CN105506054A - Listeria monocytogenes selective medium - Google Patents
Listeria monocytogenes selective medium Download PDFInfo
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- CN105506054A CN105506054A CN201610079557.8A CN201610079557A CN105506054A CN 105506054 A CN105506054 A CN 105506054A CN 201610079557 A CN201610079557 A CN 201610079557A CN 105506054 A CN105506054 A CN 105506054A
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- listeria monocytogenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Abstract
The invention discloses a listeria monocytogenes selective medium, and belongs to the field of detection of microculture. The listeria monocytogenes selective medium is characterized in that the prepared 1000 ml culture medium comprises the following components: yeast extract powder, beef powder, casein peptone, agar, glucose, sodium chloride, zinc gluconate, sodium dihydrogen phosphate, optical, polymyxin,bacitracin, gamma-arginine hydroxy and sterile horse blood protein, and distilled water is added into the components to 1000 ml. Compared with the prior art, the selective medium has the characteristics of being reliable in detection result, and high in listeria monocytogenes separation rate.
Description
Technical field
The present invention relates to inspection microorganism culturing field, be specifically related to a kind of Selective agar medium of Listeria monocytogenes.
Background technology
Microorganism culturing is a kind of technology making microbial growth by artificial means, and the substratum that selective medium is used to promote or suppress the organism (as cell or bacterium etc.) of certain type and designs, utilize this substratum required microorganism can be separated from the microorganism mixed.Listeria monocytogenes (Listeriamonocytogenes) is a kind of facultative anaerobic bacteria, extensively be distributed in physical environment, as soil, sewage, feed, animal, Healthy People and variously eat raw with in instant food, comprise raw meat and cooked meat product, quick-frozen rice and flour food, cheese, vegetables, salad and sea-food etc., mankind's listeriosis can be caused, normally cause because people eat by the food of this fungi pollution.After infecting, main clinical manifestation is heating, upper respiratory tract infection, pneumonia, diarrhoea, bloodstream infection, meningitis, or infects foetus via pregnant woman's private parts, cause meningitis, even dead.Similar to influenza in clinical symptom, easy mistaken diagnosis.
In recent years, listeriosis is unrare, and all can occur in the crowd of newborn infant, gravid woman, more than 40 years old and immune function depression, especially gravid woman infects 30 ~ 100 times that listerial risk is general population.Early diagnosis early treatment, can 100% to cure.If delay in diagnosis, case fatality rate can reach 20 ~ 30%, and the correct and Rapid identification therefore as the clinical bacteria inspection of making a definite diagnosis unique foundation is very important.At present, this Pseudomonas identify gold standard be PCR qualification, but PCR test set require and running cost all higher, the therefore multiplex trypticase soy agar (TSA-YE) of clinical cultivation.。But it is low that Listeria monocytogenes cultivates positive rate, mainly in clinical culturing process, this bacterium is easily considered to the miscellaneous bacteria (diphtheroid) of pollution and is abandoned, and the intestinal bacteria, diphtheroid etc. in stool sample are faster than Listeria monocytogenes growth on current substratum, cover substratum very soon, Listeria monocytogenes cannot grow, and result incurs loss through delay consultation hours.
Summary of the invention
Technical assignment of the present invention is for above the deficiencies in the prior art, provides a kind of Listeria monocytogenes Selective agar medium.
The technical scheme that the present invention solves its technical problem is: a kind of Listeria monocytogenes Selective agar medium, is characterized by, and contains in the formula of preparation 1000ml substratum:
Yeast extract powder 5 ~ 8g;
Beef powder 6 ~ 10g;
Casein peptone 6 ~ 10g;
Agar 15 ~ 20g;
Glucose 3 ~ 5g;
Sodium-chlor 5 ~ 5.5g;
Zinc Gluconate 0.01 ~ 0.05g;
SODIUM PHOSPHATE, MONOBASIC 1 ~ 3g;
The sweet peptide 0.1 ~ 0.5g of optical valley;
Polymyxin 0.001 ~ 0.005g;
Bacitracin 0.001 ~ 0.003g;
γ-hydroxyarginine 0.1 ~ 0.3g;
Aseptic horse haemproteins 20 ~ 50g;
Distilled water adds to 1000ml.
The preparation method of above-mentioned substratum is: take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, γ-hydroxyarginine, mixing distilled water dissolves, 210 DEG C, sterilizing 15min, be cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Wherein: described beef powder, casein peptone, glucose provide carbon source, nitrogenous source, the nutritive ingredients such as yeast extract powder rich in proteins, amino acid, polypeptide, Nucleotide, VITAMIN, somatomedin, trace element, the Growth and Reproduction for Listeria monocytogenes provides balanced nutritive element; Sodium-chlor maintains balanced osmotic pressure; Agar is the peptizer of substratum; Zinc Gluconate is nutrition agent, and contriver finds, add certain density zine ion in the medium, the growth conditions of object bacteria is better than control group, and therefore inference zine ion contributes to the growth of this bacterium; SODIUM PHOSPHATE, MONOBASIC is buffer reagent, and polymyxin suppresses the growth of Gram-negative bacteria; Bacitracin (Bacitracin), laboratory study shows that bacitracin can suppress the growth of the miscellaneous bacteria such as gram-positive bacteria and negative cocci, pneumococcus, staphylococcus, gonococcus, meningococcus, but under this concentration due to listeria without antibacterial and bacteriostasis, therefore improve the separation rate of Listeria monocytogenes.By experiment same Listeria monocytogenes is inoculated on 2 TSA-YE substratum, one of them TSA-YE substratum add the sweet peptide of 0.01% sterilizing optical valley in, cultivate at 37 DEG C, bacterial strain is observed after 48 hours, find that the TSA-YE substratum adding the sweet peptide of optical valley has little Listeria monocytogenes bacterium colony, the TSA-YE substratum not adding the sweet peptide of optical valley does not find Listeria monocytogenes bacterium colony, and the sweet peptide of optical valley can reduce redox-potential, is conducive to Growth of Cells, differentiation, aseptic horse haemproteins provides energy environment, γ-hydroxyarginine is activator, can promote that Listeria monocytogenes is to the picked-up of mineral substance, apiolin is fungistat, Fructus Corni is the fruit of Cornaceae plant skunk bush, modern medicine study shows, Fructus Corni to be tanned hair containing skunk bush, skunk bush splits glucoside, iron, aluminium, copper, zinc, boron, 21 kinds of elements such as phosphorus, Threonine, α-amino-isovaleric acid, leucine etc. 14 seed amino acid, vitamin A, glucose, fructose, sucrose, ursolic acid, gallic acid, oxysuccinic acid, the carbohydrates such as tartrate and organic acid, Fructus Corni Aqueous extracts is good nutrition-fortifying agent, and to streptococcus aureus, Nai Shi micrococcus catarrhalis, tubercule bacillus, pneumococcus, hemophilus influenzae, polioviruses etc. all have bacteriostatic action in various degree.
The present invention compared with prior art, has and detects the feature reliable, Listeria monocytogenes separation rate is high.
Embodiment
Below in conjunction with practical situation, the specific embodiment of the present invention is elaborated.
Embodiment 1, contains in the formula of preparation 1000ml substratum: yeast extract powder 5g; Beef powder 10g; Casein peptone 6g; Agar 15g; Glucose 4g; Sodium-chlor 5g; SODIUM PHOSPHATE, MONOBASIC 1g; The sweet peptide 0.3g of optical valley; Zinc Gluconate 0.01g; Polymyxin 0.001g; Bacitracin 0.003g; Aseptic horse haemproteins 20g, distilled water adds to 1000ml.Preparation method: take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, mixing distilled water dissolves, and 210 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 2, contains in the formula of preparation 1000ml substratum: yeast extract powder 5g; Beef powder 6g; Casein peptone 8g; Agar 15g; Glucose 3g; Sodium-chlor 5g; SODIUM PHOSPHATE, MONOBASIC 3g; The sweet peptide 0.1g of optical valley; Zinc Gluconate 0.05g; Polymyxin 0.003g; Bacitracin 0.001g; γ-hydroxyarginine 0.1g; Aseptic horse haemproteins 40g, distilled water adds to 1000ml.Preparation method: take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, γ-hydroxyarginine, SODIUM PHOSPHATE, MONOBASIC, mixing distilled water dissolves, and 210 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 3, contains in the formula of preparation 1000ml substratum: yeast extract powder 5g; Beef powder 8g; Casein peptone 6g; Agar 18g; Glucose 5g; Sodium-chlor 5.5g; SODIUM PHOSPHATE, MONOBASIC 2g; The sweet peptide 0.5g of optical valley; Zinc Gluconate 0.03g; Polymyxin 0.005g; Bacitracin 0.001g; Apiolin 0.03g; Aseptic horse haemproteins 50g, distilled water adds to 1000ml.Preparation method: take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, apiolin, mixing distilled water dissolves, and 210 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 4, contains in the formula of preparation 1000ml substratum: yeast extract powder 6g; Beef powder 10g; Casein peptone 8g; Agar 18g; Glucose 4g; Sodium-chlor 5g; SODIUM PHOSPHATE, MONOBASIC 2g; The sweet peptide 0.1g of optical valley; Zinc Gluconate 0.03g; Polymyxin 0.005g; Bacitracin 0.002g; Aseptic horse haemproteins 30g; 200g/L Fructus Corni Aqueous extracts 200ml; Distilled water adds to 1000ml.Preparation method: (1) is got 40g Fructus Corni and added water boil 30min, filters, gets filtrate, according to filtrate volume, taking the concentrated or adjustment volume that adds water to be 200g/L to concentration, obtaining Fructus Corni Aqueous extracts 200ml; (2) take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, be dissolved in the Fructus Corni extracting solution of step (1) gained and mix, 210 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 5, contains in the formula of preparation 1000ml substratum: yeast extract powder 6g; Beef powder 6g; Casein peptone 8g; Agar 20g; Glucose 3g; Sodium-chlor 5.5g; SODIUM PHOSPHATE, MONOBASIC 3g; The sweet peptide 0.5g of optical valley; Zinc Gluconate 0.01g; Polymyxin 0.001g; Bacitracin 0.003g; γ-hydroxyarginine 0.2g; Apiolin 0.03g; Aseptic horse haemproteins 40g, distilled water adds to 1000ml.Preparation method: take yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, γ-hydroxyarginine, apiolin, mixing distilled water dissolves, 210 DEG C, sterilizing 15min, be cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 6, contains in the formula of preparation 1000ml substratum: yeast extract powder 8g; Beef powder 8g; Casein peptone 10g; Agar 20g; Glucose 3g; Sodium-chlor 5.5g; SODIUM PHOSPHATE, MONOBASIC 3g; The sweet peptide 0.5g of optical valley; Zinc Gluconate 0.01g; Polymyxin 0.001g; Bacitracin 0.003g; γ-hydroxyarginine 0.3g; Aseptic horse haemproteins 50g; The Fructus Corni Aqueous extracts 250ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 50g Fructus Corni and added water boil 45min, filters, gets filtrate, according to filtrate volume, taking the concentrated or adjustment volume that adds water to be 200g/L to concentration, obtaining Fructus Corni Aqueous extracts 250ml; (2) yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, γ-hydroxyarginine is taken, be dissolved in the Fructus Corni extracting solution of step (1) gained and mix, 210 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 7, contains in the formula of preparation 1000ml substratum: yeast extract powder 8g; Beef powder 8g; Casein peptone 10g; Agar 15g; Glucose 4g; Sodium-chlor 5g; SODIUM PHOSPHATE, MONOBASIC 3g; The sweet peptide 0.3g of optical valley; Zinc Gluconate 0.03g; Polymyxin 0.003g; Bacitracin 0.001g; Apiolin 0.01g; Aseptic horse haemproteins 20g; The Fructus Corni Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 60g Fructus Corni and added water boil 60min, filters, gets filtrate, according to filtrate volume, taking the concentrated or adjustment volume that adds water to be 200g/L to concentration, obtaining Fructus Corni Aqueous extracts 300ml; (2) yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, apiolin is taken, be dissolved in the Fructus Corni extracting solution of step (1) gained and mix, 210 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 8, contains in the formula of preparation 1000ml substratum: yeast extract powder 8g; Beef powder 10g; Casein peptone 6g; Agar 18g; Glucose 3g; Sodium-chlor 5.5g; SODIUM PHOSPHATE, MONOBASIC 2g; The sweet peptide 0.3g of optical valley; Zinc Gluconate 0.01g; Polymyxin 0.005g; Bacitracin 0.001g; γ-hydroxyarginine 0.3g; Apiolin 0.03g; Aseptic horse haemproteins 30g; The Fructus Corni Aqueous extracts 250ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 60g Fructus Corni and added water boil 60min, filters, gets filtrate, according to filtrate volume, taking the concentrated or adjustment volume that adds water to be 200g/L to concentration, obtaining Fructus Corni Aqueous extracts 300ml; (2) yeast extract powder, beef powder, casein peptone, agar, glucose, sodium-chlor, γ-hydroxyarginine, SODIUM PHOSPHATE, MONOBASIC, apiolin is taken, be dissolved in the Fructus Corni extracting solution of step (1) gained and mix, 210 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterile dextrose acid zinc; The sweet peptide of sterilizing optical valley; Sterilizing polymyxin; Sterilizing bacitracin; Aseptic horse haemproteins mixing, packing after sterile purified water constant volume to 1000ml.
Described Fructus Corni Aqueous extracts concentration 200g/L refers to often liter containing crude drug 200g.
Gained Listeria monocytogenes Selective agar medium of the present invention is used for stool sample, and have and detect the feature reliable, Listeria monocytogenes separation rate is high, be clinical data sufficient proof, pertinent data is as follows.
1 object and method.
1.1 medium preparing: experimental group establishes 4 groups altogether, are respectively embodiment 1 scheme group; Embodiment 2 scheme group, embodiment 6 scheme group, embodiment 8 scheme group, control group is junket peptone soy agar (TSA-YE) substratum, and its formula is: containing peptone 17.0g in often liter; Multivalence peptone 3.0g; Sodium-chlor 5.0g; Dipotassium hydrogen phosphate 2.5g; Glucose 2.5g; Agar 15.0.
1.2 fecal sample process, cultural method: outpatient service or 156 examples of being in hospital are suffered from diarrhoea and with similar flu-like symptom patient fresh excreta, are suspended in 5ml0.9% physiological saline, are inoculated in embodiment 1 scheme group with transfering loop sectional streak; Embodiment 2 scheme group; Embodiment 6 scheme group; Embodiment 8 scheme group and control group substratum.The substratum inoculated is placed in heat insulating culture 48h under 35 DEG C of atmospheric condition, and the tiny bacterium colony of picking carries out identification of bacteria.Each sample number censorship PCR does molecular biology identification.
1.3 detect positive criteria: take out culture dish, carry out object bacteria selection and qualification: observe colony growth situation, select object bacteria smear, gramstaining, sediments microscope inspection according to " clinical microbiology diagnosis and diagram ".Judge that net result gold standard is fluorescent quantitative PCR result.
2 results: what 156 parts of diarrhea patient stool samples detected that bacterium colony is finally accredited as the Listeria monocytogenes positive through PCR is 19 examples.Each group of 48h detects the positive, false positive, feminine gender, false negative bacterial strain number of cases see the following form,
| Grouping | Sum | PCR is positive | Positive | False positive | Negative | False negative |
| Embodiment 1 | 156 | 19 | 16 | 3 | 140 | 6 |
| Embodiment 2 | 156 | 19 | 17 | 1 | 139 | 3 |
| Embodiment 6 | 156 | 19 | 17 | 1 | 139 | 3 |
| Embodiment 8 | 156 | 19 | 18 | 0 | 138 | 1 |
| Control group (TSA-YE) | 156 | 19 | 6 | 1 | 150 | 14 |
The above results can be found out, cultivate after 48 hours, each embodiment group and control group positive rate more all have notable difference (P<0.05), and the false negative rate of each embodiment group is all lower than control group, have pole notable difference (P<0.001).The control group positive detects result and compares with PCR detected result, the huge (χ of positive rate difference
2=7.35, P<0.01), and each embodiment group compares with PCR detected result, positive rate no significant difference (P>0.05).
Above result shows, stool sample is after embodiment of the present invention culture medium culturing, Listeria monocytogenes positive rate and PCR checkout discrepancy no difference of science of statistics, and separation rate is apparently higher than existing substratum, general T SA-YE substratum selectivity is not enough, in ight soil, miscellaneous bacteria ramp covers the Growth positive that substratum affects Listeria monocytogenes, substratum of the present invention can be tried one's best bacterial strain positive rate accurately, Listeria monocytogenes detects reliably, positive separation rate is high, thus the time is made a definite diagnosis in shortening.
Claims (1)
1. a Listeria monocytogenes Selective agar medium, is characterized in that containing in the formula of preparation 1000ml substratum:
Yeast extract powder 5 ~ 8g;
Beef powder 6 ~ 10g;
Casein peptone 6 ~ 10g;
Agar 15 ~ 20g;
Glucose 3 ~ 5g;
Sodium-chlor 5 ~ 5.5g;
Zinc Gluconate 0.01 ~ 0.05g;
SODIUM PHOSPHATE, MONOBASIC 1 ~ 3g;
The sweet peptide 0.1 ~ 0.5g of optical valley;
Polymyxin 0.001 ~ 0.005g;
Bacitracin 0.001 ~ 0.003g;
γ-hydroxyarginine 0.1 ~ 0.3g;
Aseptic horse haemproteins 20 ~ 50g;
Distilled water adds to 1000ml.
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Cited By (1)
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| CN110343646A (en) * | 2019-08-12 | 2019-10-18 | 北京市食品安全监控和风险评估中心（北京市食品检验所） | A method of promoting Listeria Monocytogenes growth |
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