CN107236781A - Fungi positive blood culture of isolated glue and chemical reagent pre-treating method - Google Patents
Fungi positive blood culture of isolated glue and chemical reagent pre-treating method Download PDFInfo
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- CN107236781A CN107236781A CN201710414323.9A CN201710414323A CN107236781A CN 107236781 A CN107236781 A CN 107236781A CN 201710414323 A CN201710414323 A CN 201710414323A CN 107236781 A CN107236781 A CN 107236781A
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Abstract
A kind of fungi positive blood culture of isolated glue and chemical reagent pre-treating method, extract 8.5ml blood culture with positive bacteria liquid into 8.5ml separation sebific ducts；Separation sebific duct is placed into centrifuge, centrifuge centrifuges 10min in the presence of 4000g centrifugal force；Centrifugation goes toward addition 1ml tweens Tween20 in EP pipes inhale repeatedly after supernatant to blow, EP pipes are stood 3min under conditions of room temperature again, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；With 1ml aseptic double-distilled waters, pressure-vaccum is precipitated repeatedly, is allowed bacterium to be suspended in aseptic double-distilled water with this and is formed bacteria suspension；The bacteria suspension of acquisition carries out direct MALDI TOF Mass Spectrometric Identifications after being handled by chemical reagent, effectively prevent the defect that time-consuming of whole process in the prior art.
Description
Technical field
The present invention relates to fungi positive blood culture technique field, and in particular to a kind of fungi positive blood culture of isolated glue and change
Learn reagent pre-treating method.
Background technology
The authentication method of the positive blood culture of existing fungi is：By traditional process processing after hemoculture instrument positive alarm, apply
Piece Grain stain, micro- sem observation, transferred species sand are protected weak flat board and cultivated under aerobic conditions after the pure bacterium colony of 24-48h acquisitions, reuse
Existing the commercialization instrument based on biochemical reaction or immunological method such as Vitek 2Compact, API 20C etc. are identified.
But such method is needed in positive blood nutrient solution transferred species to culture medium at present, culture is incubated visible to naked eyes
Single pure bacterium colony after, then select single pure bacterium colony and identified by follow-up method, whole process takes longer (about 48h-
72h)。
The content of the invention
To solve the above problems, the invention provides a kind of fungi positive blood culture of isolated glue and chemical reagent pre-treatment side
Method, effectively prevent the defect that time-consuming of whole process in the prior art.
In order to overcome deficiency of the prior art, the invention provides a kind of fungi positive blood culture of isolated glue and chemistry examination
The solution of agent pre-treating method, it is specific as follows：
A kind of fungi positive blood culture of isolated glue and chemical reagent pre-treating method, are comprised the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge is centrifuged in the presence of 4000g centrifugal force
10min；
Step 3：Blood culture with positive bacteria liquid is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct
Supernatant is abandoned in suction；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed with this
It is suspended in aseptic double-distilled water and forms bacteria suspension；
Step 5：1ml bacteria suspensions are transferred in 1.5ml EP pipes, EP pipes are put into centrifuge, centrifuge exists
Centrifugation 2min is carried out under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge is in 13000rpm
Rotating speed under conditions of carry out centrifugation 2min, not touching EP ttom of pipe precipitation under conditions of inhale abandon supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then the handle under conditions of room temperature
EP pipes stand 3min, and centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, are not touching the precipitation of EP ttom of pipe
Under conditions of inhale abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP is managed
It is placed on vortex oscillation instrument and be vortexed after concussion 1min, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700,
Then pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP pipes
Inhaled under conditions of the precipitation at bottom and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stood at ambient temperature
The residual ethanol in material in 5min, drying EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of EP ttom of pipe and percentage forms the second mixing for 70% formic acid
Thing, then carries out pressure-vaccum mixing to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：15 μ l acetonitriles are added to form the 3rd mixture into EP pipes, then carrying out pressure-vaccum to the 3rd mixture mixes
It is even, then EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano-
4- hydroxycinnamic acids (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
With the number of times of the 1ml aseptic double-distilled waters mycoderm on pressure-vaccum separation gel surface repeatedly it is 5-10 times in the step 4.
The time that the EP pipes are stood under conditions of room temperature again in the step 14 is 5-10min.
Beneficial effects of the present invention are：
The method of the fungi positive blood culture of isolated glue of the present invention need not be by positive blood nutrient solution transferred species to culture medium
Identified to growing single bacterium colony, then by follow-up method, but positive blood nutrient solution is subjected to separation gel pre-treatment
Direct Mass Spectrometric Identification afterwards, whole process takes about 30-40min, identity process is substantially reduced, while having saved manpower and materials.
Brief description of the drawings
Fig. 1 is the structural representation of the interlocking equipment of the present invention；
Fig. 2 is the structural representation of the first scarfing unit of the present invention；
Fig. 3 is the partial sectional schematic view of the second scarfing unit of the present invention；
Fig. 4 is the sectional view of the interlocking equipment of the present invention.
Embodiment
The present invention is described further below in conjunction with drawings and examples.
Embodiment 1
Fungi positive blood culture of isolated glue and chemical reagent pre-treating method, are comprised the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge is centrifuged in the presence of 4000g centrifugal force
10min；
Step 3：Blood culture with positive bacteria liquid is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct
Supernatant is abandoned in suction；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed with this
It is suspended in aseptic double-distilled water and forms bacteria suspension；
Step 5：1ml bacteria suspensions are transferred in 1.5ml EP pipes, EP pipes are put into centrifuge, centrifuge exists
Centrifugation 2min is carried out under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge is in 13000rpm
Rotating speed under conditions of carry out centrifugation 2min, not touching EP ttom of pipe precipitation under conditions of inhale abandon supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then the handle under conditions of room temperature
EP pipes stand 3min, and centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, are not touching the precipitation of EP ttom of pipe
Under conditions of inhale abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP is managed
It is placed on vortex oscillation instrument and be vortexed after concussion 1min, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700,
Then pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP pipes
Inhaled under conditions of the precipitation at bottom and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stood at ambient temperature
The residual ethanol in material in 5min, drying EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of each EP ttom of pipe and percentage forms second for 70% formic acid
Mixture, then carries out pressure-vaccum mixing to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：15 μ l acetonitriles are added to form the 3rd mixture into EP pipes, then carrying out pressure-vaccum to the 3rd mixture mixes
It is even, then EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano-
4- hydroxycinnamic acids (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
In the step 4 with 1ml aseptic double-distilled waters repeatedly the mycoderm on pressure-vaccum separation gel surface number of times be 5 times.
The time that EP pipes are stood under conditions of room temperature again in the step 14 is 5min.
The present embodiment has the beneficial effect that：
The method of the fungi positive blood culture of isolated glue of the present embodiment need not be by positive blood nutrient solution transferred species to culture medium
Single bacterium colony is up to grown, then is identified by follow-up method, but positive blood nutrient solution is carried out before separation gel to locate
Direct Mass Spectrometric Identification after reason, whole process takes 30min, substantially reduces identity process, while having saved manpower and materials.
Embodiment 2
Fungi positive blood culture of isolated glue and chemical reagent pre-treating method, are comprised the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge is centrifuged in the presence of 4000g centrifugal force
10min；
Step 3：Blood culture with positive bacteria liquid is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct
Supernatant is abandoned in suction；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed with this
It is suspended in aseptic double-distilled water and forms bacteria suspension；
Step 5：1ml bacteria suspensions are transferred in a 1.5ml EP pipe, EP pipes are put into centrifuge, centrifuge exists
Centrifugation 2min is carried out under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge is in 13000rpm
Rotating speed under conditions of carry out centrifugation 2min, not touching EP ttom of pipe precipitation under conditions of inhale abandon supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then the handle under conditions of room temperature
EP pipes stand 3min, and centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, are not touching the precipitation of EP ttom of pipe
Under conditions of inhale abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP is managed
It is placed on vortex oscillation instrument and be vortexed after concussion 1min, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700,
Then pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP pipes
Inhaled under conditions of the precipitation at bottom and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stood at ambient temperature
The residual ethanol in material in 5min, drying EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of EP ttom of pipe and percentage forms the second mixing for 70% formic acid
Thing, then carries out pressure-vaccum mixing to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：15 μ l acetonitriles are added to form the 3rd mixture into EP pipes, then carrying out pressure-vaccum to the 3rd mixture mixes
It is even, then EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano-
4- hydroxycinnamic acids (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
In the step 4 with 1ml aseptic double-distilled waters repeatedly the mycoderm on pressure-vaccum separation gel surface number of times be 8 times.
The time that EP pipes are stood under conditions of room temperature again in the step 14 is 8min.
The present embodiment has the beneficial effect that：
The method of the fungi positive blood culture of isolated glue of the present embodiment need not be by positive blood nutrient solution transferred species to culture medium
Single bacterium colony is up to grown, then is identified by follow-up method, but positive blood nutrient solution is carried out before separation gel to locate
Direct Mass Spectrometric Identification after reason, whole process takes 35min, substantially reduces identity process, while having saved manpower and materials.
Embodiment 3
Fungi positive blood culture of isolated glue and chemical reagent pre-treating method, are comprised the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge is centrifuged in the presence of 4000g centrifugal force
10min；
Step 3：Blood culture with positive bacteria liquid is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct
Supernatant is abandoned in suction；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed with this
It is suspended in aseptic double-distilled water and forms bacteria suspension；
Step 5：1ml bacteria suspensions are transferred in 1.5ml EP pipes, EP pipes are put into centrifuge, centrifuge exists
Centrifugation 2min is carried out under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge is in 13000rpm
Rotating speed under conditions of carry out centrifugation 2min, not touching EP ttom of pipe precipitation under conditions of inhale abandon supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then the handle under conditions of room temperature
EP pipes stand 3min, and centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, are not touching the precipitation of EP ttom of pipe
Under conditions of inhale abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP is managed
It is placed on vortex oscillation instrument and be vortexed after concussion 1min, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700,
Then pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP pipes
Inhaled under conditions of the precipitation at bottom and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stood at ambient temperature
The residual ethanol in material in 5min, drying EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of EP ttom of pipe and percentage forms the second mixing for 70% formic acid
Thing, then carries out pressure-vaccum mixing to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：15 μ l acetonitriles are added to form the 3rd mixture into EP pipes, then carrying out pressure-vaccum to the 3rd mixture mixes
It is even, then EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano-
4- hydroxycinnamic acids (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
In the step 4 with 1ml aseptic double-distilled waters repeatedly the mycoderm on pressure-vaccum separation gel surface number of times be 10 times.
The time that EP pipes are stood under conditions of room temperature again in the step 14 is 10min.
The present embodiment has the beneficial effect that：
The method of the fungi positive blood culture of isolated glue of the present embodiment need not be by positive blood nutrient solution transferred species to culture medium
Single bacterium colony is up to grown, then is identified by follow-up method, but positive blood nutrient solution is carried out before separation gel to locate
Direct Mass Spectrometric Identification after reason, whole process takes 40min, substantially reduces identity process, while having saved manpower and materials.
Embodiment 4
Fungi positive blood culture of isolated glue and chemical reagent pre-treating method, are comprised the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge is centrifuged in the presence of 4000g centrifugal force
10min；
Step 3：Blood culture with positive bacteria liquid is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct
Supernatant is abandoned in suction；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed with this
It is suspended in aseptic double-distilled water and forms bacteria suspension；
Step 5：1ml bacteria suspensions are transferred in 1.5ml EP pipes, EP pipes are put into centrifuge, centrifuge exists
Centrifugation 2min is carried out under conditions of 13000rpm rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge is in 13000rpm
Rotating speed under conditions of carry out centrifugation 2min, not touching EP ttom of pipe precipitation under conditions of inhale abandon supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then the handle under conditions of room temperature
EP pipes stand 3min, and centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, are not touching the precipitation of EP ttom of pipe
Under conditions of inhale abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP is managed
It is placed on vortex oscillation instrument and be vortexed after concussion 1min, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700,
Then pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed,
Inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandon supernatant；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP pipes
Inhaled under conditions of the precipitation at bottom and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stood at ambient temperature
The residual ethanol in material in 5min, drying EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of EP ttom of pipe and percentage forms the second mixing for 70% formic acid
Thing, then carries out pressure-vaccum mixing to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：15 μ l acetonitriles are added to form the 3rd mixture into EP pipes, then carrying out pressure-vaccum to the 3rd mixture mixes
It is even, then EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano-
4- hydroxycinnamic acids (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
In the step 4 with 1ml aseptic double-distilled waters repeatedly the mycoderm on pressure-vaccum separation gel surface number of times be 10 times.
The time that EP pipes are stood under conditions of room temperature again in the step 14 is 10min.
The present embodiment has the beneficial effect that：
The method of the fungi positive blood culture of isolated glue of the present embodiment need not be by positive blood nutrient solution transferred species to culture medium
Single bacterium colony is up to grown, then is identified by follow-up method, but positive blood nutrient solution is carried out before separation gel to locate
Direct Mass Spectrometric Identification after reason, whole process takes 40min, substantially reduces identity process, while having saved manpower and materials.
Also it is exactly that the centrifuge includes support, and the support is rectangular-shape, and support is directly placed on ground
Tend to make moist, for the roof of protection against the tide, the often stainless steel base of the same rectangular-shape for being placed on ground of the bottom wall of support
It is connected, the bottom wall of bearing is with the bottom wall that the structure that the roof of the stainless steel base of rectangular-shape is connected is by the bearing
It is connected with the roof of the stainless steel base, the knot that the bottom wall of the bearing is connected with the roof of the stainless steel base
Structure is generally must set positioning port on the roof of the stainless steel base, then sequentially pass through from top to bottom by screw mandrel described
The bottom wall of base and the screw being welded and fixed in positioning port screw connection, completed with this base bottom wall and it is described not
The roof of rust steel base is connected, and such a structure not only increases manufacturing expense, and with Assembly veracity is low, visual effect not
It is good.
The bottom wall of the bearing of the centrifuge is connected with the roof of the stainless steel base for the rectangular-shape for being placed on ground
Structure be connected for the roof of bottom wall and the stainless steel base of the bearing via equipment S00 is rabbeted；
The interlocking equipment S00 contains the first scarfing unit S0 and the second scarfing unit T0；
The first scarfing unit S0 connects firmly the roof in the stainless steel base, and the second scarfing unit T0 is connected firmly described
The bottom wall of bearing；
The top of the first scarfing unit S0 is cuboid plate, and the bottom of the first scarfing unit S0 is arch shaped body, described
The top of arch shaped body and the bottom wall of cuboid plate are mutually connected firmly, and the bottom of the arch shaped body and the roof of the stainless steel base are mutually solid
Connection, is provided with the boarding hole S1 until inside the arch shaped body, the top of the boarding hole S1 on the roof of the cuboid plate
Interface S11 is on the roof of the cuboid plate；
The top of the second scarfing unit T0 is cylindric cylinder, and the top of the cylindric cylinder is connected firmly in the base
Bottom wall, the bottom of the cylindric cylinder is the scarf joint T1 of glass bronze material, and the scarf joint T1 rabbets into the interlocking
In the S1 of hole, the top joint S11 of the boarding hole S1 stops to the scarf joint T1 in addition, the appearance of the scarf joint T1
Face can close up during rabbeting into or exiting the boarding hole S1.
The boarding hole S1 of the first scarfing unit S0 is ball arc structure, the profile of the outer surface of the scarf joint T1
Also it is ball arc structure.
The boarding hole S1 of first scarfing unit S0 profile can also arch upward for some sections of arc shapes and be smoothly connected
Form, can also be to be round table-like, and the boarding hole S1 of the outer surface of the scarf joint T1 also with the first scarfing unit S0
Contour shape it is identical.
The scarf joint T1 contains some around the glass being connected on the cylindric cylinder bottom of the second scarfing unit T0
Arcuation the scarfer T11, the arcuation scarfer T11 of bronze material are equidistantly looped around the cylindrical strut of the second scarfing unit T0
On body bottom, all arcuation scarfer T11 outer surface forms the outer surface of the scarf joint T1 together.
The boarding hole S1 contains the edge table in the lower surface S12 and boarding hole S1 in the boarding hole S1
Lower surface S12 in face S13, the boarding hole S1 is opposite and right with the top joint S11 of the boarding hole S1, the interlocking
Edge surface in the S1 of hole is connected with the lower surface S12 in the boarding hole S1, in addition the edge in the boarding hole S1
Surface S13 to the top joint S11 of boarding hole S1 structure is gradually-reducing shape structure.
It is suitable that lower surface S12 in the boarding hole S1 is that edge surface S13 in ball arcuation, the boarding hole S1 contains
The connected cylindric branch S13S of sequence and round table-like branch S13T, the bottom of the cylindric branch S13S and the boarding hole S1
Interior lower surface S12 is connected, and the top of the round table-like branch S13T is connected with the top joint S11 of the boarding hole S1,
The round table-like branch S13T is exactly the gradually-reducing shape structure.
In addition, the roof of the first scarfing unit S0 and the stainless steel base is one-shot forming overall structure, described the
Two scarfing unit T0 are one-shot forming overall structure with the base.
During the scarf joint T1 of the glass bronze material rabbets into the boarding hole S1, the interlocking of the glass bronze material
Head T1 outer surface progressively closes up to be put in by the top joint S11 of the boarding hole S1, and in the embedding of the glass bronze material
During joint T1 exits the boarding hole S1 of the first scarfing unit S0, the scarf joint T1 of glass bronze material outer surface is progressively
Close up to be exited by the top joint S11 of the boarding hole S1, thus the first scarfing unit S0 combined with the second scarfing unit T0 it is more firm
Lean on, Assembly veracity is high in addition, it is easy to efficiently assembling and dismounting.
The present invention is described in the way of brief description of the drawings above, it will be understood by those of skill in the art that the disclosure
Embodiments described above is not limited to, in the case of without departing from the scope of the present invention, can make a variety of changes, change and replace
Change.
Claims (4)
1. a kind of fungi positive blood culture of isolated glue and chemical reagent pre-treating method, it is characterised in that comprise the following steps that：
Step 1：Blood culture with positive bacteria liquid is extracted into 8.5ml separation sebific ducts；
Step 2：Separation sebific duct is placed into centrifuge, centrifuge centrifuges 10min in the presence of 4000g centrifugal force；
Step 3：Suction is carried out under conditions of the surface mycoderm of the separation gel in not touching separation sebific duct to blood culture with positive bacteria liquid to abandon
Supernatant；
Step 4：With the mycoderm on separation gel surface in 1ml aseptic double-distilled waters repeatedly pressure-vaccum separation sebific duct, mycoderm is allowed to suspend with this
Bacteria suspension is formed in aseptic double-distilled water；
Step 5：1ml bacteria suspensions are transferred in 1.5ml EP pipes, EP pipes are put into centrifuge, centrifuge is 13000rpm's
Centrifugation 2min is carried out under conditions of rotating speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 6：Pressure-vaccum washing is carried out repeatedly after 1ml aseptic double-distilled waters toward adding in EP pipes again, centrifuge turns 13000rpm's
Centrifugation 2min is carried out under conditions of speed, is inhaled under conditions of the not precipitation of touching EP ttom of pipe and abandons supernatant；
Step 7：Then blow toward adding in EP pipes to be inhaled repeatedly after 1ml tweens Tween20, then EP managed under conditions of room temperature
3min is stood, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, in the bar of the not precipitation of touching EP ttom of pipe
Inhaled under part and abandon supernatant；
Step 8：The SDS that 1ml aseptic double-distilled waters and 200 μ l and percentage are 10% is added into EP pipes, EP pipes are placed
Carry out being vortexed after concussion 1min on vortex oscillation instrument, then EP pipes standing 5min under conditions of room temperature；
Step 9：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, not
Supernatant is abandoned in suction under conditions of touching the precipitation of EP ttom of pipe；
Step 10：Percentage is added into the precipitation of EP ttom of pipe and constitutes the first mixture for the 75% μ l of ethanol 700, then
Pressure-vaccum mixing is carried out to first mixture；
Step 11：Then the EP pipes are stood 10min at ambient temperature；
Step 12：EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed, not
Supernatant is abandoned in suction under conditions of touching the precipitation of EP ttom of pipe；
Step 13：Continue to allow centrifuge to carry out centrifugation 1min under conditions of 13000rpm rotating speed, in not touching EP ttom of pipe
Inhaled under conditions of precipitation and abandon supernatant；Then the EP pipes are placed in Biohazard Safety Equipment and stand 5min at ambient temperature, blown
The residual ethanol in material in dry EP pipes causes residual ethanol to volatilize completely；
Step 14：15 μ l are added into the precipitation of EP ttom of pipe and percentage forms the second mixture for 70% formic acid,
Then pressure-vaccum mixing is carried out to the second mixture, then EP pipes standing under conditions of room temperature；
Step 15：Add 15 μ l acetonitriles to form the 3rd mixture into EP pipes, pressure-vaccum mixing then is carried out to the 3rd mixture, then
EP pipes are put into centrifuge, centrifuge carries out centrifugation 2min under conditions of 13000rpm rotating speed；
Step 16：Take the supernatant in 1 μ l EP pipes to drip in target plate, target plate is dried at ambient temperature；Plus 1 μ l alpha-cyano -4- hydroxyl
Base cinnamic acid (CHCA) matrix, target plate is put into Bruker mass spectrographs and carries out mass spectral analysis.
2. fungi positive blood culture of isolated glue according to claim 1 and chemical reagent pre-treating method, it is characterised in that
The amount that blood culture with positive bacteria liquid is extracted in the step 1 is 8.5ml.
3. fungi positive blood culture of isolated glue according to claim 1 and chemical reagent pre-treating method, it is characterised in that
With the number of times of the 1ml aseptic double-distilled waters mycoderm on pressure-vaccum separation gel surface repeatedly it is 5-10 times in the step 4.
4. fungi positive blood culture of isolated glue according to claim 1 and chemical reagent pre-treating method, it is characterised in that
The time that EP pipes are stood under conditions of room temperature again in the step 14 is 5-10min.
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| Application Number | Priority Date | Filing Date | Title |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102395680A (en) * | 2009-03-03 | 2012-03-28 | 公共救济事业局-巴黎医院 | Method for identifying germs in a liquid medium |
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2017
- 2017-06-05 CN CN201710414323.9A patent/CN107236781A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102395680A (en) * | 2009-03-03 | 2012-03-28 | 公共救济事业局-巴黎医院 | Method for identifying germs in a liquid medium |
Non-Patent Citations (2)
| Title |
|---|
| 陈峰等: "分离胶促凝管联合MALDI-TOF MS直接检测血培养阳性细菌", 《检验医学》 * |
| 陈飞等: "MALDI-TOF MS在临床微生物样本直接检测中的应用", 《检验医学》 * |
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