CN104846059A - Nocardia braziliensis culture medium suitable for pleural effusion - Google Patents
Nocardia braziliensis culture medium suitable for pleural effusion Download PDFInfo
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Abstract
The invention discloses a Nocardia braziliensis culture medium suitable for pleural effusion, belonging to the field of inspection microbe culture. The invention is characterized in that every 1000ml of culture medium contains ox liver powder, malt extract powder, yeast extract powder, sodium chloride, agar, ferrous lactate, zinc calcium gluconate, nalidixic acid, argutone, asparamide, sterile defibered sheep blood, casein phosphopeptide and the balance of distilled water. Compared with the prior art, the culture medium disclosed by the invention has the characteristics of high detection reliability and high separation rate of Nocardia braziliensis.
Description
Technical field
The present invention relates to inspection microorganism culturing field, be specifically related to a kind of Nocardia brasiliensis Selective agar medium for hydrothorax sample.
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.Nocardia brasiliensis (N.braziliensis) is aerobic soil saprophytic microorganism, belong to Nocardia, actinomycetes door, this bacterium is extensively present in air, dust, soil and domestic animal body surface, Nocardia brasiliensis toxicity is comparatively strong, and virulence is strong, can cause outbreak of epidemic, many by respiratory system or primary infection, cause the chronic suppurative infection at subcutis and the position such as lung, brain.Nocardia brasiliensis invades subcutis by the skin of damage, direct contaminated wound, produce chronic purulent granuloma, show as swelling, abscess and multiple fistula, as the so-called mycetoma of foot and leg, clinically show as acute, subacute or chronic process, sickly to pulmonary tuberculosis, tuberculosis of skin, actinomycosis, mycomycete in symptom be similarly easy to mistaken diagnosis.
In recent years, Nocardia brasiliensis disease is unrare, have or without the crowd of dysimmunity in all can occur.Nocardia brasiliensis poor growth, the course of disease are long, although sickness rate only has 1%, mortality ratio but up to 86%, early diagnosis and time appropriate treatment, can 100% to cure, therefore as the clinical bacteria making a definite diagnosis unique foundation check correctly and Rapid identification very important.At present, this Pseudomonas identify that gold standard is PCR qualification, but PCR test set requires and running cost is all higher, therefore multiplex common blood agar culture plate (BAP) cultivation of clinical cultivation.And Nocardia brasiliensis belongs to severe bacteria, namely harsher to growing environment, nutritional requirement bacterium, or can not be difficult to growth in conventional environment.Nocardia brasiliensis first culture poor growth, hydrothorax sample is easy to by saccharomyces albicans on blood agar culture-medium, the fungies such as streptococcus aureus, miscellaneous bacteria cover, Nocardia brasiliensis cannot grow, positive rate is low, traditional method laboratory culture is used within 48 hours, to cause 60% Nocardia brasiliensis undetected, cultivate more than 96 hours positive performance, within about 1 week, qualification can be made more exactly, biochemical identification needs 4 ~ 6 time-of-weeks, the result delay time at stop, cause many places focus transfer, case fatality rate raises.
Summary of the invention
Technical assignment of the present invention is for above the deficiencies in the prior art, provides a kind of Selective agar medium being suitable for the growth of hydrothorax sample Nocardia brasiliensis.
The technical scheme that the present invention solves its technical problem is: a kind of Nocardia brasiliensis substratum being applicable to hydrothorax, is characterized in that, contains in the formula of preparation 1000ml substratum:
Beef liver powder 5.0 ~ 15.0g;
Fructus Hordei Germinatus leaching powder 2.0 ~ 5.0g;
Yeast extract powder 5.0 ~ 10.0g;
Sodium-chlor 5.0g;
Agar 15.0 ~ 20.0g;
Iron lactate 0.01 ~ 0.05g;
Zinc Gluconate calcium 0.01 ~ 0.05g;
Nalidixic acid 0.01 ~ 0.05g;
2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.001 ~ 0.005g;
L-asparagine 0.01 ~ 0.1g;
Aseptic defiber Sheep Blood 80 ~ 100ml;
Phosphopeptide caseinate 0.1 ~ 0.5g;
Distilled water adds to 1000ml.
The preparation method of above-mentioned substratum is: take beef liver powder; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing distilled water dissolves, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Sterilizing network protein phosphatase polypeptide; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Wherein: described beef liver powder, Fructus Hordei Germinatus leaching powder provides carbon source, nitrogenous source, the nutritive ingredients such as yeast extract powder rich in proteins, amino acid, polypeptide, Nucleotide, multivitamin, somatomedin, trace element, for microorganism provides balanced nutritious, these materials are all very important factors to the Growth and Reproduction promoting Nocardia brasiliensis; Sodium-chlor maintains balanced osmotic pressure; Agar is the peptizer of substratum; Iron lactate is nutrition agent, for Nocardia brasiliensis flourish provides required ferro element; Zinc Gluconate calcium, except doing nutrient intensifier, can also suppress Gram-positive bacteria growing; Nalidixic acid suppresses the growth of Gram-negative bacteria; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone (2-Ethoxymethylene-3,5-dihydroxy-y-pyrone), laboratory study shows that the growth of 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone to miscellaneous bacterias such as streptococcus aureus, diphtheria corynebacterium, anthrax bacillus, Corynebacterium diphtheriae, paratyphoid bacillus A, Pseudomonas aeruginosa, intestinal bacteria, bacterium flexneri, dysentery bacterium, meningococcus, Bacillus subtilus, human nature tubercule bacillus has restraining effect, but under this concentration due to Nocardia without antibacterial and bacteriostasis, therefore improve the separation rate of Nocardia brasiliensis.Aseptic defiber Sheep Blood provides energy environment; L-asparagine is nutrition-fortifying agent, promote that Nocardia brasiliensis is to amino acid whose picked-up, by experiment same Nocardia brasiliensis is inoculated on 2 BAP substratum, one of them BAP substratum adds 0.1g/L l-asparagine, cultivate at 37 DEG C, bacterial strain is observed after 48 hours, find that the BAP substratum adding l-asparagine has little Nocardia brasiliensis bacterium colony, the BAP substratum not adding l-asparagine does not find Nocardia brasiliensis bacterium colony, confirms that the accelerating growth for this severe bacteria of Nocardia brasiliensis is most important thus; Phosphopeptide caseinate is activator, can promote that Nocardia brasiliensis is to the picked-up of ferro element, calcium constituent; Radix Rehmanniae is the fresh of goatweed Radix Rehmanniae or dried root, and modern medicine study shows, containing more than 20 kind of glycoside in Radix Rehmanniae rhizome; 8 kinds of carbohydrates; More than 20 seed amino acids and more than 20 plant inorganic elements, and Radix Rehmanniae Aqueous extracts is good nutrition-fortifying agent, and have the effect suppressing staphylococcus aureus growth.
The present invention compared with prior art, has and detects the feature reliable, Nocardia brasiliensis 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: beef liver powder 15.0g; Fructus Hordei Germinatus leaching powder 2.0g; Yeast extract powder 5.0g; Sodium-chlor 5.0g; Agar 15.0g; Iron lactate 0.01g; Zinc Gluconate calcium 0.05g; Nalidixic acid 0.05g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.001g; L-asparagine 0.1g; Aseptic defiber Sheep Blood 100ml; Distilled water adds to 1000ml.Preparation method takes beef liver powder; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing distilled water dissolves, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 2, contains in the formula of preparation 1000ml substratum: beef liver powder 10.0g; Fructus Hordei Germinatus leaching powder 5.0g; Yeast extract powder 8.0g; Sodium-chlor 5.0g; Agar 15.0g; Iron lactate 0.01g; Zinc Gluconate calcium 0.03g; Nalidixic acid 0.02g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.003g; L-asparagine 0.01g; Network protein phosphatase polypeptide 0.1g; Aseptic defiber Sheep Blood 80ml; Distilled water adds to 1000ml.Preparation method takes beef liver powder; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixes, and distilled water dissolves, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Sterilizing network protein phosphatase polypeptide; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 3, contains in the formula of preparation 1000ml substratum: beef liver powder 15.0g; Fructus Hordei Germinatus leaching powder 3.0g; Yeast extract powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Iron lactate 0.02g; Zinc Gluconate calcium 0.05g; Nalidixic acid 0.05; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.005g; L-asparagine 0.05g; Vitamin K
10.01g; Aseptic defiber Sheep Blood 90ml; Distilled water adds to 1000ml.Preparation method takes beef liver powder; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixes, and distilled water dissolves, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Sterilizing vitamin K
1; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 4, contains in the formula of preparation 1000ml substratum: beef liver powder 5.0g; Fructus Hordei Germinatus leaching powder 5.0g; Yeast extract powder 8.0g; Sodium-chlor 5.0g; Agar 20.0g; Iron lactate 0.03g; Iron lactate; Zinc Gluconate calcium 0.03g; Nalidixic acid 0.01g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.001g; L-asparagine 0.1g; Aseptic defiber Sheep Blood 90ml; 200g/L Radix Rehmanniae Aqueous extracts 200ml; Distilled water adds to 1000ml.Preparation method: (1) is got 40g Radix Rehmanniae 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 Radix Rehmanniae Aqueous extracts 200ml; (2) beef liver powder is taken; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing is dissolved in the Radix Rehmanniae extracting solution of step 1 gained, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 5, contains in the formula of preparation 1000ml substratum: beef liver powder 15.0g; Fructus Hordei Germinatus leaching powder 2.0g; Yeast extract powder 5.0g; Sodium-chlor 5.0g; Agar 15.0g; Iron lactate 0.05g; Zinc Gluconate calcium 0.05g; Nalidixic acid 0.03g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.003g; L-asparagine 0.05g; Network protein phosphatase polypeptide 0.5g; Vitamin K
10.1g; Aseptic defiber Sheep Blood 90ml; Distilled water adds to 1000ml.Preparation method takes beef liver powder; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixes, and distilled water dissolves, and 280 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds sterilizing nalidixic acid; Sterilizing network protein phosphatase polypeptide; Sterilizing vitamin K
1; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 6, contains in the formula of preparation 1000ml substratum: beef liver powder 5.0g; Fructus Hordei Germinatus leaching powder 3.0g; Yeast extract powder 5.0g; Sodium-chlor 5.0g; Agar 20.0g; Iron lactate 0.02g; Zinc Gluconate calcium 0.03g; Nalidixic acid 0.01g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.001g; L-asparagine 0.05g; Network protein phosphatase polypeptide 0.3g; Aseptic defiber Sheep Blood 90ml; The Radix Rehmanniae Aqueous extracts 250ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method preparation method: (1) is got 50g Radix Rehmanniae 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 Radix Rehmanniae Aqueous extracts 250ml; (2) beef liver powder is taken; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing is dissolved in the Radix Rehmanniae extracting solution of step (1) gained and mixes, and 280 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterilizing iron lactate; Sterilizing nalidixic acid; Sterilizing network protein phosphatase polypeptide; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 7, contains in the formula of preparation 1000ml substratum: beef liver powder 10.0g; Fructus Hordei Germinatus leaching powder 5.0g; Yeast extract powder 8.0g; Sodium-chlor 5.0g; Agar 15.0g; Iron lactate 0.05g; Zinc Gluconate calcium 0.01g; Nalidixic acid 0.03g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.003g; L-asparagine 0.1g; Vitamin K
10.05g; Aseptic defiber Sheep Blood 100ml; The Radix Rehmanniae Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 60g Radix Rehmanniae 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 Radix Rehmanniae Aqueous extracts 300ml; (2) beef liver powder is taken; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing is dissolved in the Radix Rehmanniae extracting solution of step (1) gained and mixes, and 280 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterilizing nalidixic acid; Sterilizing vitamin K
1; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Embodiment 8, contains in the formula of preparation 1000ml substratum: beef liver powder 10.0g; Fructus Hordei Germinatus leaching powder 3.0g; Yeast extract powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Iron lactate 0.01g; Zinc Gluconate calcium 0.05g; Nalidixic acid 0.05g; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.005g; L-asparagine 0.01g; Network protein phosphatase polypeptide 0.3g; Vitamin K
10.05g; Aseptic defiber Sheep Blood 100ml; The Radix Rehmanniae Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 60g Radix Rehmanniae 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 Radix Rehmanniae Aqueous extracts 300ml; (2) beef liver powder is taken; Fructus Hordei Germinatus leaching powder; Yeast extract powder; Sodium-chlor; Agar; Iron lactate; Zinc Gluconate calcium; 2-Ethoxymethylene-3,5-dihydroxy-y-pyrone; L-asparagine mixing is dissolved in the Radix Rehmanniae extracting solution of step (1) gained and mixes, and 280 DEG C of sterilizing 15min, are cooled to 45 DEG C, add sterilizing nalidixic acid; Sterilizing vitamin K
1; Sterilizing network protein phosphatase polypeptide; Aseptic defiber Sheep Blood mixing, packing after sterile purified water constant volume to 1000ml.
Described Radix Rehmanniae Aqueous extracts concentration 200g/L refers to often liter containing crude drug 200g.
Gained Nocardia brasiliensis Selective agar medium of the present invention is used for hydrothorax sample to be had and detects the feature reliable, Nocardia brasiliensis separation rate is high, and 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 and control group, control group is common blood agar culture plate (BAP) substratum, its formula is often liter and contains beef powder 5g, peptone 12g, agar 15g, sodium-chlor 5g, aseptic defiber Sheep Blood 100ml, sterile purified water constant volume.
1.2 hydrothoraxs gather cultural method: highly doubtful Nocardia brasiliensis patients with pneumonia 13 example, get chest hydrops, 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.37 DEG C of insulations, insulation can is cultivated 48h, 96h picking object bacteria and is dropped into row identification of bacteria.Each sample number censorship PCR does molecular biology identification.
1.3 detect positive criteria: take out culture dish, observe bacterium colony on substratum, in white, protruding, smooth, wax sample, surface drying, the colony growth situation not easily swept, select object bacteria smear, gramstaining, sediments microscope inspection goes out the positive, checks according to " clinical microbiology diagnosis and diagram ".Net result gold standard is PCR qualification.
2 results: what 13 parts of highly doubtful Nocardia brasiliensis patients with pneumonia hydrothorax samples were finally accredited as the Nocardia brasiliensis positive through PCR is 12 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 | 13 | 12 | 9 | 1 | 4 | 4 |
| Embodiment 2 | 13 | 12 | 10 | 0 | 3 | 2 |
| Embodiment 6 | 13 | 12 | 10 | 1 | 3 | 3 |
| Embodiment 8 | 13 | 12 | 11 | 0 | 2 | 1 |
| Control group (BAP) | 13 | 12 | 3 | 0 | 10 | 9 |
The above results can be found out, cultivate after 48 hours, each embodiment group and control group positive rate more all have pole notable difference (P<0.05), and false negative rate is starkly lower than control group (P<0.05).Each embodiment group compares with PCR detected result, positive rate no significant difference (P>0.05).
Each group of 96h 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 | 13 | 12 | 13 | 1 | 0 | 0 |
| Embodiment 2 | 13 | 12 | 12 | 1 | 1 | 1 |
| Embodiment 6 | 13 | 12 | 12 | 0 | 1 | 0 |
| Embodiment 8 | 13 | 12 | 12 | 0 | 1 | 0 |
| Control group (BAP) | 13 | 12 | 8 | 2 | 5 | 6 |
The above results can be found out, cultivate after 96 hours, each embodiment group compares with control group positive rate, all has notable difference (P<0.05), and false negative rate is starkly lower than control group (P<0.05).Each embodiment group compares with PCR detected result, positive rate no significant difference (P>0.05).
Above result shows, hydrothorax sample is after embodiment of the present invention culture medium culturing, Nocardia brasiliensis positive rate and PCR checkout discrepancy no difference of science of statistics, and 48 hours separation rates are apparently higher than existing substratum, after 96 hours, control group (BAP) substratum positive bacteria detects quantity increasing degree and is greater than each embodiment, and each embodiment and control group positive rate comparing difference reduce, but difference still has statistical significance.Above result confirms: common BAP substratum under-nutrition bacterial growth is slow, need more than 96h just to occur bacterial strain Growth positive, substratum 48h of the present invention just can obtain as far as possible bacterial strain positive rate accurately, and Nocardia brasiliensis detects reliably, separation rate is high, thus the time is made a definite diagnosis in shortening.
Claims (1)
1. be applicable to a Nocardia brasiliensis substratum for hydrothorax, it is characterized in that containing in the formula of preparation 1000ml substratum:
Beef liver powder 5.0 ~ 15.0g;
Fructus Hordei Germinatus leaching powder 2.0 ~ 5.0g;
Yeast extract powder 5.0 ~ 10.0g;
Sodium-chlor 5.0g;
Agar 15.0 ~ 20.0g;
Ferrous porphyrin 0.01 ~ 0.05g;
Calcium lactate 0.01 ~ 0.05g;
Nalidixic acid 0.01 ~ 0.05g;
2-Ethoxymethylene-3,5-dihydroxy-y-pyrone 0.001 ~ 0.005g;
L-asparagine 0.01 ~ 0.1g;
Aseptic defiber Sheep Blood 80 ~ 100ml;
Phosphopeptide caseinate 0.1 ~ 0.5g;
Distilled water adds to 1000ml.
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Citations (1)
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| CN102559841A (en) * | 2012-03-06 | 2012-07-11 | 王京军 | Nocardia culture medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102559841A (en) * | 2012-03-06 | 2012-07-11 | 王京军 | Nocardia culture medium |
Non-Patent Citations (3)
| Title |
|---|
| 张媛等: "诺卡菌的培养和染色特征研究", 《中国人兽共患病学报》 * |
| 杨模坤等: "马桶花抑菌新活性成分马桶花酮的分离及化学机构", 《药学学报》 * |
| 马乐好等主编: "《微生物培养基实用手册》", 31 March 2006, 吉林科学技术出版社 * |
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Application publication date: 20150819 |