CN105039202A - Selective medium for multiplex proliferation of salmonella, listeria monocytogenes and vibrio parahaemolyticus and preparation method of selective medium - Google Patents
Selective medium for multiplex proliferation of salmonella, listeria monocytogenes and vibrio parahaemolyticus and preparation method of selective medium Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 241000607272 Vibrio parahaemolyticus Species 0.000 title claims abstract description 36
- 239000006152 selective media Substances 0.000 title claims abstract description 25
- 230000035755 proliferation Effects 0.000 title abstract 4
- 241000894006 Bacteria Species 0.000 claims abstract description 85
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- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229960000210 nalidixic acid Drugs 0.000 claims abstract description 47
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- 238000001514 detection method Methods 0.000 claims abstract description 18
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Abstract
The invention relates to the technical field of detection of pathogenic bacterium, and discloses a selective medium for multiplex proliferation of salmonella, listeria monocytogenes and vibrio parahaemolyticus. The selective medium is prepared from tryptone, peptone, sodium chloride, sodium dihydrogen phosphate, glucose, mannitol, esculin hydrate, sodium citrate, skim milk powder, sterile purified water, potassium tellurite solution, acriflavine solution and nalidixic acid solution. A preparation method of the selective medium comprises the following steps: preparation of potassium tellurite solution; preparation of acriflavine solution; preparation of nalidixic acid solution; preparation of a semi-finished product medium; addition of a sample; and addition of potassium tellurite solution, acriflavine solution and nalidixic acid solution in the medium. The selective medium can carry out multiplex proliferation on the target bacteria and inhibit non-target bacteria, is small in inhibiting effect on the target bacteria in the sub-lethal state so that the target bacteria in the sub-lethal state can realize effective proliferation. The preparation method of the selective medium is easy to operate and high in efficiency.
Description
Technical field
The present invention relates to the detection technique field of pathogenic bacterium, particularly relate to a kind of Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound and increase selective medium of bacterium and preparation method thereof.
Background technology
Food safety is a global great public health problem, and food contamination and food origin disease are at developed country and developing country's still ubiquity.China's Bacterial foodborne diseases is primarily of Salmonellas (Salmonella) and Vibrio parahaemolyticus (Vibrioparahaemolyticus)) cause.Listeria monocytogenes (ListeriaMonocytogenes) is though sickness rate is not high, and its lethality rate is far above other common foodborne bacterial pathogenses.Marine products based food as high protein, low-fat " plain boiled pork ", delicious flavour, nutritious, deeply like by consumers in general, but be subject to bacterial contamination and cause putrid and deteriorated, affect product safety.Therefore Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes import and export fishery products often to examine project.
At present cellar culture, biochemical identification are mainly adopted for Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes, mostly will expend 4-6 days time, and program is complicated, agents useful for same is various, bothersome effort, and detector efficiency is low, detection sensitivity degree is low, and false negative is more serious.Enzyme connection fluorescence immunoassay detects (VIDAS) polymerase chain reaction (PCR), gold test strip method, API method, polysaccharase immunologic detection method (EIA) and DNA probe innovation technology and is applied to microorganism detection gradually in recent years, greatly improves the sensitivity of detection and simplifies operation.But the detection sensitivity that can meet the demands still needs to be unable to do without front increasing bacterium or selective enrichment.Existing increasing bacterium method mainly carries out respective increasing bacterium process according to the bacterial strain that will detect, namely different bacterial strains adopts respective selective enrichment medium to carry out the process of increasing bacterium, waste time and energy, while not meeting present detection method one platform, detect the development trend of various pathogens.In domestic and international existing research, the research relating to common enrichment medium technology mainly comprises: Salmonellas and Shigellae increase technology, Salmonellas, intestinal bacteria and streptococcus aureus altogether and increase technology (SEL), Salmonellas, intestinal bacteria and Listeria monocytogenes altogether and increase technology and wide spectrum altogether and increase bacterial context soup (UPB) etc., except SEL, all the other all belong to non-selectivity enrichment medium, can not meet multiple background microorganism many time carry out increasing the requirement of bacterium to specific objective bacterium.In current detection method, different pathogenic bacterium have independently detection method, need to carry out increasing bacterium respectively.Therefore obtain a kind of can Sync enrichment Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes common increasing substratum, to realization altogether inspection have great importance.Up to now, the report increasing bacterium technology about Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes selectivity is altogether had no.
In addition, chilled fishery products fishing for, process, various damage can be subject in preserving process, make thalline be in sub-lethal state, easily produce false negative, easily cause undetected.Although the thalline of sub-lethal state has certain activity, but lose the Some Physiological Characters of normal thalline, this can cause when compound Zengjing Granule, selective substances in selective medium is when suppressing non-targeted bacterium, the object bacteria being in sub-lethal state is also suppressed simultaneously, the object bacteria being in sub-lethal state cannot be grown, thus cause detected result and reality inconsistent.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound and increasing selective medium of bacterium and preparation method thereof.Substratum of the present invention can carry out compound to object bacteria Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes simultaneously increases bacterium and suppress other non-targeted bacterium, and it is little to the object bacteria restraining effect being in sub-lethal state, make the object bacteria of sub-lethal state also can carry out increasing bacterium, enriching effect is good; Medium preparation method provided by the invention is simple to operate, and efficiency is high.
Concrete technical scheme of the present invention is: a kind of Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the selective medium of bacterium, and meter comprises following component by weight:
Tryptones 15-20 part, peptone 2-4 part, sodium-chlor 8-12 part, SODIUM PHOSPHATE, MONOBASIC 2-3 part, glucose 2-3 part, N.F,USP MANNITOL 2-3 part, Vitamin C2 0.01-0.03 part, Trisodium Citrate 0.5-1.5 part, skim-milk 4-6 part, sterile purified water 1000 parts, potassium hydroxide solution 0.1-1 part of 1mol/L, hydrochloric acid soln 0.1-1 part of 1mol/L, potassium tellurite solution 0.8-1.2 part, trypaflavine solution 0.8-1.2 part, nalidixic acid solution 0.8-1.2 part.
Described potassium tellurite solution is made up of 0.00005 part of potassium tellurite and 10 parts of sterile purified waters; Described trypaflavine solution is made up of 0.1 part of trypaflavine and 10 parts of sterile purified waters; The sodium hydroxide solution that described nalidixic acid solution is 0.05mol/L by 0.01 part of nalidixic acid and 10 parts of concentration forms.
In substratum of the present invention, with Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus for object bacteria, based on Tryptones, peptone, sodium-chlor and SODIUM PHOSPHATE, MONOBASIC, substratum provides nutrition source for mushroom.Glucose, N.F,USP MANNITOL, as growth stimulant, can promote fungus grown.Skim-milk, as recovery promotor, can promote the object bacteria recovery being in sub-lethal state.Trisodium Citrate is as the utilizable carbon source of object bacteria, and it then cannot be utilized for other numerous mushrooms.Vitamin C2, potassium tellurite solution, trypaflavine solution and nalidixic acid solution are as inhibitor, and they have optionally restraining effect, less to the restraining effect of object bacteria, stronger to the inhibition of other numerous mushrooms.
As preferably, the component counting described selective medium is by weight: Tryptones 17 parts, peptone 3 parts, 10 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, 2.5 parts, N.F,USP MANNITOL, Vitamin C2 0.02 part, Trisodium Citrate 1 part, skim-milk 5 parts, sterile purified water 1000 parts, potassium hydroxide solution 0.1-1 part of 1mol/L, hydrochloric acid soln 0.1-1 part of 1mol/L, potassium tellurite solution 1 part, trypaflavine solution 1 part, nalidixic acid solution 1 part.
As preferably, described selective medium also comprises 0.1-0.5 part tea saponin.The gold-coloured staphylococci, yeast, mould etc. of tea saponin to routine have stronger restraining effect, and not obvious to object bacteria restraining effect of the present invention, are suitable for the component making substratum of the present invention.
Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase a preparation method for the selective medium of bacterium, comprise the steps:
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use;
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use; The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use;
Take Tryptones 15-20 part, peptone 2-4 part, sodium-chlor 8-12 part, SODIUM PHOSPHATE, MONOBASIC 2-3 part, glucose 2-3 part, N.F,USP MANNITOL 2-3 part, Vitamin C2 0.01-0.03 part, Trisodium Citrate 0.5-1.5 part, skim-milk 4-6 part, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 0.16-0.24 part is added in mixture, trypaflavine solution 0.16-0.24 part, nalidixic acid solution 0.16-0.24 part, after mixing, obtained work in-process substratum,
In an aseptic environment, sample to be detected is added in work in-process substratum after homogenizer process, after mixing 2min, 4-8 hour is cultivated at 37 DEG C, then in work in-process substratum, potassium tellurite solution 0.64-0.96 part is evenly added, trypaflavine solution 0.64-0.96 part, nalidixic acid solution 0.64-0.96 part; Continue again to cultivate 16-20 hour at 37 DEG C.
In the preparation process of substratum of the present invention, the preparation of substratum is divided into two steps, in work in-process substratum, in substratum except adding nutrition source material, only with the addition of a small amount of inhibitor, while this inhibits non-targeted bacteria growing to a certain extent, reduce inhibitor to the restraining effect of the object bacteria of the lethal state in Asia as far as possible, add the exclusive nutrition source of the object bacteria of dosage, the object bacteria of sub-lethal state can be made to recover, grow.After the object bacteria recovery of the lethal state in Asia, then add the inhibitor of larger dose, suppress largely non-targeted bacterium, now the object bacteria of original sub-lethal state is recovered, and have stronger vigor, the restraining effect of inhibitor to it is not obvious.Object bacteria can be made effectively to carry out increasing bacterium at this substratum after cultivating, suppress the growth of non-targeted bacterium simultaneously.
As preferably, the amount of adding potassium tellurite solution when preparing work in-process substratum is 0.2 part, and the amount of adding trypaflavine solution is 0.2 part, and the amount of adding nalidixic acid solution is 0.2 part; After band detection sample adds work in-process substratum to, incubation time is 6 hours, and the amount of adding potassium tellurite solution to work in-process substratum is 0.8 part, and the amount of adding trypaflavine solution is 0.8 part, and the amount of adding nalidixic acid solution is 0.8 part; Incubation time is 18 hours.
As preferably, when evenly adding potassium tellurite solution, trypaflavine solution and nalidixic acid solution in described work in-process substratum, be also added with 0.1-0.5 part tea saponin simultaneously.
Be compared with the prior art, the invention has the beneficial effects as follows:
Substratum of the present invention can carry out compound to object bacteria Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes simultaneously increases bacterium and suppress other non-targeted bacterium, and it is little to the object bacteria restraining effect being in sub-lethal state, make the object bacteria of sub-lethal state also can carry out increasing bacterium, enriching effect is good.
Medium preparation method provided by the invention is simple to operate, and efficiency is high, effective.
Accompanying drawing explanation
Fig. 1 is Salmonellas fluorescence PCR detection reagent kit detected result in the present invention;
Fig. 2 is Vibrio parahaemolyticus fluorescence PCR detection reagent kit detected result in the present invention;
Fig. 3 is Listeria monocytogenes fluorescence PCR detection reagent kit detected result in the present invention.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the preparation of the selective medium of bacterium:
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use.
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use.
The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use.
Take Tryptones 17 parts, peptone 3 parts, 10 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, 2.5 parts, N.F,USP MANNITOL, Vitamin C2 0.02 part, Trisodium Citrate 1 part, skim-milk 5 parts, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 0.2 part is added in mixture, trypaflavine solution 0.2 part, nalidixic acid solution 0.2 part, after mixing, obtained work in-process substratum.
In an aseptic environment, 100 parts of samples to be detected are added in work in-process substratum after homogenizer process, after mixing 2min, cultivate 6 hours at 37 DEG C, then in work in-process substratum, evenly potassium tellurite solution 0.8 part is added, trypaflavine solution 0.8 part, nalidixic acid solution 0.8 part; Continue again to cultivate 18 hours at 37 DEG C.
Embodiment 2
Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the preparation of the selective medium of bacterium:
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use.
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use.
The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use.
Take Tryptones 17 parts, peptone 3 parts, 10 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, 2.5 parts, N.F,USP MANNITOL, Vitamin C2 0.02 part, Trisodium Citrate 1 part, skim-milk 5 parts, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 0.2 part is added in mixture, trypaflavine solution 0.2 part, nalidixic acid solution 0.2 part, after mixing, obtained work in-process substratum.
In an aseptic environment, 100 parts of samples to be detected are added in work in-process substratum after homogenizer process, after mixing 2min, cultivate 6 hours at 37 DEG C, then in work in-process substratum, evenly potassium tellurite solution 0.8 part is added, trypaflavine solution 0.8 part, nalidixic acid solution 0.8 part, tea saponin 0.3 part; Continue again to cultivate 18 hours at 37 DEG C.
Embodiment 3
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use.
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use.
The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use;
Take Tryptones 20 parts, peptone 4 parts, 8 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2 parts, glucose 3 parts, 3 parts, N.F,USP MANNITOL, Vitamin C2 0.003 part, Trisodium Citrate 1.5 parts, skim-milk 4 parts, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 0.16 part is added in mixture, trypaflavine solution 0.16 part, nalidixic acid solution 0.16 part, after mixing, obtained work in-process substratum.
In an aseptic environment, 100 parts of samples to be detected are added in work in-process substratum after homogenizer process, after mixing 2min, cultivate 8 hours at 37 DEG C, then in work in-process substratum, evenly potassium tellurite solution 0.84 part is added, trypaflavine solution 0.84 part, nalidixic acid solution 0.84 part, tea saponin 0.2 part; Continue again to cultivate 16 hours at 37 DEG C.
Comparative example
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use.
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use.
The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use.
Take Tryptones 17 parts, peptone 3 parts, 10 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, 2.5 parts, N.F,USP MANNITOL, Vitamin C2 0.02 part, Trisodium Citrate 1 part, skim-milk 5 parts, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 1 part is added in mixture, trypaflavine solution 1 part, nalidixic acid solution 1 part, after mixing, obtained substratum.
In an aseptic environment, 100 parts of samples to be detected are added in substratum after homogenizer process, after mixing 2min, cultivate 24 hours at 37 DEG C.
Bacterial number in embodiment 1-3 and comparative example substratum is measured, find that embodiment 1-3 and comparative example difference are, the many 10-15% of quantity of the number ratio comparative example of the 3 kinds of object bacteria detected in embodiment 1-3, major cause is the object bacteria that is in sub-lethal state in comparative example because the high inhibition effect of inhibitor cannot be recovered, in false negative, and embodiment 1-3 can make the object bacteria of a large amount of sub-lethal state recover and grow.
Pure bacterium detects:
Prepare multiple identical substratum by preparation method of the present invention and access respectively and be diluted to 10
-4salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes three kinds of object bacteria and miscellaneous bacteria bacterium liquid 0.2mL at 37 DEG C, cultivate 24h.The OD value under 540nm is measured with visible spectrophotometer.Do blank with buffered peptone water, the results are shown in Table 1:
Table 1: object bacteria and the growing state of non-targeted bacterium separately in the substratum of optimum proportioning
| Bacterial strain | Blank | 1 | 2 | 3 |
| Salmonellas | 0.59 | 1.41 | 1.16 | 1.32 |
| Vibrio parahaemolyticus | 0.65 | 0.85 | 0.74 | 0.86 |
| Listeria monocytogenes | 0.62 | 0.89 | 0.76 | 0.89 |
| Intestinal bacteria | 0.56 | 0.23 | 0.21 | 0.19 |
| Escherichia coli O 157 | 0.56 | 0 | 0 | 0 |
| Shigella flexneri | 0.67 | 0 | 0 | 0 |
| Bacillus ceylonensis A | 0.59 | 0 | 0 | 0 |
| Vibrio cholerae | 0.61 | 0.11 | 0.09 | 0.13 |
| Vibrio vulnificus | 0.67 | 0 | 0 | 0 |
| Vibrio alginolyticus | 0.56 | 0 | 0 | 0 |
| Streptococcus aureus | 0.54 | 0 | 0 | 0 |
Note: 1-3 is three and repeats experiment
In above-mentioned substratum, grow enriching effect after 24 hours better than the buffering protein peptone of non-selectivity for three kinds of object bacteria Salmonellass, Vibrio parahaemolyticus and Listeria monocytogenes as can be seen from Table 1; The growth of major part non-targeted bacterium receives suppression, intestinal bacteria and vibrio cholerae bacterium liquid is only had to become muddy, but these two kinds of bacterium grow and obviously have received suppression in described substratum compared with the buffered peptone water of blank non-selectivity, their growth is also obvious slow than 3 kinds of object bacteria.
Mixed bacterium detects:
According to Salmonellas: Vibrio parahaemolyticus: the ratio of Listeria monocytogenes is that 1:1:1 accesses in substratum, cultivates 24 hours, bacterium liquid is diluted to 10 for 37 DEG C
-6, be applied to BS agar plate respectively, on TCBS agar plate and PALCAM flat board, carry out separation and Culture.Salmonellas is blackish green circular colonies on BS agar, and Vibrio parahaemolyticus and Listeria monocytogenes do not grow; Vibrio parahaemolyticus is blue-greenish colour bacterium colony on TCBS flat board, and bacterium colony is rounded, neat in edge, moistening; Slightly muddy, translucent, most tool the point heart, bamboo hat shape, diameter 2-4mm, Salmonellas and Listeria monocytogenes do not grow; Listeria monocytogenes is circular greyish-green bacterium colony on PALCAM flat board, and have brownish black to be hydrolyzed circle around, some bacterium colony has black to cave in, and Salmonellas and Vibrio parahaemolyticus do not grow.Growth result is in table 2.Result shows, entered after within 24 hours, increasing bacterium, Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes can be bred to 10 simultaneously
6, the bacteria concentration requirement of late detection can be met.
Table 2 compound enrichment medium enriching effect
| Object bacteria | Inoculum size (CFU/mL) | Bacterium amount (CFU/mL) after increasing bacterium |
| Salmonellas | 10-100 | 2300000 |
| Vibrio parahaemolyticus | 10-100 | 3500000 |
| Listeria monocytogenes | 10-100 | 3600000 |
Artificial application of sample detects:
Aseptically, by peeled shrimp (through fluorescent PCR qualification not containing three kinds of object bacteria), be prepared into 25g respectively and pulverize.The culture of about 100CFU/g is inoculated in sample, under room temperature, processes 15min, be the other uniform pickup of bacterium liquid, then put it in the self-made medium containing 225mL, mixing 2min.The sample that homogeneous is good is cultivated 24 hours in 37 DEG C.DNA of bacteria is extracted by DNA extraction kit, detect with Salmonellas, Vibrio parahaemolyticus and Listeria monocytogenes fluorescence PCR detection reagent kit, detected result as shown in Figure 1, Figure 2, Figure 3 shows, all there is obvious DNA cloning curve in visible three kinds of object bacteria, illustrate that meeting enrichment medium makes the Salmonellas in sample, Vibrio parahaemolyticus and Listeria monocytogenes three kinds of object bacteria increase simultaneously, and reach the bacteria concentration requirement of PCR detection.Prove after compound enrichment medium is cultivated, can be used for PCR and detect.
The above; it is only preferred embodiment of the present invention; not the present invention is imposed any restrictions, every above embodiment is done according to the technology of the present invention essence any simple modification, change and equivalent transformation, all still belong to the protection domain of technical solution of the present invention.
Claims (6)
1. Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase a selective medium for bacterium, it is characterized in that counting by weight comprising following component:
Tryptones 15-20 part, peptone 2-4 part, sodium-chlor 8-12 part, SODIUM PHOSPHATE, MONOBASIC 2-3 part, glucose 2-3 part, N.F,USP MANNITOL 2-3 part, Vitamin C2 0.01-0.03 part, Trisodium Citrate 0.5-1.5 part, skim-milk 4-6 part, sterile purified water 1000 parts, potassium hydroxide solution 0.1-1 part of 1mol/L, hydrochloric acid soln 0.1-1 part of 1mol/L, potassium tellurite solution 0.8-1.2 part, trypaflavine solution 0.8-1.2 part, nalidixic acid solution 0.8-1.2 part;
Described potassium tellurite solution is made up of 0.00005 part of potassium tellurite and 10 parts of sterile purified waters; Described trypaflavine solution is made up of 0.1 part of trypaflavine and 10 parts of sterile purified waters; The sodium hydroxide solution that described nalidixic acid solution is 0.05mol/L by 0.01 part of nalidixic acid and 10 parts of concentration forms.
2. Salmonellas as claimed in claim 1, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the selective medium of bacterium, it is characterized in that, the component counting described selective medium is by weight: Tryptones 17 parts, peptone 3 parts, 10 parts, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC 2.5 parts, glucose 2.5 parts, 2.5 parts, N.F,USP MANNITOL, Vitamin C2 0.02 part, Trisodium Citrate 1 part, skim-milk 5 parts, sterile purified water 1000 parts, potassium hydroxide solution 0.1-1 part of 1mol/L, hydrochloric acid soln 0.1-1 part of 1mol/L, potassium tellurite solution 1 part, trypaflavine solution 1 part, nalidixic acid solution 1 part.
3. Salmonellas as claimed in claim 1, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the selective medium of bacterium, and it is characterized in that, described selective medium also comprises 0.1-0.5 part tea saponin.
4. Salmonellas, Listeria monocytogenes and Vibrio parahaemolyticus compound increase a preparation method for the selective medium of bacterium, it is characterized in that comprising the steps:
The preparation of potassium tellurite solution: take potassium tellurite 0.00005 part and join in 10 parts of sterile purified waters, obtains potassium tellurite solution and for subsequent use;
The preparation of trypaflavine solution: take trypaflavine 0.1 part and join in 10 parts of sterile purified waters, obtains trypaflavine solution and for subsequent use;
The preparation of nalidixic acid solution: taking nalidixic acid 0.01 part, to join 10 parts of concentration be in the sodium hydroxide solution of 0.05mol/L, obtains nalidixic acid solution and for subsequent use;
Take Tryptones 15-20 part, peptone 2-4 part, sodium-chlor 8-12 part, SODIUM PHOSPHATE, MONOBASIC 2-3 part, glucose 2-3 part, N.F,USP MANNITOL 2-3 part, Vitamin C2 0.01-0.03 part, Trisodium Citrate 0.5-1.5 part, skim-milk 4-6 part, successively join in 1000 parts of sterile purified waters, mix the potassium hydroxide solution of rear 1mol/L and the hydrochloric acid soln of 1mol/L by gained mixture pH regulator to 7.5, autoclaving 15min, then wait for that mixture is cooled to 50 DEG C, aseptically, potassium tellurite solution 0.16-0.24 part is added in mixture, trypaflavine solution 0.16-0.24 part, nalidixic acid solution 0.16-0.24 part, after mixing, obtained work in-process substratum,
In an aseptic environment, sample to be detected is added in work in-process substratum after homogenizer process, after mixing 2min, 4-8 hour is cultivated at 37 DEG C, then in work in-process substratum, potassium tellurite solution 0.64-0.96 part is evenly added, trypaflavine solution 0.64-0.96 part, nalidixic acid solution 0.64-0.96 part; Continue again to cultivate 16-20 hour at 37 DEG C.
5. Salmonellas as claimed in claim 4, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the preparation method of the selective medium of bacterium, it is characterized in that, the amount of adding potassium tellurite solution when preparing work in-process substratum is 0.2 part, the amount of adding trypaflavine solution is 0.2 part, and the amount of adding nalidixic acid solution is 0.2 part; After band detection sample adds work in-process substratum to, incubation time is 6 hours, and the amount of adding potassium tellurite solution to work in-process substratum is 0.8 part, and the amount of adding trypaflavine solution is 0.8 part, and the amount of adding nalidixic acid solution is 0.8 part; Incubation time is 18 hours.
6. Salmonellas as claimed in claim 4, Listeria monocytogenes and Vibrio parahaemolyticus compound increase the preparation method of the selective medium of bacterium, it is characterized in that, when evenly adding potassium tellurite solution, trypaflavine solution and nalidixic acid solution in described work in-process substratum, be also added with 0.1-0.5 part tea saponin simultaneously.
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