WO2012000309A1 - 一种无菌检查方法及其使用的全封闭集菌安瓿培养器 - Google Patents
一种无菌检查方法及其使用的全封闭集菌安瓿培养器 Download PDFInfo
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- WO2012000309A1 WO2012000309A1 PCT/CN2011/001062 CN2011001062W WO2012000309A1 WO 2012000309 A1 WO2012000309 A1 WO 2012000309A1 CN 2011001062 W CN2011001062 W CN 2011001062W WO 2012000309 A1 WO2012000309 A1 WO 2012000309A1
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- ampoule
- bacterial
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
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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
- C12Q1/22—Testing for sterility conditions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
- G01N25/20—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity
Definitions
- the invention relates to the inspection field of aseptic products for medicines, foods, biological products and medical instruments, and particularly relates to a sterilizing inspection method and a fully enclosed ampoules incubator for use thereof. Background technique
- Sterility testing is a must-have item to ensure the safe use of sterile products and is one of the important steps in determining the production cycle of a sterile product.
- the Pharmacopoeia of all countries has strict requirements for the bactericidal examination of injections, and basically forms internationally consistent inspection standards and operating procedures, which effectively improves the sterility assurance level of preparations.
- microcalorimetry is a sensitive, fast, easy-to-use, multi-channel, real-time online monitoring instrument system.
- the inventor's research group applied microcalorimetry to detect thermal effects during microbial growth, for drug quality control and The utility evaluation has achieved certain experience and results. Studies have shown that under appropriate conditions, the growth of microorganisms exhibits obvious regularity and characteristics, and it is suggested that a new method of sterility inspection can be established by using microcalorimetry.
- the principle of the invention is that the function of the thermal effect in the process of microbial growth can be detected by using a microcalorimeter, and the thermogram of the fingerprint characteristic of the microorganisms of different kinds of living conditions is recorded in a microcalorimeter, and a standard file for data analysis is established. Then record the thermal spectrum of the sample to be tested. If the sample is not sterilized or incompletely sterilized, it will be contaminated by microorganisms. The tendency of microbial growth will appear in the thermal spectrum of the sample, which is compared with the established standard file. , the sample that has been contaminated can be quickly selected, and the microbial contamination of the sample can be initially identified.
- Microcalorimeters are usually operated by placing microbial strains into a small amount of thermal ampoule in a specific medium, and then placing the ampoule in a microcalorimeter detection channel to record the change in heat generated by microbial growth.
- a microcalorimeter for sterility testing, there is a major defect in the operation, that is, when the sterility test is performed, the sealing operation of the sample and the medium injection cannot be achieved due to the ampule structure used in the microcalorimeter.
- the design principles of the fully enclosed ampoules incubator of the present invention include: (1) sterility: ⁇ use appropriate sterilization methods to ensure the sterility of the sterilizer itself; (2) sealing: ensuring the internal and external environment of the system Effective Isolation; (3) Inoculation: Configure the necessary collection device to achieve microbial enrichment and bacteriostatic elimination of the sample, and configure a suitable filter according to the nature of the sample to be tested; (4) Thermal sensitivity: System materials can make Microbial growth and metabolism of heat can be sensitively detected by calorimeter; (5) Pressure resistance: The system can meet the negative pressure requirements of the collection process and avoid microbial damage; (6) Tolerance: The system should be able to meet the sterility test sample size Need, have sufficient inspection ability; (7) Simplicity: The system should be easy to operate, with good automation performance and automatic prompting result function; (8) Economical: The system should be easy to operate, with good automation performance and automatic prompting result function; (8) Economical: The system should be easy to operate, with good automation performance and automatic prompting
- the object of the present invention is to solve the defects that the prior sterility test method has a long cycle, low sensitivity, subjective influence of the test result by the observer, and the ampule in the micro calorimeter cannot achieve full-closed aseptic operation.
- the technical solution of the present invention for achieving the above object is a sterilizing inspection method, which comprises the following steps:
- step (2) Recording the fingerprint characteristic thermogram of each strain as the identification feature: the bacterial liquid obtained in step (1) is placed in a microcalorimeter, and the thermal spectrum of the bacterial liquid of different concentrations and different living conditions of each strain is recorded. Curve, obtaining the fingerprint characteristic thermogram of different strains;
- steps (1) to (3) are steps for establishing a detection standard.
- fingerprint characteristic thermograms and related thermodynamic parameters of each strain are obtained through experiments, and the positive indicators of the strains are established, these patterns and data formulas can be used as criteria for future testing work. That is to say, step (1) to step (3) only need to establish the standard operation once. After the standard is established, the test of the sample to be tested only needs to implement the operation procedure of step (4) and compare with the standard.
- the method for obtaining the different concentration of the bacterial liquid of each strain is to filter and elute the fresh culture of the strain to obtain an eluate, and to dilute the eluate by a 10-fold serial dilution with a 0.9% sterile sodium chloride solution.
- the method for obtaining the bacterial liquid of different living conditions of each strain is that the culture is filtered and eluted to obtain an eluate, and the eluate is separately placed in a -70 ° C refrigerator and a 60 ° C water bath for 2 h, and then used.
- the 0.9% sterile sodium chloride solution was diluted 10 times in series.
- step (2) The specific steps of placing the bacterial liquid in the microcalorimeter in step (2) are:
- step (1) The method (2-1) in step (1) to take each of the strains 10- 3, 10- 5, 10- 7 , 1 '10' ⁇ ( ⁇ °, 10- 11 -fold dilution concentration series of culture;
- thermodynamic parameters of the thermogram include: the detection channel thermal power Pi as a function of time and the blank control channel thermal power P simultaneously engraved therewith.
- the maximum heating power P max the maximum heating power time T tribe ax , the total calorific value H t tal , and the slope k of each curve exponential growth section every 15 min.
- the positive judgment index of the strain (3) may be: recording the occurrence time of k 0 , and taking k 0 as a necessary condition for detecting the microbial contamination of the sample, and establishing a positive judgment time index of the microbial growth.
- the temperature of the detection channel is set according to the temperature suitable for the growth of the strain, and the temperature of the detection channel can be set to 2 3 ° C ⁇ 37 ° C.
- the present invention also includes a fully enclosed ampoules incubator for use in carrying out the above method, the ampoules cultivator consisting of a collection ampoules system, a sample addition system and a peristaltic drainage system, the sample addition
- the ampoules cultivator consisting of a collection ampoules system, a sample addition system and a peristaltic drainage system, the sample addition
- the liquid system and the bacterium ampoules system are connected together through the inlet pipe, and the sterilizing system and the peristaltic drainage system are connected by a drain pipe.
- the ampoules system includes an ampoule bottle body, and a rubber sealing plug is sealed on the bottle mouth of the ampoule bottle body.
- the liquid inlet pipe, the liquid discharging pipe and the exhaust pipe penetrate the rubber sealing plug and extend into the ampoule bottle body, and the ampoule bottle body is built therein.
- Filter the bottom of the filter is covered with a filter membrane.
- the top of the filter is connected to the inlet of the ampoule.
- the drain of the ampoule is extended to the bottom of the ampoule.
- the inlet tube outside the ampoule.
- An inlet control valve and a drain control valve are respectively arranged on the drain pipe, and an air filter is connected to the top of the exhaust pipe.
- the dosing system includes a sample/medium container and a liquid inlet device with an air filter.
- the peristaltic drainage system includes a peristaltic pump, and the outlet of the peristaltic pump is connected to the waste collector.
- a liquid inlet pipe connector is installed on the liquid inlet pipe between the liquid inlet control valve and the sample dosing system, and when the pipe connector is disconnected, the sample dosing system can be separated from the bacteria collecting ampoule system;
- a drain pipe connector is installed on the drain pipe between the liquid control valve and the peristaltic drain system, and when the pipe connector is disconnected, the peristaltic drain system can be separated from the bacteria collection and ampoules system.
- the inlet conduit connector and the drain conduit connector are plug-type, and the inlet ports of the inlet conduit connector and the drain conduit connector can be docked to form a sealed conduit connector.
- the ampoule is made of glass or hard plastic and is marked with a tick mark on the ampoule.
- the inlet pipe, the drain pipe and the exhaust pipe are silicone hoses, and the inlet control valve, the drain control valve and the exhaust control valve are bayonet valves.
- the exhaust pipe is a stainless steel needle with an air filter at the top.
- the part of the inlet pipe that protrudes into the ampoule can be a thick and thin conical tube, and the filter is fixed.
- the upper surface of the upper end of the conical tube is a threaded structure, and the lower surface of the rubber sealing plug is fixed with an internal thread interface, and the conical tube can be connected to the internal thread through a threaded structure.
- the microcalorimetric sterilizing detection method of the invention has the following advantages compared with the prior art bacteriological observation method: 1 The detection time is shorter than the microscopic calorimetry method: the microcalorimetric detection time is concentrated in the 0 ⁇ 18h, and the positive time of the observation method is concentrated in 10 - 36h; 2 sensitivity, the microcalorimetry is higher than the collection observation method: the microcalorimetry can detect less than 10 - ia ⁇ 10 - 11 dilutions of microbial growth, while the observation method did not detect the growth of the positive bacteria under the same conditions; 3 quantitative and fingerprint characteristics, microcalorimetry is better than observation: microcalorimetry can provide The growth thermogram curve and the quantifiable thermodynamic parameters and the positive bacteria detection standard equations of the microbial strain fingerprint characteristics are observed, and the observation method only relies on the naked eye to observe the turbidity of the medium, and the characteristics cannot be quantitatively and aseptically judged; 4 In terms of automation and accuracy, the microcalorimetry method is superior
- thermodynamic parameters reports positive results, and the degree of automation is high. At the same time, it can avoid the increase of workload and the risk of re-contamination caused by repeated artificial intervention observations of the collection observation method; avoid the non-microbial growth that may exist in the conventional observation method. False positive judgment of turbidity (such as turbidity caused by mixing of drug and medium) and false negative judgment that microbial growth is difficult to cause turbidity of culture medium (eg, Candida albicans and Bacillus subtilis are difficult to cause culture in a short period of time The base is significantly turbid, and the result is more difficult to determine).
- turbidity such as turbidity caused by mixing of drug and medium
- false negative judgment that microbial growth is difficult to cause turbidity of culture medium eg, Candida albicans and Bacillus subtilis are difficult to cause culture in a short period of time The base is significantly turbid, and the result is more difficult to determine.
- the fully-enclosed ampoules incubator of the invention can realize microbial enrichment (bactericidal function) of the sample in the fully-closed sterile system, and eliminate the antibacterial activity interference (anti-interference function) of the rinsing film.
- microbial enrichment bactericidal function
- anti-interference function antibacterial activity interference
- Adding culture medium to cultivate resuscitation microorganisms (culture function) and placing calorimeter channels to record the microbial growth and heat metabolism status (recording function) the whole process from sampling to cultivation eliminates the possibility of external factors polluting samples or culture medium. Eliminating the possibility of misjudging the sample as positive for microbial contamination (false positive) significantly improves the accuracy of the test.
- the fully enclosed ampoules incubator of the present invention is compared with the existing sterilized inoculum culture incubator
- the advantages are as follows: 1
- the invention can be used for micro-calorimetric sterility inspection, and the existing sterilizing inspection bacteria collection device is only suitable for common observation method; 2 compared with the existing collection culture incubator, it is necessary to repeat observations by observing with the naked eye ( 14 days), the labor is heavy, the labor cost is high; the invention records the change of the biological growth and metabolism heat in the sample in real time, online, multi-channel and automatic by the microcalorimeter, the degree of automation is high, and the labor can be reduced Strength and labor cost; 3 Using the invention to check the caloric metabolism of the sample microbial contaminants by microcalorimetry, the microbial contamination can be judged more sensitively and quickly; the turbid medium is observed by the naked eye compared with the existing existing collection culture incubator.
- the method is more sensitive, and can detect microbial contamination at an early stage, thereby saving inspection time; 4 using the invention to check the caloric metabolism of the sample microbial contaminants by microcalorimetry, and can combine the aseptic state of the sample to determine the discriminant equation, thereby accurately and quantitatively Judging the sterility of the sample; visually observing the medium compared to the existing ones
- the method of turbidity method for judging the aseptic state of the sample is more accurate, and can effectively avoid the misjudgment of the results which may be caused by the naked eye observation of the prior method; 5 using the present invention to record the microbial contamination of the sample by microcalorimetry
- the heat metabolism curve of the object has certain characteristic fingerprinting; it can provide more comprehensive information than the existing visual observation method which only provides the result judgment, and can be used for preliminary identification of contaminated microorganism species.
- Figure 1 is a graph showing the growth of different concentrations of Staphylococcus aureus recorded by the method of the present invention
- Figure 2 is a graph showing the growth of Escherichia coli at different concentrations recorded by the method of the present invention
- Figure 3 is a graph of different concentrations of patina recorded by the method of the present invention
- Figure 4 is a growth map of different concentrations of Clostridium sporogenes recorded by the method of the present invention
- Figure 5 is a growth map of different concentrations of Shigella dysenteriae recorded by the method of the present invention
- Figure 6 is a growth map of different concentrations of Bacillus subtilis recorded by the method of the present invention
- A sterile thioglycolate fluid culture group
- B 1 ( ⁇ 3 dilution; C: 1 0- 5 dilution; D: 1 0- 7 dilution; ⁇ : 1 ( ⁇ 8 dilution;
- F 1 ( ⁇ 9 dilution; G: 1 0 _1 ° dilution; H: 1 G- 11 dilution;
- FIG 7 is a graph showing the growth of Aspergillus niger in different concentrations recorded by the method of the present invention
- FIG 8 is a recording method of the present invention, different concentrations of Candida albicans pattern
- A a sterile medium modified Martin
- B 10_ 3 dilution
- C 10- 5 dilution
- D 1 (T dilution
- ⁇ 1 ( ⁇ 8 dilution
- F 10- 9 dilution
- G 10- lfl dilution
- H 1Q- 11 dilution
- Figure 9 is a growth map of S. aureus in different states recorded by the method of the present invention; in the figure, A: sterile thioglycolate fluid medium; B: sterile physiological saline; C: 35 ° C 10_ 5 dilution; : 35 ° C1 (T 7 dilution; E: - 70 ° C10- 5 dilution; F: -7 (TC10- 7 dilution; G: 60 ° C10_ 5 dilution; H: 6 (TC10- 7 dilution ;
- Figure 10 is a thermogram showing the sterility test of the compound sterilized injection of different sterilization conditions using the method of the present invention
- A normal sample + thioglycolate fluid medium
- B Staphylococcus aureus + thioglycolate fluid medium
- C unsterilized sample + thioglycolate fluid medium
- D sterilization Incomplete sample + thioglycolate fluid medium
- E normal sample + modified Martin medium
- F Candida albicans + modified Martin medium
- G unsterilized sample + modified Martin medium
- H sterilization not Thorough sample + modified Martin medium
- Figure 11 is a schematic view showing the relationship of various parameters of the septic test positive judgment index of the present invention.
- Pi test sample heat power
- P thermal power of sterile medium engraved with Pi
- k slope per 15 min of the thermogram
- L time of exponential growth of exponential growth
- T d positive for bioassay of sample t Time of Detect ion;
- Figure 12 is a structural view of the fully enclosed bacteria ampoules incubator of the present invention.
- Figure 13 is a schematic view showing the structure of the fully enclosed bacteria ampoules incubator after the inlet pipe connector and the drain pipe connector plug are butt jointed to form a sealed pipe connector;
- Figure 14 is a schematic structural view showing a portion of the inlet pipe extending into the ampoule body into a tapered tube
- Figure 15 is a schematic view showing the structure of the conical tube
- the method for sterility testing of a sterile preparation provided by the present invention is carried out according to the following steps:
- Drugs and reagents compound Yinchen injection (specification 50mL/bottle, batch number 20100120), including normal samples (Norm-sterilized Samples, Norm- SS), non-sterile samples (Non-steri 1 ized Samples, Non-SS) Sub-sterilized Samples (Sub-SS) (100 °C steam sterilization 10Omin) were provided by the Pharmacy Department of the 302th Hospital of the People's Liberation Army.
- TAM Air Isothermal microcalorimeter (TA instrument, US), TAM Assistant workstation, detection limit is 4 ⁇ , 24h baseline drift is less than ⁇ 20 ⁇ , detection range is ⁇ 600mW, work The temperature is 5 ⁇ 90 °C.
- SW-CT- 2FD double single-sided purification station (Suzhou purification equipment factory); NS01-2 type fully enclosed sterile test filter culture device (Beijing Niu Niu Gene Technology Co., Ltd., batch number 20090910); TH2-22 desktop constant temperature oscillator (Jiangsu Taicang Experimental Equipment Factory); ⁇ - ⁇ Intelligent Collecting Instrument (Hangzhou Tailin Medical Instrument Co., Ltd.); 303AB- 6 Type Waterproof Incubator (Shanghai Shuli Instrument and Meter Company), G.45 ⁇ cellulose acetate micropores Filter membrane (Beijing Chemical Factory), 0.9% sterile sodium chloride solution (Shijiazhuang Si Yao Group).
- TM Thioglycollate medium
- MMM Modified Martin Medium
- Peptone Peptone, 4 to 090708
- Fresh cultures of Aspergillus niger were inoculated into the modified Martin agar medium slant, cultured at 23 ⁇ 28 °C for 5-7 days, and 3 ⁇ 5mL 0.9% sterile sodium chloride solution was added to elute the spores. Eluate was diluted spore suspension containing spores made from a number less than 100cfu. Ml / 1 with sterile 0.9% sodium chloride solution was 10-fold serial line.
- Each of the above-mentioned microbial dilutions of different microorganisms is used as a positive control for sterility testing.
- the thioglycolate fluid medium is added to the diluted culture of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium spp., and Shigella dysenteriae, and the amount of heat is 35 ° C.
- the heat flow curve of each strain was recorded; the modified Martin medium was added to the diluted culture of Candida albicans and Aspergillus niger, and the growth thermogram of each strain was recorded in a 28 °C microcalorimeter. As shown in Figure 1 to Figure 8.
- thermodynamic parameters of the thermogram determine the positive judgment index of the strain, and extract the thermodynamic parameters of each curve: the thermal power Pi of the detection channel and the blank control channel thermal power P0 and maximum heating power simultaneously with the time P max , the maximum heating power time T max , and the total calorific value H t .
- Ul extract the slope k value every 15 min of each curve exponential growth segment, and record the time of exponential growth (T e ).
- the parameters extracted under different concentrations of each strain are as follows: where, Dilution: dilution; cfu: colony forming unit, colony forming unit; T e : k ⁇ O occurrence time; k: slope of the thermal spectrum curve every 15 minutes; d : microbial detection time; Pi: growth power of different dilutions of bacterial liquid; P. : thermal power with sterile medium at the same time; P max , maximum heating power; T max , maximum heating power time; H tQtal , total calorific value.
- Table 1A Thermodynamic parameters of growth of different concentrations of Staphylococcus aureus
- the method for determining the microbial contamination of the method of the present invention is that k 0 is a necessary condition for detecting the test species, and Check the channel thermal power P i value and simultaneously engrave the blank channel thermal power P. The difference is greater than P.
- the growth thermogram of each strain has obvious fingerprint characteristics, which can be used for the identification of different strains; among them, the maximum heating power (P raa J, total calorific value (H t ul ) and curve peak structure It can represent the difference in characteristics between various strains and has stability.
- the detection time of different states of Staphylococcus aureus is: fresh culture ( ⁇ 18h) ⁇ frozen deposit culture ( ⁇ 24h) ⁇ high temperature preservation culture (>36h);
- the recovery time of microorganisms in different states under the detection conditions is prolonged with the degree of damage, and the method of the present invention can sensitively detect microorganisms grown in different states.
- the viable count results show that: the method of the invention can detect various microorganisms lower than lcuf, and the method has high sensitivity; wherein, microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus niger can be detected to be less than 10 - 11 dilution culture.
- microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus niger can be detected to be less than 10 - 11 dilution culture.
- thermogram of the sample with the positive indicator of the strain obtained in step (4), when the characterization of the positive indicator of the strain appears in the sample thermogram, it can be determined that there is microbial contamination in the sample, and then according to the step ( 3)
- the obtained thermographic curve of the fingerprint characteristics of the strain can be used to determine the type of contaminated species in the sample.
- the unsterilized sample of the compound Yinchen injection is selected in the following inspection procedure, and the sterilization is not performed. Thorough samples, normal sterilized samples + less than 100 cfu of S. aureus, normal sterilized samples + less than 100 cfu of Candida albicans were used as controls for the test samples, data were collected and data analyzed.
- the specific operation method is as follows:
- thermogram shows that the normal sample channel (normal sample + thioglycolate fluid medium, normal sample + modified Martin medium) has a gentle downward trend, and the thermodynamic parameters show that the normal sample k value is continuously negative. Value status, indicating that the normal sample is free of microbial contamination and the medium is aseptic;
- thermogram shows that the positive control channel (Staphylococcus aureus + thioglycolate fluid medium, Candida albicans + modified Martin medium) has good microbial growth, indicating that the condition is suitable for sterility testing of compound Yinchen injection. Has better sensitivity;
- thermodynamic parameters showed that the Pmax of the unsterilized sample was higher than the sterilized sample, indicating that the contamination was higher. It also indicates the sensitivity of the sterility test method to differences in the degree of contamination of the sample.
- the detection time of other microorganisms using the collection observation method was less than 36h, but the average detection time was longer than the microcalorimetry method.
- the thermal method is concentrated in 0 ⁇ 18 hours, and the collection observation method is concentrated in 10 ⁇ 36 hours);
- the minimum dilution is 1 (°), and the microbial 10-11 dilution is not detected.
- the lowest concentration detected by each microorganism is higher than that of microcalorimetry, and the detection sensitivity is lower than that of microcalorimetry;
- Candida albicans does not cause obvious turbidity of the culture medium during the inspection, and the collection observation method is difficult to be accurate.
- the method of the invention is faster and more sensitive than the method of collecting bacteria.
- Microbiological contamination Candida albicans, Bacillus subtilis, etc.
- turbidity in the growth medium is relatively more sensitive to examination.
- the above comparison indicates that the sterility test using the microcalorimetry method of the present invention is faster and more sensitive than the conventional bacteriological observation method, and has a high degree of automation and objectivity, and can be used as a new method for sterility examination.
- Embodiment 2 Steps (1) to (4) of the present embodiment are the same as Embodiment 1 .
- a fully enclosed ampoules incubator is used, and the structure and use method thereof are as follows:
- the fully enclosed bacteria ampoules incubator is composed of a bacterium ampoules system, a sample addition system and a peristaltic drainage system, and the inlet and the ampoules system pass through the inlet tube 4 Connected together, the collection ampoules system and the peristaltic drainage system are connected by a drain tube 5.
- the collection and ampoules system comprises an ampoule bottle 1 , and the rubber sealing plug 3 is sealed and fixed on the bottle mouth of the ampoule bottle.
- the inlet pipe 4, the drain pipe 5 and the exhaust pipe 6 penetrate the rubber seal plug and extend into the ampoule bottle.
- the ampoule has a built-in filter 2, and the bottom of the filter is covered with a filter 15 .
- the top of the filter is connected to the inlet of the ampoule.
- the drain of the ampoule extends to the bottom of the ampoule.
- the filter can be pre-set to different materials depending on the filter object.
- An inlet control valve 7, a drain control valve 8 and an exhaust control valve 17 are respectively installed on the inlet pipe and the drain pipe outside the ampoule body, and the air filter 16 is connected to the top of the exhaust pipe.
- the dosing system includes a sample/medium container 12 and a liquid inlet device 10 with an air filter.
- the peristaltic drainage system comprises a peristaltic pump 1 3 and the outlet of the peristaltic pump is connected to the waste collector 14 .
- the liquid inlet pipe connector 11 is installed on the liquid inlet pipe between the liquid inlet control valve and the sample filling system, and when the pipe connector is disconnected, the sample adding liquid system and the bacteria collecting ampoule can be installed.
- the system is separated; a drain pipe connector 18 is installed on the drain pipe between the drain control valve and the peristaltic drain system, and when the pipe connector is disconnected, the peristaltic drain system can be separated from the bacteria collection and ampoules system.
- the inlet conduit connector and the drain conduit connector are of the plug type, and the plugs of the inlet conduit connector and the drain conduit connector are mateable to form a sealed conduit connector 19.
- the sample addition system is separated from the collection ampoules system from the plug of the inlet conduit connector 11; from the plug of the drain conduit connector 18 Separating the peristaltic drainage system from the collection ampoules system; then docking the inlet conduit connector plug and the drain conduit connector plug to form a sealed conduit connector 19
- the collection culture container is in a sealed state as shown in FIG.
- the ampule is marked with a scale line 9.
- the marking accuracy can be determined as needed, such as 5mL, 10mL, 15mL and other scale lines.
- the ampoule is made of a glass structure or a rigid plastic structure, and the transparent material can ensure the accuracy of external observation.
- the inlet control valve, the drain control valve and the exhaust control valve are bayonet valves.
- the inlet pipe, the drain pipe and the exhaust pipe are silicone hoses.
- the exhaust pipe is a stainless steel needle with an air filter at the top end connected by a silicone hose and a hollow side wall opening at the tail end.
- the present invention also provides a deformed structure of the inlet pipe.
- the portion of the inlet pipe which extends into the ampoule body may be a tapered tube 20 which is thick and thick, and the filter is fixed at the lower end of the conical tube.
- the upper surface of the upper end of the conical tube is a threaded structure 21, and the lower surface of the rubber sealing plug is fixed with an internal thread interface 22, and the conical tube can be connected to the internal thread through a threaded structure 21.
- the sample dosing system, the collecting ampoules system, and the peristaltic drainage system are sequentially connected together, that is, the liquid inlet device 10 is connected to the sample/medium container 12, and the plug of the inlet pipe connector 11 is inserted.
- the mouth is docked, the sample/medium container 12 is the sample to be inspected; and the plug of the drain pipe connector 18 is also docked;
- the method for sterilizing the test preparation compound Yinchen injection by using the fully enclosed ampoules is:
- the liquid inlet device 10 is connected to the sample/medium container 12, and the plug of the inlet pipe connector 11 is butted.
- the sample/medium container 12 is a sample of the compound Yinchen injection to be inspected;
- the plug of the drain pipe connector 18 is also butted;
- the sealed ampoules incubator was placed in a microcalorimeter to obtain the thermal curve of the compound Yinchen injection sample.
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US13/807,558 US8778629B2 (en) | 2010-06-29 | 2011-06-28 | Sterility test method and totally enclosed bacterial ampoule incubator used by it |
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WO2013117782A1 (es) * | 2012-02-06 | 2013-08-15 | Universidad De Vigo | Método y aparato para la detección de microorganismos |
CN112666087A (zh) * | 2021-01-21 | 2021-04-16 | 上海菁一科技有限公司 | 一种分光光度测试法样品处理测试胶囊 |
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CN101744914A (zh) * | 2010-02-05 | 2010-06-23 | 中国人民解放军第三〇二医院 | 一种快速检测茵栀黄注射液质量的方法 |
CN201756550U (zh) * | 2010-06-29 | 2011-03-09 | 中国人民解放军第三○二医院 | 一种全封闭集菌安瓿培养器 |
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CN1772917A (zh) * | 2004-08-13 | 2006-05-17 | 米利波尔公司 | 用于在抗生素存在下恢复微生物的培养基 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013117782A1 (es) * | 2012-02-06 | 2013-08-15 | Universidad De Vigo | Método y aparato para la detección de microorganismos |
CN112666087A (zh) * | 2021-01-21 | 2021-04-16 | 上海菁一科技有限公司 | 一种分光光度测试法样品处理测试胶囊 |
CN112666087B (zh) * | 2021-01-21 | 2022-11-25 | 上海菁一科技有限公司 | 一种分光光度测试法样品处理测试胶囊 |
Also Published As
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US8778629B2 (en) | 2014-07-15 |
KR20130004771U (ko) | 2013-08-07 |
US20130109052A1 (en) | 2013-05-02 |
CN101893589B (zh) | 2012-10-17 |
KR200474522Y1 (ko) | 2014-09-22 |
CN101893589A (zh) | 2010-11-24 |
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