CN102703565B - Chromogenic culture medium for separating and detecting shigella - Google Patents
Chromogenic culture medium for separating and detecting shigella Download PDFInfo
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- 241000607768 Shigella Species 0.000 title abstract description 9
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- 229930006000 Sucrose Natural products 0.000 claims abstract description 17
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a chromogenic culture medium for separating and detecting shigella; the culture medium comprises yeast powder, tryptone, soy peptone, sodium chloride, lactose, cane sugar, 2-deoxy-D-ribose, agar powder, beta-glucosaccharase chromogenic substrate, beta-pyranfucosidase chromogenic substrate, beta-galactosidase chromogenic substrate, phenol red, 3# cholate, novobiocin sodium salt, cefsulodin and cefixime; residue is water; the chromogenic culture medium provided by the invention can be used for separating and detecting four species of shigella and has advantages of high specificity, high sensitivity, easy operation, simple result judgment and the like; the chromogenic culture medium is suitable for detecting each sample and has wide application prospect; and detecting effect of the chromogenic culture medium can achieve level of like products in an import R&F company and is superior to inland like products.
Description
Technical field
The present invention relates to a kind of substratum of microorganism detection, relate in particular to a kind of for separating of the color developing culture medium with detecting Shigellae.
Background technology
Shigella (Shigella) belongs to enterobacteriaceae, and four kinds are arranged, be respectively shigella dysenteriae (
shigella dysenteriae), shigella flexneri (
shigellaflexneri), Shigella bogdii (
shigellaboydii), Shigella sonnei (
shigellasonnei).The ulcer colon that four kinds of Shigella all can produce bacillary dysentery in human body infects, and is most important pathogenic bacteria in bacillary dysentery.This pathogenic bacteria is the food by manure contamination or water-borne generally.All have the report that causes in a large number disease because of shigella infection every year all over the world, and China is subject to the threat of this bacterium also increasing.Therefore, how fast, separate and identify Shigellae from the samples such as food that contain a large amount of other bacteriums accurately, specifically and have important practical significance.
At present the detection of Shigellae be take to selective medium as main, its principle is to utilize Shigellae do not utilize the glucides such as lactose, sucrose to produce sour characteristic and distinguish with other enterobacteria.But because biological characteristics and other bacterium of enterobacteriaceae of Shigellae are as closely similar as intestinal bacteria, Salmonellas etc., thereby cause a large amount of false positives and false negative result occurring for detection of traditional selective medium of Shigellae.Some detection methods once advised detecting single sample with three kinds of different selective mediums simultaneously, and to increase the accuracy of substratum, this makes sepn process expensive and loaded down with trivial details.Although having in recent years some occurs based on molecular biology and immunologic method for quick, but on the one hand,, these methods can not be directly used in the detection of object bacteria, on the other hand, these methods need technical professional or expensive instrument, and its application is limited by very large.Compare, utilizing enzymic activity detection and identification bacterium is a kind of effective means, and this technology can be quantitatively and the preliminary evaluation object bacteria, and easy and simple to handle, increased substantially working efficiency.
Yet do not find at present the specific enzymes that Shigellae is expressed temporarily, make the color developing culture medium exploitation of Shigellae be restricted.Therefore the Shigellae color developing culture medium utilizes specific enzymes to make other bacterium colour developing mostly at present, thereby distinguishes mutually with the Shigellae of not developing the color.United States Patent (USP) (Pat. No. 5,871,944) described a kind of color developing culture medium of differentiating Salmonellas and Shigellae from a mixt bacteria sample, the characteristic that this substratum utilizes Shigellae not produce beta-galactosidase enzymes is added corresponding substrate in substratum.Yet on the one hand, this substratum can not be distinguished Salmonellas and Shigellae; On the other hand, other bacterium that does not produce beta-galactosidase enzymes can not be distinguished mutually with Shigellae, thereby cause false positive.Although at present existing, utilize Salmonellas to produce hydrogen sulfide to show that on substratum evil mind distinguishes the Shigellae color developing culture medium of Salmonellas, but the Salmonellas that does not produce hydrogen sulfide still can not distinguish with Shigellae, and it is not obvious that the Salmonellas of some product hydrogen sulfide produces evil mind in substratum, also can cause false positive results.
In sum, the technical bottleneck for separating of the substratum with detecting Shigellae is at present: on the one hand, a kind of substratum is difficult to detect four kinds of Shigellae simultaneously, causes undetected phenomenon; On the other hand, the enterobacteriaceae lactobacteriaceaes such as Shigellae and Salmonellas are difficult to distinguish, and cause false positive results.
Summary of the invention
The object of the present invention is to provide a kind of for separating of the color developing culture medium with detecting Shigellae.
The technical solution used in the present invention is:
A kind of for separating of the color developing culture medium with detecting Shigellae, every 1000 mL substratum contain yeast powder 2～10 g, Tryptones 2～7 g, soy peptone 2～10 g, sodium-chlor 4～7 g, lactose 3～15g, sucrose 3～15 g, DRI 3～15 g, agar powder 12～20 g, beta-glucosidase chromogenic substrate 0.05～0.5 g, β-pyrans fucosidase chromogenic substrate 0.05～0.5 g, beta-galactosidase enzymes chromogenic substrate 0.05～0.5 g, pH indicator 0.03～0.09 g, cholate 2～9 g, Novobiocin sodium salt 1～5 mg, cefsulodin 1～10 mg, Cefixime Micronized 0.01～0.1 mg, surplus is water.
Preferably, the beta-glucosidase chromogenic substrate is the chloro-3-indoles-beta-glucoside of the bromo-4-of 5-.
Preferably, β-pyrans fucosidase chromogenic substrate is the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside.
Preferably, the beta-galactosidase enzymes chromogenic substrate is the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides.
Preferably, cholate is at least one in sodium deoxycholate, pig cholate, mixing cholate, bovine bile and no. 3 bile salt.
Preferably, cholate is no. 3 bile salt.
Preferably, the pH indicator is phenol red.
Preferably, comprise yeast powder 4 g, Tryptones 3 g, soy peptone 6 g, sodium-chlor 5 g, lactose 5 g, sucrose 5 g, DRI 5 g, chloro-3-indoles-beta-glucoside 0.4 g of agar powder 15 g, the bromo-4-of 5-, the chloro-3-indoles of the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.2 g, bromo-4-of 5--N-acetyl-beta galactose glycosides 0.4 g, phenol red 0.08 g, no. 3 bile salt 6 g, Novobiocin sodium salt 4 mg, cefsulodin 9 mg, Cefixime Micronized 0.03 mg in every 1000 mL substratum, surplus is water.
It is phenol red that the present invention has added carbohydrate and indicator that Shigellae do not utilize in substratum.The carbohydrate that Shigellae does not utilize comprises: sorbyl alcohol, Pentitol, DRI, inositol, wood sugar, cellobiose, lactose, galactitol, sucrose, raffinose, salicin, can utilize one or more in these carbohydrate to produce sour bacterium, the color shown under the effect of pH indicator, this bacterium and Shigellae can be made a distinction, the present invention makes fermenting carbohydrate produce sour bacterium and demonstrates yellow bacterium colony under the preferred phenol red effect of pH indicator, thereby distinguishes with Shigellae.The preferred lactose of the present invention, sucrose and DRI.
The present invention has added the chromogenic substrate of beta-glucosidase, β-pyrans fucosidase and beta-galactosidase enzymes in substratum, the bacterium of at least one in these three kinds of enzymes of energy expression generation is decomposed substrate under the effect of enzyme, chromophoric group dissociates, make bacterium colony present the color and luster of chromophoric group, thereby distinguish mutually with Shigellae.
The present invention has added cholate in substratum, can suppress most gram positive bacterium in sample.
Substratum of the present invention has also added microbiotic.Microbiotic can suppress some bacteria growing, and as tellurite can be used to delay the growth of colon bacillus, Novobiocin sodium salt and Cefixime Micronized can suppress the growth of Bacillus proteus, and cefsulodin can suppress the bacterium of Rhodopseudomonas one class.The preferred Novobiocin sodium salt of the present invention, cefsulodin and Cefixime Micronized.
The feature that bacterium presents in substratum of the present invention has:
1) do not utilize lactose, sucrose and DRI to produce acid and the microbes that do not produce beta-glucosidase, β-pyrans fucosidase and beta-galactosidase enzymes forms the first color bacterium colony (white), Shigellae presents the first color bacterium colony;
2) can utilize at least one material in lactose, sucrose and DRI to produce sour microorganism, form the second color bacterium colony (yellow) under the phenol red effect of pH indicator;
3) can produce in beta-glucosidase, β-pyrans fucosidase and beta-galactosidase enzymes the microorganism of at least one, form the third color bacterium colony (green);
4) can utilize at least one material in lactose, sucrose and DRI to produce acid and produce in beta-glucosidase, β-pyrans fucosidase and beta-galactosidase enzymes at least one microorganism, form the blend color of the second and the third color, thereby form the bacterium colony (blue-greenish colour or blackish green) of the 4th kind of color; Above four kinds of colors are easily distinguished.
Color developing culture medium of the present invention, for separating of the method with detecting Shigellae, comprises the steps:
1) the dull and stereotyped preparation that develops the color: each component by above-mentioned color developing culture medium except Novobiocin sodium salt, cefsulodin, Cefixime Micronized, DRI, join in deionized water, stir, heated and boiled, to dissolving fully, is regulated pH to 6.8 ± 0.2, to be cooled to 45～55 ℃, the Novobiocin sodium salt, cefsulodin, Cefixime Micronized, the DRI that add filtration sterilization, mix, be down flat plate, standby;
2) sample preparation: the sample processing method according to each field regulation and stipulation is processed;
3) inoculation culture: by sample or to be inoculated into colour developing containing the line of the enrichment liquid of sample or coating dull and stereotyped upper, cultivate 20～24 h for 37 ℃;
4) interpretation of result: if the dull and stereotyped upper substratum of colour developing reddens, occur that clear white or colourless translucent, diameter are 1～3 mm, there is no Pigmented bacterium colony, illustrate that this bacterium colony is suspicious Shigellae bacterium colony, this bacterium colony can be chosen and further done biochemical identification, other bacterium or suppressed or present yellow, green, blue-greenish colour, blackish green.
The invention has the beneficial effects as follows:
Color developing culture medium of the present invention can be used for the separation and detection of four kinds of Shigella, has the advantages such as high specificity, highly sensitive, easy handling, result judgement are simple, is suitable for the detection of various samples, has wide practical use.Color developing culture medium detects effect can reach import R& F company like product level, be better than domestic existing like product.
Color developing culture medium of the present invention greatly reduces the appearance of false positive and false negative result, and application prospect is very widely arranged.The separation and detection of Shigellae in the sample that color developing culture medium of the present invention is applicable to comprise a large amount of other enterobacteriaceaes, especially, substratum of the present invention can be distinguished Salmonellas and Shigellae well.
Color developing culture medium of the present invention than substratum colony colour in the past still less, more easily detects Shigellae reliably, and Shigellae presents white in substratum of the present invention, the bright-coloured yellow can be clearly presented with other bacterium or the color differentiating such as green.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but do not limit to so.
embodiment 1
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 4 g, Tryptones 3 g, soy peptone 6 g, sodium-chlor 5 g, lactose 5 g, sucrose 5 g, DRI 5 g, agar powder 15 g, chloro-3-indoles-beta-glucoside 0.4 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.2 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.4 g, phenol red 0.08 g, no. 3 bile salt 6 g, Novobiocin sodium salt 4 mg, cefsulodin 9 mg, Cefixime Micronized 0.03 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 2
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 6 g, Tryptones 3 g, soy peptone 7 g, sodium-chlor 5 g, lactose 3 g, sucrose 8 g, DRI 7 g, agar powder 18 g, chloro-3-indoles-beta-glucoside 0.2 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.1 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.2 g, phenol red 0.06 g, no. 3 bile salt 5 g, Novobiocin sodium salt 3 mg, cefsulodin 3 mg, Cefixime Micronized 0.05 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 3
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 8 g, Tryptones 2 g, soy peptone 6 g, sodium-chlor 5 g, lactose 5 g, sucrose 5 g, DRI 10 g, agar powder 15 g, chloro-3-indoles-beta-glucoside 0.1 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.2 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.4 g, phenol red 0.04 g, no. 3 bile salt 7 g, Novobiocin sodium salt 2 mg, cefsulodin 8 mg, Cefixime Micronized 0.07 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 4
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 7 g, Tryptones 6 g, soy peptone 3 g, sodium-chlor 5 g, lactose 8 g, sucrose 3 g, DRI 12 g, agar powder 15 g, chloro-3-indoles-beta-glucoside 0.2 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.4 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.3 g, phenol red 0.08 g, no. 3 bile salt 5 g, Novobiocin sodium salt 3 mg, cefsulodin 7 mg, Cefixime Micronized 0.015 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 5
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 2 g, Tryptones 5 g, soy peptone 4 g, sodium-chlor 4 g, lactose 3 g, sucrose 9 g, DRI 15 g, agar powder 17 g, chloro-3-indoles-beta-glucoside 0.3 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.5 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.05 g, phenol red 0.09 g, no. 3 bile salt 9 g, Novobiocin sodium salt 1 mg, cefsulodin 6 mg, Cefixime Micronized 0.1 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 6
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 5 g, Tryptones 4 g, soy peptone 10 g, sodium-chlor 6 g, lactose 10 g, sucrose 15 g, DRI 8 g, agar powder 12 g, chloro-3-indoles-beta-glucoside 0.5 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.05 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.1 g, phenol red 0.03 g, no. 3 bile salt 4 g, Novobiocin sodium salt 5 mg, cefsulodin 10 mg, Cefixime Micronized 0.01 mg, surplus is deionized water, pH is 6.8 ± 0.2.
embodiment 7
A kind of for detection of the color developing culture medium with separating Shigellae, every 1000 mL substratum contain yeast powder 10 g, Tryptones 7 g, soy peptone 2 g, sodium-chlor 7 g, lactose 15 g, sucrose 3 g, DRI 3 g, agar powder 20 g, chloro-3-indoles-beta-glucoside 0.05 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.3 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.5 g, phenol red 0.05 g, no. 3 bile salt 2 g, Novobiocin sodium salt 3 mg, cefsulodin 1 mg, Cefixime Micronized 0.05 mg, surplus is deionized water, pH is 6.8 ± 0.2.
the specificity experiment
22 strain reference cultures described in table 1 are made respectively to 10
8～10
9the bacteria suspension of CFU/mL, get respectively a ring streak inoculation on the flat board (being called for short HKM) that described in case study on implementation 1, substratum is made, and cultivates 24 h for 37 ℃, observes growing state and the colony characteristics of each bacterium, the results are shown in Table 2.Annotate: in table 2, the colony growth situation means with several districts of growth, and 4th district mean well-grown, the bacterium colony normal in size, and 1st～3 district means to grow suppressed, and bacterium colony is less than normal or grow and be less than 4th district.
As shown in Table 2,7 strain Shigellaes all form white, bacterium colony clearly on substratum, and bacillus ceylonensis A has the transparent ring of clear white, all shows positive findings, except shigella dysenteriae CMCC (B) 51056 bacterium colonies are less than normal, all the other 6 strain Shigellae well-growns.Non-object bacteria or suppressed, or the bacterium colony of formation and Shigellae different colours, can distinguish with Shigellae preferably.Experimental result shows that the color developing culture medium for the Shigellae separation and detection of the present invention has higher specificity.
The described 22 strain reference cultures of table 1 are prepared into to 10
7～10
8the bacteria suspension of CFU/mL, get respectively flat board (being called for short HKM) and contrast that ring streak inoculation substratum described in the case study on implementation 1 makes dull and stereotyped upper, cultivates 24 h for 37 ℃, the colour developing situation of observation bacterium colony, and experimental result is shown in Table 3.
Control medium has: R& The Shigellae color developing culture medium of F company (is called for short R& F), the HE agar (being called for short HE) of the wood sugar-Methionin of the Shigellae color developing culture medium of domestic manufacturer (being called for short domestic manufacturer), OXOID company-deoxycholate agar (being called for short XLD), OXOID company.Shigellae feature in above-mentioned control medium is respectively, R& F is that substratum becomes redness, forms clear white or colourless translucent bacterium colony; Domestic manufacturer is that substratum becomes redness, and bacterium colony is white or colourless translucent; XLD is that substratum becomes redness, colourless translucent colony; HE is that substratum becomes to become deep green, and bacterium colony is green or light green.
As shown in Table 3, Shigellae all shows positive findings in above-mentioned 5 kinds of substratum.For other non-Shigellae, at HKM and R& On the F flat board, except proteus vulgaris and Shigellae, phase region is exceptionally well, other bacterium all can be distinguished preferably with Shigellae, and at home on producer, XLD and HE flat board, not producing the Salmonellas of hydrogen sulfide can not distinguish with Shigellae as Salmonella paratyphi A, Pseudomonas aeruginosa can not be distinguished with Shigellae in addition, and false positive rate is higher.Show Shigellae color developing culture medium of the present invention and R& The F flat board is equal no significant difference aspect specificity and colour developing, all is better than the like product of XLD, HE and domestic manufacturer.
sensitivity experiment
7 strain Shigellae reference cultures described in table 1 are made respectively to 10
2～10
3the CFU/mL bacteria suspension, get 100 μ L and be inoculated into respectively substratum described in case study on implementation 1, R& with the spiral inoculation method; On the flat board that 5 kinds of substratum such as the HE agar of the wood sugar-Methionin of F company Shigellae color developing culture medium, domestic manufacturer's Shigellae color developing culture medium, OXOID company-deoxycholate agar, OXOID company are made, cultivate 24 h for 37 ℃, calculate the concentration of bacteria suspension by colony number, the results are shown in Table 4.
As shown in Table 4, aspect detection sensitivity, XLD and HE's is better, is secondly HKM and R& F, both no significant differences, all be better than the like product of domestic manufacturer.Aspect the bacterium colony size, shigella dysenteriae CMCC (B) 51056 is slightly less than at R& on HKM; On F, XLD and HE, shigella flexneri ATCC 12022 is less on XLD and HE, and bacterium colony is not of uniform size, and suppressed phenomenon is arranged, and other 5 strain Shigellae is at HKM, R& Big or small no significant difference on F, XLD, tetra-kinds of substratum of HE, all be better than domestic manufacturer's like product.Shigellae color developing culture medium of the present invention and R& The F like product is no significant difference aspect detection sensitivity, is better than domestic manufacturer's like product.
In sum, described in the invention have the advantages such as high specificity, highly sensitive, easy handling, result judgement are simple for separating of the color developing culture medium with detecting Shigellae.Detect effect and can reach import R& F company like product level, be better than domestic existing like product.In addition, the color developing culture medium for the Shigellae separation and detection described in the invention is compared with traditional selective medium, greatly reduces the appearance of false positive and false negative result, and application prospect is very widely arranged.
Although the present invention is described concrete case study on implementation, the present invention is not limited thereto, and industry technician should be appreciated that under principle of the present invention can make multiple modification and change to the present invention.Therefore, this invention is intended to be encompassed in all these modifications and the change in claims and coordinator scope thereof.
Claims (1)
1. one kind for separating of the color developing culture medium with detecting Shigellae, every 1000 mL substratum contain yeast powder 4 g, Tryptones 3 g, soy peptone 6 g, sodium-chlor 5 g, lactose 5 g, sucrose 5 g, DRI 5 g, agar powder 15 g, chloro-3-indoles-beta-glucoside 0.4 g of the bromo-4-of 5-, the chloro-3-indoles-β of the bromo-4-of 5--pyrans fucoside 0.2 g, the chloro-3-indoles of the bromo-4-of 5--N-acetyl-beta galactose glycosides 0.4 g, phenol red 0.08 g, no. 3 bile salt 6 g, Novobiocin sodium salt 4 mg, cefsulodin 9 mg, Cefixime Micronized 0.03 mg, surplus is deionized water, pH is 6.8 ± 0.2.
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