rate be 95.60%；The test piece made by the Listeria monocytogenes chromogenic culture medium of the present invention and OXOID colour developing trainings
The coincidence rate of foster base is 102.39%.With the coincidence rate of the Listeria chromogenic culture medium in National Standard Method more than 80%, explanation
Effect is good.
Shelf-life tests
The packaged Listeria monocytogenes chromogenic culture medium of production and Listeria monocytogenes test piece is taken, 4- is respectively placed in
10 DEG C of refrigerators, 25 DEG C, 45 DEG C are stored respectively.According to 3 days, 7 days, 15 days, 30 days, 90 days, 180 days, 270 days, 360 days,
It is inoculated with Quality-control strains Listeria monocytogenes ATCC19115 standard bacteria suspensions respectively and is tested within 540 day, 720 days, while with
OXOID Listeria chromogenic culture mediums are contrasted, and under different temperatures, the change curve result of coincidence rate is shown in Fig. 3.Single increasing Li Si
Special bacterium chromogenic culture medium and the test of test piece shelf-life are as shown in table 4.
Table 4, Listeria monocytogenes chromogenic culture medium and the test of test piece shelf-life
As can be seen from Table 4, Listeria monocytogenes chromogenic culture medium is preserved 720 days at 4-10 DEG C；25 DEG C of preservation 180d；45
DEG C preserve 15 days and OXOID chromogenic culture mediums coincidence rate all more than 80%.Illustrate the culture medium storage requirement be 4-10 DEG C
Refrigerator store 2 years.Listeria chromogenic culture medium, can be with short time (less than 15 days) under conditions of not possessing cold chain distribution
Normal temperature is transported.Listeria monocytogenes are tested piece and are preserved 540 days at 4-10 DEG C；25 DEG C of preservation 90d；45 DEG C of preservations are shown for 7 days with OXOID
Color culture medium coincidence rate is all more than 80%.Illustrate the culture medium storage requirement be 4-10 DEG C of Refrigerator store a year and a half.The product
Product can be transported with short time (less than 7 days) normal temperature under conditions of not possessing cold chain distribution.
Claims (10)
1. a kind of Listeria monocytogenes chromogenic culture medium, contains peptone 7-25 g, yeast extract powder in every 1000 mL culture mediums
4-10 g, sodium chloride 4-15 g, Sodium Pyruvate 0.5-3 g, lithium chloride 0.5-5 g, magnesium glycerophosphate 0.5-3 g, enzyme-specific
Chromogenic substrate 0.02-0.15 g, miscellaneous bacteria inhibitor 20-200 mg.
2. Listeria monocytogenes chromogenic culture medium according to claim 1, it is characterised in that：In every 1000 mL culture mediums
Containing peptone 8-18 g, dusty yeast 5-8 g, sodium chloride 5-9 g, Sodium Pyruvate 1-3 g, lithium chloride 0.5-3 g, phosphoglycerol
Magnesium 1-3 g, 0.04-0.12 g of enzyme-specific chromogenic substrate, miscellaneous bacteria inhibitors 4 0-150 mg.
3. Listeria monocytogenes chromogenic culture medium according to claim 1 and 2, it is characterised in that：Enzyme-specific is inositol
Phosphoric acid phosphatidase.
4. Listeria monocytogenes chromogenic culture medium according to claim 3, it is characterised in that：Enzyme-specific chromogenic substrate is
The bromo- 4- chloro-3-hydroxyls indoles inositolophosphates of 5-.
5. Listeria monocytogenes chromogenic culture medium according to claim 1 and 2, it is characterised in that：Miscellaneous bacteria inhibitor is by head
Spore his pyridine, sulfuric acid polymyxin, nalidixic acid, anphotericin composition.
6. Listeria monocytogenes chromogenic culture medium according to claim 5, it is characterised in that：Cefotaxime, sulfuric acid are sticked more
Rhzomorph, nalidixic acid, anphotericin mass ratio are 1：（1-4）：（2-6）：（1-3）.
7. Listeria monocytogenes chromogenic culture medium according to claim 1 and 2, it is characterised in that：Every 1000 mL culture mediums
In be also added with agar 12-18 g.
8. a kind of Listeria monocytogenes test piece, are followed successively by base plate from down to up, water absorbent gel layer, filter paper layer and upper epiphragma, its
It is characterised by：Filter paper layer is adsorbed with the Listeria monocytogenes chromogenic culture medium described in claim 1～6 any one.
9. Listeria monocytogenes according to claim 8 test piece, it is characterised in that：Water absorbent gel layer is consisted of：Sea
Algae sugar：Sodium Polyacrylate：Guar gum：Sodium carboxymethylcellulose mass ratio is=1：（1-4）：（1-5）：（20-60）.
10. a kind of Listeria monocytogenes detection method, including sample drop to be added to the Listeria monocytogenes described in claim 7
In chromogenic culture medium, or on the Listeria monocytogenes test piece of claim 8 and 9,37 DEG C incubated 24～36 hours, shows
Occur glaucous bacterium point on color culture medium or test piece and be Listeria monocytogenes positive bacterium colony.
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