CN103898192A - Blood culture method - Google Patents
Blood culture method Download PDFInfo
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- CN103898192A CN103898192A CN201410165196.XA CN201410165196A CN103898192A CN 103898192 A CN103898192 A CN 103898192A CN 201410165196 A CN201410165196 A CN 201410165196A CN 103898192 A CN103898192 A CN 103898192A
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- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000009640 blood culture Methods 0.000 title abstract description 5
- 210000004369 blood Anatomy 0.000 claims abstract description 46
- 239000008280 blood Substances 0.000 claims abstract description 46
- 238000001556 precipitation Methods 0.000 claims abstract description 26
- 239000003219 hemolytic agent Substances 0.000 claims abstract description 12
- 238000007789 sealing Methods 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 29
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 230000001717 pathogenic effect Effects 0.000 claims description 10
- 210000003677 hemocyte Anatomy 0.000 claims description 9
- 229940000351 hemocyte Drugs 0.000 claims description 9
- 238000012364 cultivation method Methods 0.000 claims description 8
- 239000006916 nutrient agar Substances 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 5
- 238000007689 inspection Methods 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000000386 microscopy Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 abstract description 7
- 229920001817 Agar Polymers 0.000 abstract description 2
- 210000000601 blood cell Anatomy 0.000 abstract 2
- 239000008272 agar Substances 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 238000004043 dyeing Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000011534 incubation Methods 0.000 abstract 1
- 244000052769 pathogen Species 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000009631 Broth culture Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 229910000831 Steel Inorganic materials 0.000 description 6
- 239000010959 steel Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 3
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 229940088592 immunologic factor Drugs 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 235000014347 soups Nutrition 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012009 microbiological test Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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- 239000007790 solid phase Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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Abstract
The invention discloses a blood culture method. The blood culture method comprises the following steps of adding a hemolytic agent to a centrifuge tube, sealing the centrifuge tube, and carrying out sterilization in the tube to achieve an aseptic condition; injecting a blood sample collected by an aseptic means into the centrifuge tube, and vibrating the centrifuge tube at room temperature to fully mix the blood sample with the hemolytic agent to crack blood cells so that pathogens in the blood cells are released; centrifuging the centrifuge tube at a centrifugal speed of 3-15krpm for 10-60 minutes, and stably taking out the centrifuge tube; separating mixed liquor in the centrifuge tube to form supernatant and precipitation concentrated liquor; extracting the precipitation concentrated liquor in the centrifuge tube; inoculating the precipitation concentrated liquor into an agar culture medium to undergo an incubation culture test, and simultaneously carrying out smear dyeing microscopic examination. The method is simple in flow, is short in test time, is low in cost and has a high positive detection rate.
Description
Technical field
The present invention relates to a kind of blood cultivation method.
Background technology
Existing blood cultivation is that the blood preparation of fresh in vitro is seeded on nutritional medium, under the condition such as certain temperature, humidity, makes the bacterial growth breeding higher to nutritional requirement, thereby detects a kind of artificial culture method of pathogenic bacteria.This culture method is usually used in the Etiologic of microbemia, septicemia and pyaemia septica.
Traditional blood cultivation process: gather blood samples of patients sample, inject flashing lightning magnetic field detector, have the liquid broth culture of tens milliliters in bottle.If meat soup hemoculture is positive within five days, need to extract out bacterium liquid, be seeded on suitable nutrient agar, then continue to hatch, obtain bacterium colony and can carry out Bacteria Identification and medicament sensitivity test; If meat soup hemoculture is negative, in the time of the 5th day, need to extract bacterium liquid out, be seeded to suitable nutrient agar, then continue to hatch, be " blind turning ".In this traditional method, there is following defect:
(1) blood preparation injects after broth culture, and the immune factor existing in blood samples of patients and the microbiotic that may exist all have restraining effect to the pathogenic agent in blood, thus the positive accuracy that detects result of impact;
(2) in blood preparation, hemocyte is complete, and intracellular pathogenic agent is limited to cytolemma, is difficult to cultivate detect, and susceptibility and specificity all can be affected, thereby affect positive rate;
(3) blood preparation is first cultivated in broth culture, no matter the consequently no positive, all needs to extract the nutrient agar that is seeded to solid phase out, proving time is long, and if the pathogenic bacteria in blood is fungi, only the broth culture of liquid phase is hatched and is easily occurred false negative and undetected;
(4) somewhat expensive for the broth culture of blood cultivation, validity period is shorter, general maximum 1 year; Particularly hemoculture instrument, tens is up to a million easily, and general middle and small hospital, backwoodsman clinic are difficult to bear;
(5) gather after blood samples of patients sample, general employing centrifuge tube extracts a certain amount of blood injection flashing lightning magnetic field detector and cultivates, normally adopt extraction utensil from centrifuge tube, to extract a certain amount of blood out, and it is very high to the requirement of gnotobasis in the culturing process of blood cultivation, and the easy contaminated blood sample of extraction utensil, introduce bacterium etc., cause inspection to disturb.
Summary of the invention
The object of the present invention is to provide a kind of blood cultivation method that flow process is simple, proving time is short, cost is low and positive rate is high.
Blood cultivation method of the present invention, comprises the following steps:
A. in centrifuge tube, add hemolytic agent, sealing centrifuge tube, the sterilizing that carries out disinfection in pipe reaches sterile state;
B. inject centrifuge tube by aseptic means sample of blood, drawn, under room temperature state, shake centrifuge tube, fully mix blood sample and hemolytic agent, make hemocyte cracking, endoglobar pathogenic agent is discharged into outside hemocyte;
C. with the centrifugal speed of 3k～15krpm by centrifugal centrifuge tube 10～60 minutes, steadily take out centrifuge tube;
D. the mixed solution in centrifuge tube presents separation, forms supernatant liquor and precipitation concentrated solution;
E. extract the precipitation concentrated solution in centrifuge tube;
F. precipitation concentrated solution is seeded to nutrient agar and hatches cultivation inspection, carry out smear staining microscopy simultaneously.
Further, described centrifuge tube is followed successively by pipe, middle pipe and collection tube from top to bottom, described middle pipe with upper pipe, collection tube is detachable is connected, described upper pipe both ends open and upper end open are provided with sealing cover assembly, described middle pipe both ends open and in establish segregaion valve, the upper end open of described collection tube and lower end are circular-arc bottom face; The injection rate of blood sample in step b so that the pipe internal volume that precipitation concentrated solution in steps d is filled below full segregaion valve be as the criterion; The process of extracting precipitation concentrated solution in step e is: first close segregaion valve, then collection tube is departed from, the solution in collection tube is precipitation concentrated solution.
This programme tool has the following advantages:
(1) hemocyte is hatching before cultivating cleavedly, and hiding pathogenic agent is released in cell, has improved the concentration of pathogenic agent, susceptibility and the specificity checked is improved, and then has improved positive rate;
(2) centrifugal treating is removed the supernatant liquor that comprises most of microbiotic and immune factor, plays the effect of effective constituent in concentrate blood sample, has improved positive rate;
(3) save broth culture and culturing process thereof, directly the blood preparation after cracking centrifugal concentrating has been inoculated on nutrient agar, for clinical diagnosis and treatment saves time, overcome the slow short slab of microbiological Test;
(4) do not need broth culture and relevant expensive, heavy hemoculture instrument, avoid installing, transport and use required severe condition, needn't worry the problem of substratum valid period, and only need room temperature storage, only use centrifuge tube and common whizzer, light and cost is low, be applicable to infirmary, clinic, army's FAMB.
And, centrifuge tube in this case adopts segregaion valve to stop supernatant liquor and precipitation concentrated solution, makes in the time extracting precipitation concentrated solution, has avoided employing extraction utensil to affect operation efficiency and has polluted sedimentary problem, accomplish pollution-freely, ensured the accuracy of positive rate.The Demountable of the upper pipe of centrifuge tube, middle pipe and collection tube, convenient in the time extracting precipitation concentrated solution, simplify operating process, ensure the quality of precipitation concentrated solution.
Brief description of the drawings
Structural state figure when Fig. 1 is the centrifuge tube inversion in the embodiment of the present invention two;
Fig. 2 is the structure iron of the centrifuge tube in the embodiment of the present invention three.
Description of reference numerals: 1-upper pipe; 2-collection tube; 3-segregaion valve; 7-sealing cover assembly; 11-middle pipe; 31-valve body switch; 32-steel ball; 33-support; 34-steel ball seat.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment mono-
A. the hemolytic agent that to add with the volume ratio of blood preparation in centrifuge tube be 1:5, by the sealing cover component sealing centrifuge tube of centrifuge tube upper end, then reaches sterile state to the sterilizing that carries out disinfection in managing; Or first by the sterilizing respectively of centrifuge tube and hemolytic agent, then hemolytic agent is joined is in the centrifuge tube of sterile state, then seal;
B. the blood sample collecting by aseptic means is injected to centrifuge tube, under room temperature state, slightly firmly shake under centrifuge tube ten, can fully mix blood sample and hemolytic agent, make hemocyte cracking, the bacterium of concealing in hemocyte is discharged;
C. with the centrifugal speed of 3krpm by centrifugal centrifuge tube 30 minutes, steadily take out centrifuge tube, not jolting;
D. wait for that the mixed solution in centrifuge tube presents separation, form supernatant liquor and precipitation concentrated solution;
E. adopt extraction utensil to take out supernatant liquor, the extraction utensil renewing extracts the precipitation concentrated solution in centrifuge tube, and extraction utensil carries out sterilization processing before using;
F. can dip easily the direct streak inoculation of precipitation concentrated solution with transfering loop or swab and on various suitable nutrient agars, hatch cultivation inspection, carry out smear staining microscopy simultaneously.
Embodiment bis-
A. prepare special centrifuge tube, centrifuge tube is formed by upper pipe 1, middle pipe 11 and collection tube 2 detachable assembleds, described upper pipe 1 both ends open and upper end open are provided with sealing cover assembly 7, described middle pipe 11 both ends opens and in establish the unidirectional segregaion valve 3 of ball type, when centrifuge tube is upright, steel ball 32 is placed on support 33, valve open; When centrifuge tube is inverted, as shown in Figure 1, steel ball 32 departs from support 33, and steel ball 32 is blocked the passage of steel ball seat 34, valve closes, and the upper end open of described collection tube 2 and lower end are circular-arc bottom face; The hemolytic agent that to add with the volume ratio of blood preparation be 1:20, sealing centrifuge tube, the sterilizing that carries out disinfection in pipe reaches sterile state;
B. the injection rate of blood sample is so that the pipe internal volume that the precipitation concentrated solution in steps d is filled below full segregaion valve is as the criterion, and all the other are identical with the step b in embodiment mono-;
C. with the centrifugal speed of 10krpm by centrifugal centrifuge tube 10 minutes, steadily take out centrifuge tube, not jolting;
D. identical with the steps d in embodiment mono-;
E. be inverted centrifuge tube, valve is closing condition, and pipe 1 is departed from; By upright collection tube 2, valve in opened condition, is twisted lower intermediate tube 11 again, and collection tube 2 departs from middle pipe 11, is precipitated concentrated solution;
F. identical with the step f in embodiment mono-.
Embodiment tri-
A. prepare special centrifuge tube, as shown in Figure 2, centrifuge tube is formed by upper pipe 1, middle pipe 11 and collection tube 2 detachable assembleds, described upper pipe 1 both ends open and upper end open are provided with sealing cover assembly 7, described middle pipe 11 both ends opens and in establish and block class segregaion valve 3 with valve body switch 31, valve body switch 31 is located at outside pipe, and by the break-make of valve body switch 31 by-pass valve controls, the upper end open of described collection tube 2 and lower end are circular-arc bottom face; The hemolytic agent that to add with the volume ratio of blood preparation be 1:10, sealing centrifuge tube, the sterilizing that carries out disinfection in pipe reaches sterile state;
B. identical with the step b in embodiment bis-;
C. with the centrifugal speed of 15krpm by centrifugal centrifuge tube 60 minutes, steadily take out centrifuge tube, not jolting;
D. identical with the steps d in embodiment mono-;
E. keep centrifuge tube erectility, by valve body switch 31 valve-offs, directly back out collection tube 2, be precipitated concentrated solution;
F. identical with the step f in embodiment mono-.
What above-described embodiment one adopted is common centrifuge tube, should note the aseptically process of extraction utensil etc. in the process of extracting precipitation concentrated solution, should avoid polluting, in order to avoid cause detecting invalid.And embodiment bis-and embodiment tri-be without the introducing of extraction utensil, more easily accomplish pollution-freely, ensure the sterile state of whole process, anti-pollution in advance without taking loaded down with trivial details step measure, improve working efficiency, for the quality time has been striven in clinical diagnosis.Therefore, in force, preferably adopt the mode of embodiment bis-and three to carry out blood cultivation.
Be below the present invention compared with the prior art and analyze, statistical study dependency, further to evaluate technical scheme of the present invention.
(1) materials and methods
Blood cultivation instrument and Blood culture bottle thereof with Mei Liai are made as control group, gather clinical thoughts and have a finger in every pie the blood of levying patient and do blood cultivation.Using novel method of the present invention as test group, the two pairing test that compares.
Record and result
Control group sample and test group are cultivated number of cases and are 227 examples, wherein, and positive 27 examples of control group (positive rate is 11.89%), positive 34 examples of test group (positive rate is 14.98%).
Assay
Two groups of statistical analysis, chi square test=2.0489, p < 0.05, refusal H0, differs remarkable.Only from positive rate, novel method result of the present invention is better than the result that existing methodical import equipment and Blood culture bottle thereof draw.Cause the reason of this result to be: a clinical patients is not often the best moment on blood drawing opportunity, but just use before microbiotic, that is to say and in blood, have higher antibiotic concentration, and novel method has been removed most antibiotics because of centrifugal concentrating, weaken the restraining effect of microbiotic to pathogenic agent, after concentrated, in sample to be checked, the concentration of pathogenic agent improves, and then has improved susceptibility and the specificity of positive inspection; The cracking of b present method hemocyte, discharged the pathogenic agent in cell, improved pathogenic agent concentration, thereby make recall rate improve.
Claims (4)
1. a blood cultivation method, is characterized in that comprising the following steps:
A. in centrifuge tube, add hemolytic agent, sealing centrifuge tube, the sterilizing that carries out disinfection in pipe reaches sterile state;
B. inject centrifuge tube by aseptic means sample of blood, drawn, under room temperature state, shake centrifuge tube, fully mix blood sample and hemolytic agent, make hemocyte cracking, endoglobar pathogenic agent is discharged into outside hemocyte;
C. with the centrifugal speed of 3k～15krpm by centrifugal centrifuge tube 10～60 minutes, steadily take out centrifuge tube;
D. the mixed solution in centrifuge tube presents separation, forms supernatant liquor and precipitation concentrated solution;
E. extract the precipitation concentrated solution in centrifuge tube;
F. precipitation concentrated solution is seeded to nutrient agar and hatches cultivation inspection, carry out smear staining microscopy simultaneously.
2. blood cultivation method according to claim 1, it is characterized in that: described centrifuge tube is followed successively by pipe, middle pipe and collection tube from top to bottom, described middle pipe with upper pipe, collection tube is detachable is connected, described upper pipe both ends open and upper end open are provided with sealing cover assembly, described middle pipe both ends open and in establish segregaion valve, the upper end open of described collection tube and lower end are circular-arc bottom face;
The injection rate of blood sample in step b so that the pipe internal volume that precipitation concentrated solution in steps d is filled below full segregaion valve be as the criterion;
The process of extracting precipitation concentrated solution in step e is: first close segregaion valve, then collection tube is departed from, the solution in collection tube is precipitation concentrated solution.
3. blood cultivation method according to claim 2, it is characterized in that: described segregaion valve is ball type one-way cock, the process of extracting precipitation concentrated solution in step e is: be inverted centrifuge tube, segregaion valve cuts out automatically, and pipe is departed from, then collection tube is upright, segregaion valve is opened automatically, twist lower intermediate tube, collection tube and middle pipe depart from, and are precipitated concentrated solution.
4. blood cultivation method according to claim 2, it is characterized in that: described segregaion valve is to block class valve with valve body switch, the process of extracting precipitation concentrated solution in step e is: keep centrifuge tube erectility, close segregaion valve by valve body switch, directly back out collection tube, be precipitated concentrated solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410165196.XA CN103898192B (en) | 2014-04-23 | 2014-04-23 | Blood culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410165196.XA CN103898192B (en) | 2014-04-23 | 2014-04-23 | Blood culture method |
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| CN103898192A true CN103898192A (en) | 2014-07-02 |
| CN103898192B CN103898192B (en) | 2015-01-28 |
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| CN201410165196.XA Active CN103898192B (en) | 2014-04-23 | 2014-04-23 | Blood culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1381239A (en) * | 2001-04-18 | 2002-11-27 | 松元司 | Method for extracting composition from cultured leukocyte |
| CN1410531A (en) * | 2002-11-20 | 2003-04-16 | 上海市第一人民医院 | Culturing method of human peripheral blood vessel endothelial ancestry cell |
| CN2575675Y (en) * | 2002-08-20 | 2003-09-24 | 李开 | Flashing lightning magnetic field detector |
| CN1516642A (en) * | 2001-06-18 | 2004-07-28 | ���˶ٵϿ�ɭ��˾ | Multilayer container and process relating to forming multilayer container |
| CN2689223Y (en) * | 2004-04-09 | 2005-03-30 | 唐明忠 | Blood culturing bottles |
| CN2921835Y (en) * | 2006-07-17 | 2007-07-11 | 丛波 | Blood culture bottle |
| CN201614375U (en) * | 2009-12-29 | 2010-10-27 | 成都威力生生物科技有限公司 | Blood culture bottle |
-
2014
- 2014-04-23 CN CN201410165196.XA patent/CN103898192B/en active Active
Patent Citations (8)
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|---|---|---|---|---|
| CN1275618A (en) * | 2000-06-21 | 2000-12-06 | 张国庆 | Method for cultivating T-lymphocytes |
| CN1381239A (en) * | 2001-04-18 | 2002-11-27 | 松元司 | Method for extracting composition from cultured leukocyte |
| CN1516642A (en) * | 2001-06-18 | 2004-07-28 | ���˶ٵϿ�ɭ��˾ | Multilayer container and process relating to forming multilayer container |
| CN2575675Y (en) * | 2002-08-20 | 2003-09-24 | 李开 | Flashing lightning magnetic field detector |
| CN1410531A (en) * | 2002-11-20 | 2003-04-16 | 上海市第一人民医院 | Culturing method of human peripheral blood vessel endothelial ancestry cell |
| CN2689223Y (en) * | 2004-04-09 | 2005-03-30 | 唐明忠 | Blood culturing bottles |
| CN2921835Y (en) * | 2006-07-17 | 2007-07-11 | 丛波 | Blood culture bottle |
| CN201614375U (en) * | 2009-12-29 | 2010-10-27 | 成都威力生生物科技有限公司 | Blood culture bottle |
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