CN104212875A - Method for measuring initial spore number of bacillus stearothermophilus in biological indicator - Google Patents
Method for measuring initial spore number of bacillus stearothermophilus in biological indicator Download PDFInfo
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Abstract
The invention discloses a method for measuring the initial spore amount of bacillus stearothermophilus in a biological indicator, which comprises the following steps: adding sterile water into a biological indicator to obtain a primary diluent, and subpackaging the primary diluent into a plurality of sterile test tubes; carrying out water bath hot shock; taking out and rapidly cooling to room temperature, and carrying out the following operations on each sterile test tube: diluting step by step to appropriate gradient by 10 times incremental dilution method to obtain secondary diluent, injecting 2 parts of the secondary diluent into two sterile plates respectively, then injecting soybean casein agar culture medium respectively, and culturing after solidification; counting the initial spores of the bacillus stearothermophilus in all the sterile plates, then carrying out average calculation on all counting results, and then dividing the counting results by the dilution grade corresponding to the appropriate gradient to obtain the number of the initial spores, thereby completing the determination. The method provided by the invention is stable and feasible through verification, solves the problem that the initial spore number of the bacillus stearothermophilus in the previous biological indicator cannot be measured, and is convenient to popularize and apply.
Description
Technical field
The present invention relates to the initial spore count quantity measuring method of bacstearothermophilus in a kind of biological indicator.
Background technology
Biological indicator is the special live microorganism goods of a class, is generally the spore of bacterium, because it has the drag determined to specific sterilising treatment, can be used for the performance confirming sterilizing device, the checking of sterilization process, the monitoring etc. of production process sterilising effect.The use of biological indicator makes sterilization monitoring means more perfect, sterilising effect is more directly perceived, effective, security height more, the every profession and trades such as medical and health-care products, health quarantine, pharmacy corporation, hospital preparation production, foodstuff production are widely used in, to assessment, the checking of the sterilising effects such as various sterile production technique, sterile product, aseptic packaging material at present.The microbial spores of some amount is comprised in biological indicator, various informative, as spore solution, gemma bar, self-contained, industrial bio indicator etc.In biological indicator, spore content is the important quality index of biological indicator.
Bacstearothermophilus (Bacillus stearothermophilus) belongs to thermophilic aerobic spore-bearing bacilli, but has the characteristic of anaerobism concurrently, and the blue Albert'stain Albert positive of bacterial propagule G is in purple, and bacterial spore Victoria Green WPB is painted.Its bacterial propagule requires low to substratum, and well-grown on purpurum bromocresolis proof agar, surface irregularity is beige.Its minimum growth temperature is 28 DEG C, maximum growth temperature 70 ~ 77 DEG C.The most suitable growth the bread worm is 56 ~ 65 DEG C, and cultivate 24h and can form bacterium colony, at 37 DEG C, 24h can't see bacterium colony.
Bacstearothermophilus is as the part in biological indicator, up to the present its initial spore quantity also do not have a set of effective measuring method, user can only understand according to the sign spore quantity of its product that dispatches from the factory, independently cannot measure according to application demand, the biological indicator especially for unknown bacstearothermophilus spore quantity is felt simply helpless especially.So market demand independently can measure the method for the initial spore quantity of bacstearothermophilus in biological indicator.
Summary of the invention
Object of the present invention is just to provide to solve the problem the initial spore count quantity measuring method of bacstearothermophilus in a kind of biological indicator.
In order to achieve the above object, present invention employs following technical scheme:
The initial spore count quantity measuring method of bacstearothermophilus in biological indicator of the present invention, comprises the following steps:
(1) biological indicator is got, add sterilized water and obtain a diluent after mixing, biological indicator in a diluent and the volume ratio between sterilized water are 1 ﹕ (3-5), a diluent is loaded multiple sterile test tube by same volume respectively, obtain trial-product, the sterilized water separately getting 1 sterile test tube loading same volume is for subsequent use;
(2) above-mentioned trial-product and the sterile test tube that sterilized water is housed are carried out water-bath thermal shock jointly, bath temperature is 95-100 DEG C, and water bath time is 10-20 minute, thermometer is inserted in the sterile test tube that sterilized water is housed to measure bath temperature in the process;
(3) water-bath thermal shock complete after taking-up trial-product, be cooled to room temperature rapidly, following operation is carried out to the sterile test tube that each is equipped with a diluent: indicate initial spore quantity according to biological indicator and adopt 10 times to increase progressively dilution method, stepwise dilution obtains secondary dilution liquid to suitable gradient, this suitable gradient makes its colony number scope be 30 ~ 300cfu/ml, under the dilution level that this suitable gradient is corresponding, secondary dilution liquid two parts is drawn with aseptic technique method sterilizing suction pipe, the volume of every part is A ml, inject respectively in two sterilized petri dishes, be the soybean casein nutrient agar dissolved that (15 ~ 20) A ml temperature is no more than 450 DEG C respectively to injected slurry volume in two sterilized petri dishes again, mix, after it solidifies, two sterilized petri dishes are inverted and cultivate, culture temperature is 55 ~ 60 DEG C, incubation time is 40-60 hour,
(4) after completing cultivation, the initial spore of the bacstearothermophilus in all sterilized petri dishes is counted, be averaged all count results calculating again, result after average is again divided by the dilution level that gradient suitable described in step (3) is corresponding, obtain final initial spore quantity, complete mensuration.
As preferably, in described step (1), the biological indicator in a diluent and the volume ratio between sterilized water are 1 ﹕ 4; In described step (2), water bath time is 15 minutes; In described step (3), incubation time is 48 hours.
In described step (1), the volume often propping up a diluent in described sterile test tube is 10ml, and the quantity that the described sterile test tube of described diluent is housed is 12; In described step (3), dilution level corresponding to described suitable gradient is 10
-4, the volume of the every part of secondary dilution liquid injected in two sterilized petri dishes is respectively 1ml, and the volume of the soybean casein nutrient agar injected in each sterilized petri dishes is 15 ~ 20ml.
Beneficial effect of the present invention is:
The method of the invention is stablized feasible through checking, solve the unmeasured problem of initial spore quantity of bacstearothermophilus in former biological indicator, be convenient to user independently measures bacstearothermophilus in biological indicator initial spore quantity according to application demand, be particularly useful for the mensuration of the biological indicator of unknown bacstearothermophilus spore quantity, easy to utilize.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described in detail:
Embodiment 1:
Test according to the following steps:
(1) biological indicator 15 of the Merck KGaA that product batch number is " VM328674 " is got, often prop up 2ml, after adopting whirlpool mixed instrument mixing 5min, 15 biological indicators are positioned in aseptic blue lid bottle, with glass stick, the ampoule of indicator is all smashed to pieces, add sterilized water 120ml and be made into a diluent, the biological indicator in a diluent and the volume ratio between sterilized water are 1:4, while add aseptic waterside to rinse glass stick; To be rinsed complete after, after a diluent in aseptic blue lid bottle being adopted whirlpool mixed instrument mixing 5min, get 13 sterile test tube, the 10ml diluent got respectively in aseptic blue lid bottle is transferred in 12 sterile test tube, be denoted as A1-A12, obtain trial-product A; Other 1 sterile test tube (being commonly referred to as " blank pipe ") is got 10ml sterilized water and is proceeded to, and is designated as I-0, and when doing water-bath thermal shock, intensification timing is used.
(2) above-mentioned trial-product A and the sterile test tube (i.e. blank pipe) that sterilized water is housed are carried out water-bath thermal shock jointly, bath temperature is 95-100 DEG C, water bath time is 15 minutes, in the process thermometer is inserted in blank pipe to measure bath temperature;
(3) water-bath thermal shock complete after taking-up trial-product A, be cooled to room temperature rapidly, following operation is carried out to the sterile test tube A1-A12 that each is equipped with a diluent: indicate initial spore quantity according to biological indicator and adopt 10 times to increase progressively dilution method, stepwise dilution obtains secondary dilution liquid to suitable gradient, this suitable gradient makes its colony number scope be 30 ~ 300cfu/ml, and dilution level corresponding to the suitable gradient in this example is 10
-4secondary dilution liquid two parts is drawn with aseptic technique method sterilizing suction pipe, the volume of every part is 1ml, inject respectively in two sterilized petri dishes, be the soybean casein nutrient agar dissolved that 15 ~ 20ml temperature is no more than 450 DEG C respectively to injected slurry volume in two sterilized petri dishes again, mix, after it solidifies, two sterilized petri dishes are inverted and cultivate, culture temperature is 55 ~ 60 DEG C, and incubation time is 48 hours;
(4) after completing cultivation, the initial spore of the bacstearothermophilus in all sterilized petri dishes is counted, be averaged all count results calculating again, result after average is again divided by the dilution level that gradient suitable described in step (3) is corresponding, obtain final initial spore quantity, complete mensuration.
After completing mensuration, the formulae discovery initial spore quantity rate of recovery by below:
In this example, the actual measurement process data of initial spore quantity and initial spore quantity rate of recovery data see below shown in table one.
Embodiment 2:
Test according to the following steps:
(1) biological indicator 15 of the Merck KGaA that product batch number is " VM373174 " is got, often prop up 2ml, after adopting whirlpool mixed instrument mixing 5min, 15 biological indicators are positioned in aseptic blue lid bottle, with glass stick, the ampoule of indicator is all smashed to pieces, add sterilized water 120ml and be made into a diluent, the biological indicator in a diluent and the volume ratio between sterilized water are 1:3, while add aseptic waterside to rinse glass stick; To be rinsed complete after, after a diluent in aseptic blue lid bottle being adopted whirlpool mixed instrument mixing 5min, get 13 sterile test tube, the 10ml diluent got respectively in aseptic blue lid bottle is transferred in 12 sterile test tube, be denoted as B1-B12, obtain trial-product B; Other 1 sterile test tube (being commonly referred to as " blank pipe ") is got 10ml sterilized water and is proceeded to, and is designated as I-0, and when doing water-bath thermal shock, intensification timing is used.
(2) above-mentioned trial-product B and the sterile test tube (i.e. blank pipe) that sterilized water is housed are carried out water-bath thermal shock jointly, bath temperature is 95-100 DEG C, water bath time is 10 minutes, in the process thermometer is inserted in blank pipe to measure bath temperature;
(3) water-bath thermal shock complete after taking-up trial-product B, be cooled to room temperature rapidly, following operation is carried out to the sterile test tube B1-B12 that each is equipped with a diluent: indicate initial spore quantity according to biological indicator and adopt 10 times to increase progressively dilution method, stepwise dilution obtains secondary dilution liquid to suitable gradient, this suitable gradient makes its colony number scope be 30 ~ 300cfu/ml, and dilution level corresponding to the suitable gradient in this example is 10
-4secondary dilution liquid two parts is drawn with aseptic technique method sterilizing suction pipe, the volume of every part is 1ml, inject respectively in two sterilized petri dishes, be the soybean casein nutrient agar dissolved that 15 ~ 20ml temperature is no more than 450 DEG C respectively to injected slurry volume in two sterilized petri dishes again, mix, after it solidifies, two sterilized petri dishes are inverted and cultivate, culture temperature is 55 ~ 60 DEG C, and incubation time is 40 hours;
(4) after completing cultivation, the initial spore of the bacstearothermophilus in all sterilized petri dishes is counted, be averaged all count results calculating again, result after average is again divided by the dilution level that gradient suitable described in step (3) is corresponding, obtain final initial spore quantity, complete mensuration.
After completing mensuration, the formulae discovery initial spore quantity rate of recovery by below:
In this example, the actual measurement process data of initial spore quantity and initial spore quantity rate of recovery data see below shown in table one.
Embodiment 3:
Test according to the following steps:
(1) biological indicator 15 of the Merck KGaA that product batch number is " VM373274 " is got, often prop up 2ml, after adopting whirlpool mixed instrument mixing 5min, 15 biological indicators are positioned in aseptic blue lid bottle, with glass stick, the ampoule of indicator is all smashed to pieces, add sterilized water 120ml and be made into a diluent, the biological indicator in a diluent and the volume ratio between sterilized water are 1:5, while add aseptic waterside to rinse glass stick; To be rinsed complete after, after a diluent in aseptic blue lid bottle being adopted whirlpool mixed instrument mixing 5min, get 13 sterile test tube, the 10ml diluent got respectively in aseptic blue lid bottle is transferred in 12 sterile test tube, be denoted as C1-C12, obtain trial-product C; Other 1 sterile test tube (being commonly referred to as " blank pipe ") is got 10ml sterilized water and is proceeded to, and is designated as I-0, and when doing water-bath thermal shock, intensification timing is used.
(2) above-mentioned trial-product C and the sterile test tube (i.e. blank pipe) that sterilized water is housed are carried out water-bath thermal shock jointly, bath temperature is 95-100 DEG C, water bath time is 20 minutes, in the process thermometer is inserted in blank pipe to measure bath temperature;
(3) water-bath thermal shock complete after taking-up trial-product C, be cooled to room temperature rapidly, following operation is carried out to the sterile test tube C1-C12 that each is equipped with a diluent: indicate initial spore quantity according to biological indicator and adopt 10 times to increase progressively dilution method, stepwise dilution obtains secondary dilution liquid to suitable gradient, this suitable gradient makes its colony number scope be 30 ~ 300cfu/ml, and dilution level corresponding to the suitable gradient in this example is 10
-4secondary dilution liquid two parts is drawn with aseptic technique method sterilizing suction pipe, the volume of every part is 1ml, inject respectively in two sterilized petri dishes, be the soybean casein nutrient agar dissolved that 15 ~ 20ml temperature is no more than 450 DEG C respectively to injected slurry volume in two sterilized petri dishes again, mix, after it solidifies, two sterilized petri dishes are inverted and cultivate, culture temperature is 55 ~ 60 DEG C, and incubation time is 60 hours;
(4) after completing cultivation, the initial spore of the bacstearothermophilus in all sterilized petri dishes is counted, be averaged all count results calculating again, result after average is again divided by the dilution level that gradient suitable described in step (3) is corresponding, obtain final initial spore quantity, complete mensuration.
After completing mensuration, the formulae discovery initial spore quantity rate of recovery by below:
In this example, the actual measurement process data of initial spore quantity and initial spore quantity rate of recovery data see below shown in table one.
Table one
As seen from the above table, the initial spore count rate of recovery of Merck KGaA biological indicator of 3 batches of different lot numbers is all between 70 ~ 100%, reach 50% ~ 300% required by standard, therefore the initial spore count measured conforms with the regulations, the method that the present invention detects biological indicator initial spore quantity is stablized feasible, can be used for the mensuration of the initial spore quantity of bacstearothermophilus in biological indicator.
Claims (3)
1. the initial spore count quantity measuring method of bacstearothermophilus in biological indicator, is characterized in that: comprise the following steps:
(1) biological indicator is got, add sterilized water and obtain a diluent after mixing, biological indicator in a diluent and the volume ratio between sterilized water are 1 ﹕ (3-5), a diluent is loaded multiple sterile test tube by same volume respectively, obtain trial-product, the sterilized water separately getting 1 sterile test tube loading same volume is for subsequent use;
(2) above-mentioned trial-product and the sterile test tube that sterilized water is housed are carried out water-bath thermal shock jointly, bath temperature is 95-100 DEG C, water bath time is 10-20 minute, thermometer is inserted in the sterile test tube that sterilized water is housed to measure bath temperature in the process;
(3) water-bath thermal shock complete after taking-up trial-product, be cooled to room temperature rapidly, following operation is carried out to the sterile test tube that each is equipped with a diluent: indicate initial spore quantity according to biological indicator and adopt 10 times to increase progressively dilution method, stepwise dilution obtains secondary dilution liquid to suitable gradient, this suitable gradient makes its colony number scope be 30 ~ 300cfu/ml, under the dilution level that this suitable gradient is corresponding, secondary dilution liquid two parts is drawn with aseptic technique method sterilizing suction pipe, the volume of every part is A ml, inject respectively in two sterilized petri dishes, be the soybean casein nutrient agar dissolved that (15 ~ 20) A ml temperature is no more than 450 DEG C respectively to injected slurry volume in two sterilized petri dishes again, mix, after it solidifies, two sterilized petri dishes are inverted and cultivate, culture temperature is 55 ~ 60 DEG C, incubation time is 40-60 hour,
(4) after completing cultivation, the initial spore of the bacstearothermophilus in all sterilized petri dishes is counted, be averaged all count results calculating again, result after average is again divided by the dilution level that gradient suitable described in step (3) is corresponding, obtain final initial spore quantity, complete mensuration.
2. the initial spore count quantity measuring method of bacstearothermophilus in biological indicator according to claim 1, it is characterized in that: in described step (1), the biological indicator in a diluent and the volume ratio between sterilized water are 1 ﹕ 4; In described step (2), water bath time is 15 minutes; In described step (3), incubation time is 48 hours.
3. the initial spore count quantity measuring method of bacstearothermophilus in biological indicator according to claim 1 and 2, it is characterized in that: in described step (1), the volume often propping up a diluent in described sterile test tube is 10ml, and the quantity that the described sterile test tube of described diluent is housed is 12; In described step (3), dilution level corresponding to described suitable gradient is 10
-4, the volume of the every part of secondary dilution liquid injected in two sterilized petri dishes is respectively 1 ml, and the volume of the soybean casein nutrient agar injected in each sterilized petri dishes is 15 ~ 20 ml.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695377A (en) * | 2016-04-27 | 2016-06-22 | 南京巨鲨显示科技有限公司 | Germ recovery growth culture medium |
| CN105779563A (en) * | 2016-04-29 | 2016-07-20 | 大连华立金港药业有限公司 | Detection reagent kit and method of heat-resistant microorganisms in elemene lipidosome injection semi-finished products |
| CN108034607A (en) * | 2017-12-27 | 2018-05-15 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method and culture medium of bacillus coagulans |
| CN110055299A (en) * | 2019-01-21 | 2019-07-26 | 中山大学 | Biological indicator and preparation method thereof for the instruction that sterilizes |
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| US20120214154A1 (en) * | 2006-09-20 | 2012-08-23 | American Sterilizer Company | Genetically engineered biological indicator |
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| US20120214154A1 (en) * | 2006-09-20 | 2012-08-23 | American Sterilizer Company | Genetically engineered biological indicator |
Non-Patent Citations (2)
| Title |
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| APEX LABORATORIES,INC.(NC) ADDITIONAL INFORMATION BY MESALABS(BO: "Recovery of Spore Populations from Inoculated Stainless Steel Biological indicators", 《互联网URL: HTTP://BIOLOGICALINDICATORS.MESALABS.COM/DOCUMENTS-MANUALS/》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695377A (en) * | 2016-04-27 | 2016-06-22 | 南京巨鲨显示科技有限公司 | Germ recovery growth culture medium |
| CN105779563A (en) * | 2016-04-29 | 2016-07-20 | 大连华立金港药业有限公司 | Detection reagent kit and method of heat-resistant microorganisms in elemene lipidosome injection semi-finished products |
| CN108034607A (en) * | 2017-12-27 | 2018-05-15 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method and culture medium of bacillus coagulans |
| CN108034607B (en) * | 2017-12-27 | 2021-11-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | Detection method and culture medium of bacillus coagulans |
| CN110055299A (en) * | 2019-01-21 | 2019-07-26 | 中山大学 | Biological indicator and preparation method thereof for the instruction that sterilizes |
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