CN105039202B - A kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious and preparation method thereof - Google Patents
A kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious and preparation method thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 85
- 241000607142 Salmonella Species 0.000 title claims abstract description 37
- 241000186779 Listeria monocytogenes Species 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 241000607598 Vibrio Species 0.000 title claims abstract description 31
- 150000001875 compounds Chemical class 0.000 title claims abstract description 20
- 239000006152 selective media Substances 0.000 title claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 50
- 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 46
- 229960000210 nalidixic acid Drugs 0.000 claims abstract description 46
- BGLGAKMTYHWWKW-UHFFFAOYSA-N acridine yellow Chemical compound [H+].[Cl-].CC1=C(N)C=C2N=C(C=C(C(C)=C3)N)C3=CC2=C1 BGLGAKMTYHWWKW-UHFFFAOYSA-N 0.000 claims abstract description 42
- BFPJYWDBBLZXOM-UHFFFAOYSA-L potassium tellurite Chemical compound [K+].[K+].[O-][Te]([O-])=O BFPJYWDBBLZXOM-UHFFFAOYSA-L 0.000 claims abstract description 41
- 239000011265 semifinished product Substances 0.000 claims abstract description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- 108010080698 Peptones Proteins 0.000 claims abstract description 16
- 239000001888 Peptone Substances 0.000 claims abstract description 15
- 235000019319 peptone Nutrition 0.000 claims abstract description 15
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- 239000008103 glucose Substances 0.000 claims abstract description 12
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 12
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 12
- 239000001509 sodium citrate Substances 0.000 claims abstract description 12
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 12
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 12
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- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims abstract description 11
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 11
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 239000012137 tryptone Substances 0.000 claims abstract description 11
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- 235000010355 mannitol Nutrition 0.000 claims abstract description 8
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- 239000000203 mixture Substances 0.000 claims description 19
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- 238000011534 incubation Methods 0.000 claims description 2
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- 238000013519 translation Methods 0.000 claims description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the detection technique field of pathogenic bacteria, discloses a kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious, including：Tryptone, peptone, sodium chloride, sodium dihydrogen phosphate, glucose, mannitol, aesculin, sodium citrate, skimmed milk power, sterile purified water, potassium tellurite solution, acridine yellow solution, nalidixic acid solution.The preparation method of the culture medium includes：The preparation of potassium tellurite solution；The preparation of acridine yellow solution；The preparation of nalidixic acid solution；The preparation of semi-finished product culture medium；The addition of sample；The addition of potassium tellurite solution, acridine yellow solution, nalidixic acid solution in culture medium.The culture medium of the present invention can carry out compound increasing bacterium to object bacteria and suppress non-targeted bacterium simultaneously, and small to the object bacteria inhibitory action in sub- lethal state, make it also being capable of Effective enrichment；The culture medium preparation method of the present invention is simple to operate, efficiency high.
Description
Technical field
The present invention relates to the detection technique field of pathogenic bacteria, more particularly to a kind of salmonella, Listeria monocytogenes and pair
Compound selective medium for increasing bacterium of hemolysis vibrion and preparation method thereof.
Background technology
Food security is a global great public health problem, and food pollution and food origin disease are in developed country
With developing country's still generally existing.China's Bacterial foodborne diseases are mainly by salmonella (Salmonella) and secondary haemolysis
Vibrios (Vibrio parahaemolyticus)) trigger.Listeria monocytogenes (Listeria Monocytogenes) though morbidity
Rate is not high, but its fatal rate is far above other common foodborne bacterial pathogenses.Marine products based food as high protein, low fat it is " white
Meat ", delicious flavour is nutritious, deep to be liked by consumers in general, but is easily caused by germ contamination putrid and deteriorated, influences product
Safety.Therefore salmonella, vibrio parahaemolytious and Listeria monocytogenes are into and out saliva product and often examine project.
Cellar culture, biochemical identification are mainly used for salmonella, vibrio parahaemolytious and Listeria monocytogenes at present, greatly
4-6 days time is expended more, and program is complicated, and agents useful for same is various, and bothersome laborious, detector efficiency is low, detection sensitivity degree
Low, false negative is than more serious.Enzyme-linked fluorescence immunoassay detection (VIDAS) PCR (PCR), gold test strip in recent years
Method, API methods, polymerization enzyme immunity detection method (EIA) and DNA probe innovation technology are gradually applied to microorganism detection, greatly
It is big to improve the sensitivity detected and simplify operation.But it disclosure satisfy that the detection sensitivity of requirement still needs and be unable to do without preceding increasing bacterium
Or selective enrichment.The existing bacterium method that increases is mainly to carry out respective increasing bacterium processing according to the bacterial strain to be detected, i.e., different
Bacterial strain using respective selective enrichment medium carry out increase bacterium processing, waste time and energy, it is flat not meet present detection method one
The development trend of various pathogens is detected while platform.In domestic and international existing research, it is related to the research of common enriched medium technology
Mainly include：Salmonella and Shigella increase technology, salmonella, Escherichia coli and staphylococcus aureus and increase technology altogether altogether
(SEL), salmonella, Escherichia coli and Listeria monocytogenes increase technology altogether and wide spectrum increases bacterial context soup (UPB) etc., in addition to SEL
Remaining belongs to non-selectivity enriched medium, it is impossible to meets specific objective bacterium is carried out when a variety of background microorganisms are more to increase bacterium
It is required that.In current detection method, different pathogenic bacteria have independent detection method, it is necessary to carry out increasing bacterium respectively.Therefore one is obtained
The common increasing culture medium of kind energy Sync enrichment salmonella, vibrio parahaemolytious and Listeria monocytogenes, to realizing, cubing has important altogether
Meaning.So far, the report for increasing bacterium technology altogether on salmonella, vibrio parahaemolytious and Listeria monocytogenes selectivity is had no
Road.
In addition, chilled aquatic products can be made thalline be in sub- lethal in fishing, processing, preserving process by various damages
State, false negative is easily produced, easily cause missing inspection.Although the thalline of sub- lethal state loses with certain activity
The Some Physiological Characters of normal thalline, this can cause in compound Zengjing Granule, and the selective substances in selective medium exist
When suppressing non-targeted bacterium, while the object bacteria in sub- lethal state is also suppressed, make the mesh in sub- lethal state
Mark bacterium can not grow, so as to cause testing result and reality inconsistent.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of salmonella, Listeria monocytogenes and secondary haemolysis arc
Compound selective medium for increasing bacterium of bacterium and preparation method thereof.The present invention culture medium can simultaneously to object bacteria salmonella,
Vibrio parahaemolytious and Listeria monocytogenes carry out compound increasing bacterium and suppress other non-targeted bacterium, and to the mesh in sub- lethal state
Mark bacterium inhibitory action is small, makes the object bacteria of sub- lethal state can also carry out increasing bacterium, and enriching effect is good；Culture medium provided by the invention
Preparation method is simple to operate, efficiency high.
The present invention concrete technical scheme be：A kind of salmonella, Listeria monocytogenes and the compound increasing bacterium of vibrio parahaemolytious
Selective medium, in parts by weight meter include following component：
Tryptone 15-20 parts, peptone 2-4 parts, sodium chloride 8-12 parts, sodium dihydrogen phosphate 2-3 parts, glucose 2-3 parts,
Mannitol 2-3 parts, aesculin 0.01-0.03 parts, sodium citrate 0.5-1.5 parts, skimmed milk power 4-6 parts, sterile purified water 1000
Part, 1mol/L potassium hydroxide solution 0.1-1 parts, 1mol/L hydrochloric acid solution 0.1-1 parts, potassium tellurite solution 0.8-1.2 parts,
Acridine yellow solution 0.8-1.2 parts, nalidixic acid solution 0.8-1.2 parts.
The potassium tellurite solution is made up of 0.00005 part of potassium tellurite and 10 parts of sterile purified waters；The acridine yellow is molten
Liquid is made up of 0.1 part of acridine yellow and 10 parts of sterile purified waters；The nalidixic acid solution is dense by 0.01 part of nalidixic acid and 10 parts
Spend the sodium hydroxide solution composition for 0.05mol/L.
In the culture medium of the present invention, using salmonella, Listeria monocytogenes and vibrio parahaemolytious as object bacteria, tryptose
Culture medium provides nutrient source for mushroom based on peptone, peptone, sodium chloride and sodium dihydrogen phosphate.Glucose, mannitol conduct
Growth promoter, fungus grown can be promoted.Skimmed milk power can promote the mesh in sub- lethal state as recovery accelerator
Mark bacterium recovery.Sodium citrate is as the utilizable carbon source of object bacteria, and it can not then be utilized for other numerous mushrooms.Seven
Leaf glycosides, potassium tellurite solution, acridine yellow solution and nalidixic acid solution are as inhibitor, and their selective suppression are made
With, it is smaller to the inhibitory action of object bacteria, it is stronger to the inhibition of other numerous mushrooms.
Preferably, the component for counting the selective medium in parts by weight is：17 parts of tryptone, 3 parts of peptone,
10 parts of sodium chloride, 2.5 parts of sodium dihydrogen phosphate, 2.5 parts of glucose, 2.5 parts of mannitol, 0.02 part of aesculin, 1 part of sodium citrate,
5 parts of skimmed milk power, 1000 parts of sterile purified water, 1mol/L potassium hydroxide solution 0.1-1 parts, 1mol/L hydrochloric acid solution 0.1-
1 part, 1 part of potassium tellurite solution, 1 part of acridine yellow solution, 1 part of nalidixic acid solution.
Preferably, the selective medium also includes 0.1-0.5 part Tea Saponins.Golden grape of the Tea Saponin to routine
Coccus, saccharomycete, mould etc. have stronger inhibitory action, and to the object bacteria inhibitory action unobvious of the present invention, it is suitable for
The component of culture medium of the present invention.
A kind of preparation side of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious
Method, comprise the following steps：
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain Asia
Telluric acid potassium solution is simultaneously standby；
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution
It is and standby；The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the hydroxide that 10 parts of concentration are 0.05mol/L
In sodium solution, nalidixic acid solution and standby is obtained；
Weigh tryptone 15-20 parts, peptone 2-4 parts, sodium chloride 8-12 parts, sodium dihydrogen phosphate 2-3 parts, glucose 2-
3 parts, mannitol 2-3 parts, aesculin 0.01-0.03 parts, sodium citrate 0.5-1.5 parts, skimmed milk power 4-6 parts, successively it is added to
In 1000 parts of sterile purified waters, gained is mixed with 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution after well mixed
Compound pH is adjusted to 7.5, autoclaving 15min, then waits mixture to be cooled to 50 DEG C, aseptically, to mixture
Middle addition potassium tellurite solution 0.16-0.24 parts, acridine yellow solution 0.16-0.24 parts, nalidixic acid solution 0.16-0.24 parts,
After well mixed, semi-finished product culture medium is made；
In an aseptic environment, sample to be detected is added in semi-finished product culture medium after homogenizer is handled, mixed
After 2min, 4-8 hours are cultivated at 37 DEG C, potassium tellurite solution 0.64-0.96 is then uniformly added into semi-finished product culture medium
Part, acridine yellow solution 0.64-0.96 parts, nalidixic acid solution 0.64-0.96 parts；It is further continued for cultivating 16-20 hours at 37 DEG C.
In the preparation process of the culture medium of the present invention, the preparation of culture medium is divided into two steps, in semi-finished product culture medium,
In culture medium in addition to adding nutrient source material, a small amount of inhibitor is only with the addition of, this inhibits non-targeted to a certain extent
While bacteria growing, inhibitory action of the inhibitor to the object bacteria of sub- lethal state is reduced as far as possible, along with the object bacteria of dosage
Exclusive nutrient source, object bacteria recovery, the growth of sub- lethal state can be made.After the object bacteria recovery of sub- lethal state, then
The inhibitor of larger dose is added, non-targeted bacterium is largely suppressed, the object bacteria of now original sub- lethal state
It has been recovered that, there are stronger vigor, inhibitory action unobvious of the inhibitor to it.After culture the culture medium can make mesh
Mark bacterium effectively carries out increasing bacterium, while suppresses the growth of non-targeted bacterium.
Preferably, the amount that potassium tellurite solution is added when preparing semi-finished product culture medium is 0.2 part, addition acridine yellow is molten
The amount of liquid is 0.2 part, and the amount of addition nalidixic acid solution is 0.2 part；Trained after band detection sample is added to semi-finished product culture medium
It is 6 hours to support the time, and the amount that potassium tellurite solution is added to semi-finished product culture medium is 0.8 part, and the amount of addition acridine yellow solution is
0.8 part, the amount of addition nalidixic acid solution is 0.8 part；Incubation time is 18 hours.
Preferably, potassium tellurite solution, acridine yellow solution and naphthyridines are uniformly being added into the semi-finished product culture medium
During ketone acid solution, while also it is added with 0.1-0.5 part Tea Saponins.
It is compared with the prior art, the beneficial effects of the invention are as follows：
The culture medium of the present invention can be answered object bacteria salmonella, vibrio parahaemolytious and Listeria monocytogenes simultaneously
Close and increase bacterium and suppress other non-targeted bacterium, and it is small to the object bacteria inhibitory action in sub- lethal state, make sub- lethal state
Object bacteria can also carry out increasing bacterium, and enriching effect is good.
Culture medium preparation method provided by the invention is simple to operate, efficiency high, and effect is good.
Brief description of the drawings
Fig. 1 is salmonella fluorescence PCR detection reagent kit testing result in the present invention；
Fig. 2 is vibrio parahaemolytious fluorescence PCR detection reagent kit testing result in the present invention；
Fig. 3 is Listeria monocytogenes fluorescence PCR detection reagent kit testing result in the present invention.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1
The preparation of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious：
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain Asia
Telluric acid potassium solution is simultaneously standby.
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution
It is and standby.
The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the hydrogen-oxygen that 10 parts of concentration are 0.05mol/L
Change in sodium solution, obtain nalidixic acid solution and standby.
Weigh 17 parts of tryptone, 3 parts of peptone, 10 parts of sodium chloride, 2.5 parts of sodium dihydrogen phosphate, 2.5 parts of glucose is sweet
Reveal 2.5 parts of alcohol, 0.02 part of aesculin, 1 part of sodium citrate, 5 parts of skimmed milk power, be successively added in 1000 parts of sterile purified waters,
Gained mixture pH is adjusted to 7.5 with 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution after well mixed, it is high
Pressure sterilizing 15min, then waits mixture to be cooled to 50 DEG C, aseptically, potassium tellurite solution is added into mixture
0.2 part, 0.2 part of acridine yellow solution, 0.2 part of nalidixic acid solution, after being well mixed, semi-finished product culture medium is made.
In an aseptic environment, 100 parts of samples to be detected are added in semi-finished product culture medium after homogenizer is handled, mixed
After closing 2min, cultivated at 37 DEG C 6 hours, 0.8 part of potassium tellurite solution, acridine are then uniformly added into semi-finished product culture medium
Yellow 0.8 part of solution, 0.8 part of nalidixic acid solution；It is further continued for cultivating 18 hours at 37 DEG C.
Embodiment 2
The preparation of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious：
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain Asia
Telluric acid potassium solution is simultaneously standby.
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution
It is and standby.
The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the hydrogen-oxygen that 10 parts of concentration are 0.05mol/L
Change in sodium solution, obtain nalidixic acid solution and standby.
Weigh 17 parts of tryptone, 3 parts of peptone, 10 parts of sodium chloride, 2.5 parts of sodium dihydrogen phosphate, 2.5 parts of glucose is sweet
Reveal 2.5 parts of alcohol, 0.02 part of aesculin, 1 part of sodium citrate, 5 parts of skimmed milk power, be successively added in 1000 parts of sterile purified waters,
Gained mixture pH is adjusted to 7.5 with 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution after well mixed, it is high
Pressure sterilizing 15min, then waits mixture to be cooled to 50 DEG C, aseptically, potassium tellurite solution is added into mixture
0.2 part, 0.2 part of acridine yellow solution, 0.2 part of nalidixic acid solution, after being well mixed, semi-finished product culture medium is made.
In an aseptic environment, 100 parts of samples to be detected are added in semi-finished product culture medium after homogenizer is handled, mixed
After closing 2min, cultivated at 37 DEG C 6 hours, 0.8 part of potassium tellurite solution, acridine are then uniformly added into semi-finished product culture medium
Yellow 0.8 part of solution, 0.8 part of nalidixic acid solution, 0.3 part of Tea Saponin；It is further continued for cultivating 18 hours at 37 DEG C.
Embodiment 3
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain Asia
Telluric acid potassium solution is simultaneously standby.
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution
It is and standby.
The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the hydrogen-oxygen that 10 parts of concentration are 0.05mol/L
Change in sodium solution, obtain nalidixic acid solution and standby；
Weigh 20 parts of tryptone, 4 parts of peptone, 8 parts of sodium chloride, 2 parts of sodium dihydrogen phosphate, 3 parts of glucose, mannitol 3
Part, 0.003 part of aesculin, 1.5 parts of sodium citrate, 4 parts of skimmed milk power, successively it is added in 1000 parts of sterile purified waters, mixes
Gained mixture pH is adjusted to 7.5 with 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution after uniformly, high pressure is gone out
Bacterium 15min, then wait mixture to be cooled to 50 DEG C, aseptically, potassium tellurite solution 0.16 is added into mixture
Part, 0.16 part of acridine yellow solution, 0.16 part of nalidixic acid solution, after being well mixed, semi-finished product culture medium is made.
In an aseptic environment, 100 parts of samples to be detected are added in semi-finished product culture medium after homogenizer is handled, mixed
After closing 2min, cultivated at 37 DEG C 8 hours, 0.84 part of potassium tellurite solution, a word used for translation are then uniformly added into semi-finished product culture medium
0.84 part of pyridine Huang solution, 0.84 part of nalidixic acid solution, 0.2 part of Tea Saponin；It is further continued for cultivating 16 hours at 37 DEG C.
Comparative example
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain Asia
Telluric acid potassium solution is simultaneously standby.
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution
It is and standby.
The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the hydrogen-oxygen that 10 parts of concentration are 0.05mol/L
Change in sodium solution, obtain nalidixic acid solution and standby.
Weigh 17 parts of tryptone, 3 parts of peptone, 10 parts of sodium chloride, 2.5 parts of sodium dihydrogen phosphate, 2.5 parts of glucose is sweet
Reveal 2.5 parts of alcohol, 0.02 part of aesculin, 1 part of sodium citrate, 5 parts of skimmed milk power, be successively added in 1000 parts of sterile purified waters,
Gained mixture pH is adjusted to 7.5 with 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution after well mixed, it is high
Pressure sterilizing 15min, then waits mixture to be cooled to 50 DEG C, aseptically, potassium tellurite solution 1 is added into mixture
Part, 1 part of acridine yellow solution, 1 part of nalidixic acid solution, after being well mixed, culture medium is made.
In an aseptic environment, 100 parts of samples to be detected are added in culture medium after homogenizer is handled, mix 2min
Afterwards, cultivated 24 hours at 37 DEG C.
Bacterial number in embodiment 1-3 and comparative example culture medium is determined, finds embodiment 1-3 and comparative example difference
Be, the quantity of 3 detected in embodiment 1-3 kind object bacteria than comparative example quantity more than 10-15%, main cause is contrast
Object bacteria in sub- lethal state in example is because the high inhibition effect of inhibitor can not recover, in false negative, and embodiment 1-3
The object bacteria of a large amount of sub- lethal states can be made to recover and grow.
Pure bacterium detection：
Multiple identical culture mediums are prepared by the preparation method of the present invention and are respectively connected to be diluted to 10-4Salmonella,
The bacterium solution 0.2mL of three kinds of object bacterias of vibrio parahaemolytious and Listeria monocytogenes and miscellaneous bacteria cultivates 24h at 37 DEG C.Use vis spectroscopy
OD values under photometric determination 540nm.Blank control is done with buffered peptone water, the results are shown in Table 1：
Table 1：The independent growing state in the culture medium of optimum proportioning of object bacteria and non-targeted bacterium
| Bacterial strain | Blank | 1 | 2 | 3 |
| Salmonella | 0.59 | 1.41 | 1.16 | 1.32 |
| Vibrio parahaemolytious | 0.65 | 0.85 | 0.74 | 0.86 |
| Listeria monocytogenes | 0.62 | 0.89 | 0.76 | 0.89 |
| Escherichia coli | 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 |
| Comma bacillus | 0.61 | 0.11 | 0.09 | 0.13 |
| Vibrio vulnificus | 0.67 | 0 | 0 | 0 |
| Vibrio alginolyticus | 0.56 | 0 | 0 | 0 |
| Staphylococcus aureus | 0.54 | 0 | 0 | 0 |
Note：1-3 is that three repetitions are tested
Three kinds of object bacteria salmonellas, vibrio parahaemolytious and Listeria monocytogenes are in above-mentioned culture medium as can be seen from Table 1
It is middle growth 24 hours after enriching effect than non-selectivity buffering protein peptone more preferably；The growth of most of non-targeted bacterium receives suppression
System, only Escherichia coli and comma bacillus bacterium solution become muddy, but compared with the buffered peptone water of blank non-selectivity this two
Kind bacterium grows in described culture medium substantially have received suppression, and also substantially than 3 kinds object bacterias are slow for their growth.
Mixed Microbes detect：
According to salmonella:Vibrio parahaemolytious:The ratio of Listeria monocytogenes is 1:1:In 1 access culture medium, 37 DEG C of trainings
Support 24 hours, bacterium solution is diluted to 10-6, it is respectively coated BS agar plates, on TCBS agar plates and PALCAM flat boards, carries out
It is separately cultured.Salmonella is blackish green circular colonies on BS agar, and vibrio parahaemolytious and Listeria monocytogenes do not grow；It is secondary
Hemolysis vibrion is blue-green bacterium colony on TCBS flat boards, and bacterium colony is rounded, neat in edge, moistening；It is slightly muddy, it is translucent, it is most
Have the sharp heart, bamboo hat shape, diameter 2-4mm, salmonella and Listeria monocytogenes do not grow；Listeria monocytogenes are in PALCAM flat boards
Upper is circular celadon bacterium colony, around there is brownish black hydrolysis circle, and some bacterium colonies have black depression, salmonella and vibrio parahaemolytious
Do not grow.Growth result is shown in Table 2.As a result show, entered 24 hours after increasing bacterium, salmonella, vibrio parahaemolytious and single increasing Li Si
Special bacterium can breed to 10 simultaneously6, the bacteria concentration requirement of late detection can be met.
The compound enriched medium enriching effect of table 2
| Object bacteria | Inoculum concentration (CFU/mL) | Bacterium amount (CFU/mL) after increasing bacterium |
| Salmonella | 10-100 | 2300000 |
| Vibrio parahaemolytious | 10-100 | 3500000 |
| Listeria monocytogenes | 10-100 | 3600000 |
Artificial sample-adding detection：
Aseptically, by peeled shrimp (being free of three kinds of object bacterias by fluorescent PCR identification), 25g crushing is prepared into respectively.
About 100CFU/g culture is inoculated in sample, handles 15min at room temperature, is the other uniform pickup of bacterium solution, then by it
It is put into the self-made medium containing 225mL, mixes 2min.The good sample of homogeneous is cultivated 24 hours in 37 DEG C.Extracted with DNA
Kit extracts DNA of bacteria, is examined with salmonella, vibrio parahaemolytious and Listeria monocytogenes fluorescence PCR detection reagent kit
Survey, testing result is as shown in Figure 1, Figure 2, Figure 3 shows, it is seen that obvious DNA cloning curve occur in three kinds of object bacterias, illustrate to meet increasing
Bacterium culture medium causes three kinds of salmonella in sample, vibrio parahaemolytious and Listeria monocytogenes object bacterias while increased, and reaches
To the bacteria concentration requirement of PCR detections.Prove after compound enriched medium culture, detected available for PCR.
It is described above, only it is presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, it is every according to the present invention
Any simple modification, change and the equivalent transformation that technical spirit is made to above example, still fall within the technology of the present invention side
The protection domain of case.
Claims (5)
1. a kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious, it is characterised in that
Meter includes following component in parts by weight：
Tryptone 15-20 parts, peptone 2-4 parts, sodium chloride 8-12 parts, sodium dihydrogen phosphate 2-3 parts, glucose 2-3 parts, sweet dew
Alcohol 2-3 parts, aesculin 0.01-0.03 parts, sodium citrate 0.5-1.5 parts, skimmed milk power 4-6 parts, 1000 parts of sterile purified water,
1mol/L potassium hydroxide solution 0.1-1 parts, 1mol/L hydrochloric acid solution 0.1-1 parts, the 8-1.2 parts of potassium tellurite solution 0., a word used for translation
Pyridine Huang solution 0.8-1.2 parts, nalidixic acid solution 0.8-1.2 parts；
The potassium tellurite solution is made up of 0.00005 part of potassium tellurite and 10 parts of sterile purified waters；The acridine yellow solution by
0.1 part of acridine yellow and 10 parts of sterile purified water compositions；The nalidixic acid solution is by 0.01 part of nalidixic acid and 10 parts of concentration
0.05mol/L sodium hydroxide solution composition；
The preparation method of the selective medium, comprises the following steps：
The preparation of potassium tellurite solution：Weigh 0.00005 part of potassium tellurite to be added in 10 parts of sterile purified waters, obtain tellurous acid
Potassium solution is simultaneously standby；
The preparation of acridine yellow solution：Weigh 0.1 part of acridine yellow to be added in 10 parts of sterile purified waters, obtain acridine yellow solution and standby
With；
The preparation of nalidixic acid solution：Weigh 0.01 part of nalidixic acid and be added to the sodium hydroxide that 10 parts of concentration are 0.05mol/L
In solution, nalidixic acid solution and standby is obtained；
Weigh tryptone 15-20 parts, peptone 2-4 parts, sodium chloride 8-12 parts, sodium dihydrogen phosphate 2-3 parts, glucose 2-3 parts,
Mannitol 2-3 parts, aesculin 0.01-0.03 parts, sodium citrate 0.5-1.5 parts, skimmed milk power 4-6 parts, successively it is added to 1000
In part sterile purified water, 1mol/L potassium hydroxide solution and 1mol/L hydrochloric acid solution are used after being well mixed by gained mixture
PH is adjusted to 7.5, autoclaving 15min, is then waited mixture to be cooled to 50 DEG C, aseptically, is added into mixture
Add potassium tellurite solution 0.16-0.24 parts, acridine yellow solution 0.16-0.24 parts, nalidixic acid solution 0.16-0.24 parts, mixing
After uniformly, semi-finished product culture medium is made；
In an aseptic environment, sample to be detected is added in semi-finished product culture medium after homogenizer is handled, after mixing 2min,
4-8 hours are cultivated at 37 DEG C, potassium tellurite solution 0.64-0.96 parts, acridine are then uniformly added into semi-finished product culture medium
Yellow solution 0.64-0.96 parts, nalidixic acid solution 0.64-0.96 parts；It is further continued for cultivating 16-20 hours at 37 DEG C.
2. salmonella as claimed in claim 1, Listeria monocytogenes and the compound selectivity culture for increasing bacterium of vibrio parahaemolytious
Base, it is characterised in that the component for counting the selective medium in parts by weight is：17 parts of tryptone, 3 parts of peptone, chlorine
Change 10 parts of sodium, 2.5 parts of sodium dihydrogen phosphate, 2.5 parts of glucose, 2.5 parts of mannitol, 0.02 part of aesculin, 1 part of sodium citrate, take off
5 parts of fat milk powder, 1000 parts of sterile purified water, 1mol/L potassium hydroxide solution 0.1-1 parts, 1mol/L hydrochloric acid solution 0.1-1
Part, 1 part of potassium tellurite solution, 1 part of acridine yellow solution, 1 part of nalidixic acid solution.
3. salmonella as claimed in claim 1, Listeria monocytogenes and the compound selectivity culture for increasing bacterium of vibrio parahaemolytious
Base, it is characterised in that the selective medium also includes 0.1-0.5 part Tea Saponins.
4. salmonella as claimed in claim 1, Listeria monocytogenes and the compound selectivity culture for increasing bacterium of vibrio parahaemolytious
Base, it is characterised in that the amount that potassium tellurite solution is added when preparing semi-finished product culture medium is 0.2 part, adds acridine yellow solution
Amount be 0.2 part, addition nalidixic acid solution amount be 0.2 part；Cultivated after detected sample is added to semi-finished product culture medium
Time is 6 hours, and the amount that potassium tellurite solution is added to semi-finished product culture medium is 0.8 part, and the amount of addition acridine yellow solution is 0.8
Part, the amount of addition nalidixic acid solution is 0.8 part；Incubation time is 18 hours.
5. salmonella as claimed in claim 1, Listeria monocytogenes and the compound selectivity culture for increasing bacterium of vibrio parahaemolytious
Base, it is characterised in that potassium tellurite solution, acridine yellow solution and nalidixic acid are uniformly being added into the semi-finished product culture medium
During solution, while also it is added with 0.1-0.5 part Tea Saponins.
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| CN105505837A (en) * | 2016-02-05 | 2016-04-20 | 李志友 | L. monocytohenes culture medium |
| CN105506054A (en) * | 2016-02-05 | 2016-04-20 | 夏淑平 | Listeria monocytogenes selective medium |
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| CN106047781B (en) * | 2016-08-22 | 2018-05-18 | 四川华汉三创生物科技有限公司 | A kind of enriched medium and preparation method thereof, Zengjing Granule method |
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