CN112608975A - Method for testing microbial limit in terramycin - Google Patents
Method for testing microbial limit in terramycin Download PDFInfo
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Abstract
The invention discloses a method for testing microbial limit in terramycin, which comprises the following steps: s1, counting the mould and the yeast in the terramycin by adopting a plate method, and preparing a terramycin test solution; s2, preparing a test group, and measuring the bacterial count according to a plate method; s3, preparing a bacterial liquid group, namely, taking candida albicans and aspergillus niger test bacteria, adding no test liquid, and operating according to the step S2; s4, preparing a test sample control group, and measuring the bacterial count according to a plate method; s5, preparing a diluent control group. After the method is changed, the film suction filtration process can not generate foam and is obviously accelerated, in addition, the detection of 1 bacterial colony before aerobic bacteria needs to be multiplied by 500 times, the accuracy is poor, the requirement that the aerobic bacteria of a product required by a client is less than 100 cfu/g can not be met, the detection of one bacterial colony is multiplied by 20 times after the improvement, the accuracy is improved by 25 times, and the accuracy and the practicability of the detection are greatly improved.
Description
Technical Field
The invention relates to the technical field of terramycin, in particular to a method for testing microbial limit in terramycin.
Background
The existing oxytetracycline is prepared by sodium chloride-peptone buffer solution with pH7.0 and containing 3% Tween 80, a large amount of foam is generated during the membrane filtration of suspension, the filtration is slow, the oxytetracycline residues are left on the membrane after centrifugation and cannot be washed away, the antibacterial activity is generated, and the content of the oxytetracycline is 1: the recovery rate of aerobic bacteria of 1000 solution is only 20%, 1 colony detected in the sample needs to be multiplied by 500 times, the accuracy is too poor, and the requirement that the product aerobic bacteria is less than 100 cfu/g can not be met by customers.
The problems that how to find a proper neutralizer to remove the bacteriostatic property of a product, how to find a solvent to inhibit the growth of microorganisms, how to find a method which has no influence on bacterial colonies in a centrifugation process and how to find a method which is superior to the existing solvent in a suction filtration process are urgently needed to be solved are solved, and therefore, a method for detecting the limit of microorganisms in oxytetracycline is provided to solve the problems.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for testing the microbial limit in terramycin.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for testing the microbial limit in terramycin comprises the following steps:
s1, counting the mould and the yeast in the terramycin by adopting a plate method, and preparing a terramycin test solution;
s2, preparing a test group, and measuring the bacterial count according to a plate method;
s3, preparing a bacterial liquid group, namely, taking candida albicans and aspergillus niger test bacteria, adding no test liquid, and operating according to the step S2;
s4, preparing a test sample control group, and measuring the bacterial count according to a plate method;
s5, preparing a diluent control group, namely injecting the diluent, candida albicans and aspergillus niger test bacteria into plates respectively, pouring an agar culture medium immediately, preparing 2 plates for each test bacteria in parallel, and measuring the bacteria number according to a plate method;
s6, preparing a blank group, and measuring the bacterial count according to a plate method;
s7, performing 3 independent parallel tests, respectively calculating the recovery rate of each test bacterium, and performing inverted culture on the counts of the mould and the yeast for 5 days by using a Saccharum sinensis dextrose agar culture medium;
s8, counting aerobic bacteria in the oxytetracycline by adopting a centrifugal precipitation and membrane filtration combined method, and preparing a test sample control group;
s9, preparing a test group, wherein the front part is operated as above, only when the last flushing fluid is added, bacterial suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger are respectively added, after filtration, the filter membrane is taken down, the filter membrane is upwards attached to an agar plate, and counting is carried out after inverted culture is carried out for 72 hours;
s10, preparing a bacterium liquid group;
s11, preparing a diluent control group;
s12, preparing a blank group;
s13, the steps S8-S12 prove that 3 independent parallel tests are tested, count of aerobic bacteria is inversely cultured for 3 days by using trypticase soytone agar medium, and recovery rate of each test bacteria is respectively calculated;
s14, obtaining a step S1-a step S7, wherein the recovery rate of mould and yeast counted by the plate method is 71% -89%, the recovery rate of the diluent control group is 74% -91%, the recovery rates are all within the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting mould and yeast in terramycin;
s15, obtaining S8-step S13, counting aerobic bacteria by adopting a method of combining a centrifugal precipitation method and a membrane filtration method, wherein the recovery rate of a test group is 69-91%, the recovery rate of a diluent control group is 67-94%, the recovery rates are all in the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting the aerobic bacteria in the oxytetracycline.
Preferably, according to step S1, the sample solution is prepared by shaking up and down manually, adding the oxytetracycline sample into a triangular flask on a clean room clean bench, accurately adding the polyethylene glycol containing calcium chloride into a sterilized measuring cylinder, shaking up sufficiently to serve as the sample solution, sucking the sample solution into a small test tube by a special sample gun, sucking the polyethylene glycol containing calcium chloride into the small test tube by a sample head, and shaking up sufficiently to serve as the sample solution.
Preferably, the test group is prepared by injecting the test solution and the candida albicans and aspergillus niger suspension into plates respectively, pouring the agar medium immediately, and preparing 2 plates in parallel for each test strain according to the step S2.
Preferably, the test solutions are separately injected into the dishes at the time of preparation, and the agar medium is immediately poured, and 2 dishes are prepared in parallel, as described in step S4.
Preferably, in the step S5, when the microbial limit of oxytetracycline is determined, the test sample is diluted with a polyethylene glycol solution containing calcium chloride as a diluent, and a diluent control group is added to examine the degree of the microbial influence during the preparation of the test solution.
Preferably, 2 plates are prepared in parallel by pouring the diluent into the plate at the time of preparation, immediately pouring the agar medium, according to step S6.
Preferably, according to step S8, the oxytetracycline sample solution in step S2 is sealed with a sealing film, and is first rotated on a vortex mixer to form a suspension, so that microorganisms contaminated in oxytetracycline are dispersed in the solution, and then centrifuged on a centrifuge, the supernatant is absorbed and added with polyethylene glycol containing calcium chloride, the mixture is uniformly mixed, all mixed solution is filtered by a film, the polyethylene glycol containing calcium chloride is used as a washing solution, the washing solution is washed for 5 times, the total washing amount is 500ml, the filter membrane is taken down, the filter membrane is upwards attached to a TSA plate, and the count is carried out after the filter membrane is inversely cultured for 72 hours.
Preferably, according to step S10, a small amount of polyethylene glycol containing calcium chloride is added during preparation, after the filter membrane is wetted, suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans, and aspergillus niger are added, respectively, and then the filter membrane is washed with the polyethylene glycol containing calcium chloride, removed, stuck upwards on an agar plate, and counted after inverted culture.
Preferably, according to step S11, when preparing, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans, and aspergillus niger bacteria liquid are added into a sterilized centrifugal tube, sterilized polyethylene glycol containing calcium chloride is added, the tube is sealed with a sealing film, the tube is rotated on a vortex mixer to uniformly distribute the bacteria, then the tube is centrifuged on a centrifuge, the upper layer solution is taken, the membrane is filtered, the tube is washed with the calcium chloride-containing polyethylene glycol solution for 5 times, the total washing amount is 500ml, the filter membrane is taken down, the filter membrane is upwards attached to an agar plate, and the number is counted after the filter membrane is inversely cultured for 72 hours.
Preferably, according to step S12, the preparation method comprises sealing the polyethylene glycol solution containing calcium chloride with a sealing film, rotating on a vortex mixer, centrifuging in a centrifuge, sucking the upper solution, adding polyethylene glycol containing calcium chloride, mixing, membrane-filtering the mixture, washing with polyethylene glycol containing calcium chloride for 5 times, washing with 500ml total amount of washing solution, removing the filter membrane, attaching the filter membrane onto a TSA plate, culturing for 72 hours in an inverted manner, and counting.
The terramycin microbe limit inspection method verifies that the method for counting the mould, the yeast and the aerobic bacteria is confirmed by a plate method and a centrifugal precipitation and film filtration combined method, the counting recovery rate of the plate method for the mould and the yeast is 71% -89%, the recovery rate of a diluent control group is 74% -91%, the recovery rates are all in the range of 50% -200%, the method meets the requirement of a scheme, the method can be used for detecting the mould and the yeast in the terramycin, the centrifugal precipitation and film filtration combined method is adopted for counting the aerobic bacteria, the recovery rate of a test group is 69% -91%, the recovery rate of the diluent control group is 67% -94%, the recovery rate is all in the range of 50% -200%, and the method can be used for detecting the aerobic bacteria in the terramycin.
After the method is changed, the film suction filtration process can not generate foam and is obviously accelerated, in addition, the detection of 1 bacterial colony before aerobic bacteria needs to be multiplied by 500 times, the accuracy is poor, the requirement that the aerobic bacteria of a product required by a client is less than 100 cfu/g can not be met, the detection of one bacterial colony is multiplied by 20 times after the improvement, the accuracy is improved by 25 times, and the accuracy and the practicability of the detection are greatly improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
The invention relates to a microorganism limit inspection method, which is a method for inspecting the microbial pollution degree of non-specified sterilization preparations, raw materials and auxiliary materials, wherein the inspection items comprise the total aerobic bacteria, the total mildew and the total saccharomycetes, the microorganism limit inspection is carried out in a local A-level unidirectional flow air area under the environment cleanliness C level, the whole inspection process is strictly in accordance with the aseptic operation to prevent the recontamination of microorganisms, and the detection of plankton, sedimentary bacteria, dust particles and surface microorganisms is carried out on a working table and the environment regularly.
| Detecting items | Detection mark |
| Total number of aerobic bacteria | ≤ |
| Mold and yeast extract | ≤200cfu/g |
In the present invention, preparation of the experimental tool:
culture medium and diluent, a Sabouraud's dextrose agar culture medium, a soybean casein agar culture medium (TSA), 20% polyethylene glycol 200 containing 0.2% calcium chloride, measuring 80ml of purified water into a triangular flask by using a 100ml measuring cylinder, adding 20ml of polyethylene glycol 200 to make the total volume 100ml, adding 0.2 g of calcium chloride, dissolving and uniformly mixing to obtain 20% polyethylene glycol 200 containing 0.2% calcium chloride, performing steam sterilization at the temperature of 121-.
In the invention, bacterial liquid preparation: inoculating fresh culture of staphylococcus aureus, pseudomonas aeruginosa and bacillus subtilis to a trypticase soy agar slant, and culturing for 18-24 hours at 30-35 ℃; inoculating fresh culture of Candida albicans into glucose agar culture medium, and culturing at 20-25 deg.C for 2-3 days. The culture is prepared into bacterial suspension with the bacterial number less than or equal to 100cfu per milliliter by using 0.9 percent sterile sodium chloride solution. Inoculating fresh culture of Aspergillus niger to Sabouraud's dextrose agar medium, culturing at 20-25 deg.C for 5-7 days, adding 3-5ml 0.9% sterile sodium chloride solution containing 0.05% (ml/ml) polysorbate 80, and eluting spore. Then sucking spore suspension (sterile capillary suction tube with thin sterile gauze at tube orifice capable of filtering hypha) into sterile test tube, preparing spore suspension containing spore number less than or equal to 100cfu per 1ml with 0.9% sterile sodium chloride solution containing 0.05% (ml/ml) polysorbate 80, and using within 2 hr if standing at room temperature after bacterial liquid preparation, or using within 24 hr if preserving at 2-8 deg.C.
In the invention, the bacterial liquid is initially counted: taking 1ml of each of 5 dilution bacterial suspensions of more than or equal to 100cfu of staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida albicans and aspergillus niger, respectively injecting the dilution bacterial suspensions into a culture dish, immediately pouring 15-20ml of agar culture medium with the temperature not more than 45 ℃, injecting the suspension of the staphylococcus aureus, the pseudomonas aeruginosa and the bacillus subtilis into tryptone soy peptone agar culture medium, inverting the plate after solidification, and culturing for 18-24 hours at the temperature of 30-35 ℃ for counting. Injecting the Candida albicans and Aspergillus niger suspension into a Sabouraud's dextrose agar culture medium, inverting the plate after solidification, culturing at 20-25 ℃, counting the Candida albicans for 2-3 days, and counting the Aspergillus niger for 5-7 days. Appendix 3 "record for confirmation of microbial count" is filled out.
In the invention, the method for testing the mould and the yeast in the terramycin is verified (a plate method):
preparation of oxytetracycline test solution
Step 1: on a clean room clean bench, 10g of oxytetracycline sample was added into a triangular flask, 100ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride was accurately metered in using a sterilization cylinder, and sufficiently shaken for 30s as a 1:10 to be tested.
Step 2: from 1:10, sucking 1ml into a 15X 150mm cuvette with a special sample-adding gun, sucking 9ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride into the cuvette with a sample-changing head, and shaking sufficiently for 30s as a 1: 100 parts of a test solution.
Second, verification process
Step 1 test group:
taking 1: 10. 1: respectively injecting 100 test solution 1ml and 1ml of Candida albicans and Aspergillus niger suspension less than or equal to 100cfu/ml into a plate, immediately pouring agar culture medium, preparing 2 plates for each test strain in parallel, and measuring the bacterial count according to the plate method.
Step 2, a bacterial liquid group:
taking 1ml of Candida albicans and Aspergillus niger test bacteria which are less than or equal to 100cfu/ml, adding no test solution, and operating according to a.
Step 3, a test article control group:
taking 1: 10. 1: 100 test solutions (1 ml) were poured into each plate, agar medium was immediately poured, 2 plates were prepared in parallel, and the number of bacteria was measured by the plate method.
Step 4 diluent control:
(in the case of the microbial limit test of oxytetracycline, the test article was diluted with a 20% polyethylene glycol 200 solution containing 0.2% calcium chloride in a diluent, so that a diluent-added control group was performed to examine the degree to which the microorganisms were affected during the preparation of the test solution).
And (3) respectively injecting 1ml of the diluent and 1ml of candida albicans and aspergillus niger test bacteria of which the concentration is less than or equal to 100cfu/ml into a plate, immediately pouring the agar culture medium, preparing 2 plates for each test bacteria in parallel, and measuring the bacterial count according to a plate method.
Step 5 blank group:
in order to investigate whether the test solution is polluted or not in the sample preparation process, a blank experiment is carried out.
1ml of the diluent was poured into a plate, the agar medium was immediately poured, 2 plates were prepared in parallel, and the number of bacteria was measured by the plate method.
Third, verification test
At least 3 independent parallel tests are carried out, the recovery rate of each test bacterium is calculated respectively, and the count of the mould and the yeast is cultivated for 5 days in an inverted mode at 20-25 ℃ by using a Sabouraud's dextrose agar culture medium. Appendix 4 test records for the methods of testing for mold and yeasts are filled in.
Fourth, acceptance criteria
Step 1: blank group should be grown aseptically
Step 2: in 3 independent parallel tests, the recovery rate of bacteria in the test group and the recovery rate of bacteria in the diluent control group are both 50-200%. The recovery rate calculation formula is as follows:
in the present invention, the method for counting aerobic bacteria in oxytetracycline (centrifugation and membrane filtration combination method)
First, the control group of the test article
Taking 10 ml of oxytetracycline test solution 1:10 in the oxytetracycline test solution preparation step 1, sealing the sample with a sealing film, firstly, spinning the sample on a vortex mixer for 3 minutes to form suspension, dispersing the microorganisms polluted in the oxytetracycline into the solution, then centrifuging the solution on a centrifugal machine for 3 minutes at 2000 rpm, sucking 1ml of supernatant, adding 99ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride, uniformly mixing, taking 50ml of mixed solution, filtering the whole membrane, using the 20% polyethylene glycol 200 containing 0.2% calcium chloride as flushing fluid, flushing the solution 100ml each time for 5 times, flushing the total flushing amount 500ml, taking down the filter membrane, upwards sticking the filter membrane on a TSA (TSA) plate, and counting the sample after culturing the sample in an inverted mode at 30-35 ℃ for 72 hours.
Second, test group
The operation of the front part is the same as that of the first washing liquid, only when the last washing liquid is added, bacterial suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger are respectively added, after filtration, the filter membrane is taken down, the filter membrane is upwards attached to an agar plate, and after inverted culture is carried out for 72 hours at the temperature of 30-35 ℃, counting is carried out.
Third, bacterium liquid group
Adding a small amount of 20% polyethylene glycol 200 containing 0.2% calcium chloride, wetting the filter membrane, respectively adding bacterial suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger which are less than or equal to 100cfu/ml, washing with 100ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride, taking down the filter membrane, upwards sticking the filter membrane on an agar plate, and carrying out inverted culture at 30-35 ℃ for 72 hours, and counting.
Control group of diluent
Respectively adding 1ml (each ml contains no more than 100cfu of upper dilution grade) of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger liquid into a sterilized centrifugal tube, adding 9ml of sterilized 20% polyethylene glycol 200 containing 0.2% calcium chloride, sealing by using a sealing film, firstly spinning on a vortex mixer for 3 minutes to uniformly distribute the bacteria, then spinning/separating on a centrifugal machine for 3 minutes, taking 1ml of upper solution, filtering by using a film, flushing by using 20% polyethylene glycol 200 solution containing 0.2% calcium chloride for 5 times with 100ml of total flushing amount each time for 500ml, taking down the filter film, upwards pasting the filter film on an agar plate, culturing for 72 hours at 30-35 ℃ in an inverted mode, and counting.
Five, blank
Taking 10 ml of 20% polyethylene glycol 200 solution containing 0.2% calcium chloride, sealing the solution with a sealing film, firstly spinning the solution on a vortex mixer for 3 minutes, then centrifuging the solution on a centrifuge for 3 minutes at 2000 rpm, absorbing 1ml of upper solution, adding 99ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride, uniformly mixing, taking 50ml of mixed solution, filtering the whole mixed solution with a film, using 20% polyethylene glycol 200 containing 0.2% calcium chloride as a flushing solution, flushing the solution for 5 times in each 100ml, totally flushing the solution with 500ml, taking down the filter film, upwards pasting the filter film on a TSA flat plate, carrying out inverted culture at 30-35 ℃ for 72 hours, and counting.
Sixth, verification test
At least 3 independent parallel tests should be performed, count of aerobic bacteria is performed by using trypticase soytone agar medium and inverted culture at 30-35 ℃ for 3 days, and recovery rate of each test bacteria is calculated, respectively, and the appendix 5 "test record of aerobic bacteria test method" is filled.
Seventh, acceptance criteria
Step 1: the blank group should be grown aseptically.
Step 2: in 3 independent parallel tests, the recovery rate of bacteria in the test group and the recovery rate of bacteria in the diluent control group are both 50-200%. The recovery rate calculation formula is as follows:
the recovery (%) of the test group was equal to (test group colony count-test control group colony count)/bacterial suspension colony count × 100%
The percent recovery of the diluent control group (percent) is equal to the colony count of the diluent control group/colony count of the bacteria liquid group multiplied by 100 percent
Eighth, abnormal situation handling
In the verification process, the operation is strictly executed according to a verification scheme and related standard operation rules, when a result which does not accord with verification acceptable standards or other abnormalities occur, the operation is recorded, investigated and analyzed according to an SMP-08-007.05 deviation processing management system, an SOP-11-00900.6 standard operation rule of out-of-standard data (OOS) processing, and evaluated according to an SMP-02-001.3 quality risk management (QRS) system, and whether the abnormalities can be accepted is determined according to a final conclusion; whether to adjust the verification scheme of the verification object or not; whether to re-carry out the work of verification and the like.
Re-validation in the present invention
One, periodic re-authentication
After the first validation, a re-validation period is given by the quality manager in accordance with the method specified in SMP-12-001.1 general plan for validation and validation of traditional products (VMP). And periodically evaluating the re-verification period organization according to the operation condition of the verification object.
Second, change re-verification
After the verification object is subjected to the change defined by SMP-13-001.1 change control management system, risk assessment is carried out according to SMP-02-001.3 quality risk management (QRS) system, and whether verification is adopted or not and the mode of re-verification is determined according to the assessment conclusion.
In the invention, a proper solvent is searched for to increase the solubility of the oxytetracycline or the film suction filtration process is superior to the existing solvent and has no inhibition on the growth of microorganisms, the oxytetracycline cannot be dissolved under a neutral condition and only exists in the form of suspension, and the solubility of 20% polyethylene glycol 200 to the oxytetracycline is slightly higher but 1: 10. 1: the 100 solution is also a suspension which can not be completely dissolved, the 20 percent polyethylene glycol 200 film suction filtration process is quick and can not generate foam, the actual operation is better than the prior sodium chloride-peptone buffer solution with the pH value of 7.0, and the 20 percent polyethylene glycol 200 is verified to have no inhibition on bacterial colonies.
In the present invention, the purpose of centrifugation is to precipitate oxytetracycline, and a sample is taken to obtain supernatant, but if the colonies to be detected are also precipitated, the true data of the product cannot be detected, so whether the colony distribution is influenced by the centrifugation process is verified.
In the invention, the addition of 0.2% of calcium chloride is proved to remove the bacteriostasis of the product and improve the recovery rate of bacterial colonies.
In the invention, the washing amount is small, the residual oxytetracycline can inhibit the growth of bacterial colonies, the bacterial colonies in a sample cannot be accurately detected, the washing amount is too large, and the microorganisms in the sample can be damaged, so that the accurate washing times and washing amounts are required to be tested, the recovery rate of the bacterial colonies is 50-200%, and 100ml of washing is carried out each time through repeated tests, and 5 times of washing is taken as the optimal washing mode.
In the present invention, the number of passages of the strain used in the test should not exceed 5 (0 is the freeze-dried strain obtained from the strain preservation center), and an appropriate strain preservation technique is employed to ensure the biological properties of the test strain.
The terramycin microbe limit inspection method verifies that the method for counting the mould, the yeast and the aerobic bacteria is confirmed by a plate method and a centrifugal precipitation and film filtration combined method, the counting recovery rate of the plate method for the mould and the yeast is 71% -89%, the recovery rate of a diluent control group is 74% -91%, the recovery rates are all in the range of 50% -200%, the method meets the requirement of a scheme, the method can be used for detecting the mould and the yeast in the terramycin, the centrifugal precipitation and film filtration combined method is adopted for counting the aerobic bacteria, the recovery rate of a test group is 69% -91%, the recovery rate of the diluent control group is 67% -94%, the recovery rate is all in the range of 50% -200%, and the method can be used for detecting the aerobic bacteria in the terramycin.
The method for detecting the microbial limit in the terramycin comprises the following steps:
1. counting mould and yeast in the terramycin by adopting a plate method, preparing a terramycin test solution, manually shaking up and down for 30s before the test solution is used, adding 10g of terramycin sample into a triangular flask on a clean room superclean bench, accurately adding 100ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride by using a sterilization measuring cylinder, and fully shaking for 30s to serve as 1:10, from 1:10, sucking 1ml into a 15X 150mm cuvette with a special sample-adding gun, sucking 9ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride into the cuvette with a sample-changing head, and shaking sufficiently for 30s as a 1: 100 of test solution;
2. test group, take 1: 10. 1: respectively injecting 100 test solution 1ml and 1ml of candida albicans and aspergillus niger suspension with the concentration of less than or equal to 100cfu/ml into a plate, immediately pouring an agar culture medium, preparing 2 plates for each test strain in parallel, and measuring the bacterial count according to a plate method;
3. a bacterial liquid group, namely taking 1ml of candida albicans and aspergillus niger test bacteria which are less than or equal to 100cfu/ml, adding no test liquid, and operating according to the step S2;
4. taking a sample control group, taking 1: 10. 1: respectively injecting 1ml of 100 test solutions into dishes, immediately pouring agar culture medium, preparing 2 dishes in parallel, and measuring the bacterial count according to a dish method;
5. a diluent control group, namely taking 1ml of the diluent and 1ml of candida albicans and aspergillus niger test bacteria which are less than or equal to 100cfu/ml, respectively injecting into a plate, immediately pouring an agar culture medium, preparing 2 plates for each test bacteria in parallel, measuring the bacteria number according to a plate method, and when the microbial limit of the oxytetracycline is measured, diluting a test sample by using 20% polyethylene glycol 200 solution of which the diluent contains 0.2% of calcium chloride, and adding the diluent control group for the purpose of observing the influence degree of microorganisms in the preparation process of the test solution;
6. a blank group, 1ml of diluent is taken and injected into a plate, agar culture medium is poured immediately, 2 plates are prepared in parallel, and the bacterial count is determined according to a plate method;
7. performing 3 independent parallel tests, respectively calculating the recovery rate of each test bacteria, and performing inverted culture on the mold and yeast at 20-25 deg.C for 5 days;
8. counting aerobic bacteria in the oxytetracycline by adopting a centrifugal precipitation and film filtration combined method, taking 10 ml of oxytetracycline test solution at a ratio of 1:10 in the step S2, sealing the solution by using a sealing film, firstly spinning the solution in a vortex mixer for 3 minutes to form suspension, dispersing the microorganisms polluted in the oxytetracycline into the solution, then centrifuging the solution on a centrifuge for 3 minutes at a rotation speed of 2000 rpm, absorbing 1ml of supernatant, adding 99ml of 20% polyethylene glycol 200 containing 0.2% of calcium chloride, uniformly mixing, taking 50ml of mixed solution, filtering the whole mixed solution by using a film, taking 20% polyethylene glycol 200 containing 0.2% of calcium chloride as flushing fluid, flushing 100ml each time for 5 times with a total flushing amount of 500ml, taking down the filter membrane, upwards sticking the filter membrane on a TSA (TSA) plate, carrying out inverted culture at a temperature of 30-35 ℃ for 72 hours, and counting;
9. the test group has the same operation on the front part, only when the last flushing fluid is added, bacterial suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger which are less than or equal to 100cfu/ml are respectively added, after filtration, the filter membrane is taken down, the filter membrane is upwards pasted on an agar plate, and after inverted culture is carried out for 72 hours at the temperature of 30-35 ℃, counting is carried out;
10. a bacterium liquid group, a small amount of 20% polyethylene glycol 200 containing 0.2% calcium chloride is added, after filter membranes are wetted, bacterium suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger which are less than or equal to 100cfu/ml are respectively added, then 100ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride is used for washing, the filter membranes are taken down, are upwards attached to agar plates, and are counted after being inversely cultured for 72 hours at the temperature of 30-35 ℃;
11. a diluent control group, 1ml (each ml contains upper dilution grade less than or equal to 100 cfu) of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger liquid are respectively added into a sterilized centrifugal tube, 9ml of sterilized 20% polyethylene glycol 200 containing 0.2% calcium chloride is added, a sealing film is used for sealing, the sterilization is firstly carried out on a vortex mixer for 3 minutes to ensure that the bacteria are uniformly distributed, then a centrifuge is used for 2000 revolutions/separation for 3 minutes, 1ml of upper solution is taken out, membrane filtration is carried out, the solution is washed by 20% polyethylene glycol 200 containing 0.2% calcium chloride for 5 times in 100ml each time, the total washing amount is 500ml, the filter membrane is taken down, the filter membrane is upwards attached to an agar plate, and the count is carried out after the inverted culture is carried out for 72 hours at the temperature of 30-35 ℃;
12. blank, take 10 ml of 20% polyethylene glycol 200 solution containing 0.2% calcium chloride and seal with sealing film, spin on the vortex mixer for 3 minutes first, then rotate/separate and centrifuge on the centrifuge for 3 minutes at 2000 revolutions, absorb 1ml of upper solution and add 99ml of 20% polyethylene glycol 200 containing 0.2% calcium chloride, mix, take 50ml of mixed solution and filter all films, use 20% polyethylene glycol 200 containing 0.2% calcium chloride as flushing fluid, 100ml each time, flush 5 times altogether, flush the amount of 500ml totally, take down the filter membrane, the filter membrane sticks to TSA flat upwardly, invert and culture for 72 hours at 30-35 ℃, count;
13. the above-mentioned steps S8-S12 confirmed that 3 independent parallel tests were conducted, the count of aerobic bacteria was inverted and cultured for 3 days at 30-35 ℃ using trypticase soy agar medium, and the recovery rate of each test bacteria was calculated separately.
14. The recovery rate of the plate method for counting the mould and the yeast is 71-89%, the recovery rate of the diluent control group is 74-91%, the recovery rates are all in the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting the mould and the yeast in the terramycin.
15. The method combining the centrifugal precipitation method and the membrane filtration method is adopted to count the aerobic bacteria, the recovery rate of the test group is 69-91%, the recovery rate of the diluent control group is 67-94%, the recovery rates are all in the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting the aerobic bacteria in the oxytetracycline.
Microorganism counting confirmation record table
| Bacterial strain | Dilution factor of bacterial liquid | Bacterial suspension concentration after dilution (cfu/ml) |
| Staphylococcus aureus | 10-7 | 76 |
| Pseudomonas aeruginosa | 10-6 | 67 |
| Bacillus subtilis | 10-7 | 58 |
| Aspergillus niger | 10-4 | 35 |
| Candida albicans | 10-4 | 56 |
Test record table of mould and yeast test method
Test record table of aerobic bacteria test method
Test record table of bacteria test method
Remarking: test group bacteria recovery rate (%) - (test group colony number-test sample control group colony number)/bacteria liquid colony number × 100%
The recovery rate (%) of the diluent control group is equal to the colony number of the diluent control group/colony number of the bacteria liquid group multiplied by 100%
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Claims (10)
1. A method for testing the microbial limit in terramycin is characterized by comprising the following steps:
s1, counting the mould and the yeast in the terramycin by adopting a plate method, and preparing a terramycin test solution;
s2, preparing a test group, and measuring the bacterial count according to a plate method;
s3, preparing a bacterial liquid group, namely, taking candida albicans and aspergillus niger test bacteria, adding no test liquid, and operating according to the step S2;
s4, preparing a test sample control group, and measuring the bacterial count according to a plate method;
s5, preparing a diluent control group, namely injecting the diluent, candida albicans and aspergillus niger test bacteria into plates respectively, pouring an agar culture medium immediately, preparing 2 plates for each test bacteria in parallel, and measuring the bacteria number according to a plate method;
s6, preparing a blank group, and measuring the bacterial count according to a plate method;
s7, performing 3 independent parallel tests, respectively calculating the recovery rate of each test bacterium, and performing inverted culture on the counts of the mould and the yeast for 5 days by using a Saccharum sinensis dextrose agar culture medium;
s8, counting aerobic bacteria in the oxytetracycline by adopting a centrifugal precipitation and membrane filtration combined method, and preparing a test sample control group;
s9, preparing a test group, wherein the front part is operated as above, only when the last flushing fluid is added, bacterial suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger are respectively added, after filtration, the filter membrane is taken down, the filter membrane is upwards attached to an agar plate, and counting is carried out after inverted culture is carried out for 72 hours;
s10, preparing a bacterium liquid group;
s11, preparing a diluent control group;
s12, preparing a blank group;
s13, the steps S8-S12 prove that 3 independent parallel tests are tested, count of aerobic bacteria is inversely cultured for 3 days by using trypticase soytone agar medium, and recovery rate of each test bacteria is respectively calculated;
s14, obtaining a step S1-a step S7, wherein the recovery rate of mould and yeast counted by the plate method is 71% -89%, the recovery rate of the diluent control group is 74% -91%, the recovery rates are all within the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting mould and yeast in terramycin;
s15, obtaining S8-step S13, counting aerobic bacteria by adopting a method of combining a centrifugal precipitation method and a membrane filtration method, wherein the recovery rate of a test group is 69-91%, the recovery rate of a diluent control group is 67-94%, the recovery rates are all in the range of 50-200%, the requirements of the scheme are met, and the method can be used for detecting the aerobic bacteria in the oxytetracycline.
2. The method of claim 1, wherein the sample solution is prepared by shaking up and down manually, loading the oxytetracycline sample into a triangular flask on a clean room clean bench, accurately weighing polyethylene glycol containing calcium chloride with a sterilized cylinder, shaking sufficiently to obtain the sample solution, sucking the sample solution into a small test tube with a special sample gun, changing a sample head to suck the polyethylene glycol containing calcium chloride into the small test tube, and shaking sufficiently to obtain the sample solution according to step S1.
3. The method of claim 1, wherein the test group is prepared by injecting test solution and suspension of Candida albicans and Aspergillus niger into plates, pouring agar medium immediately, and preparing 2 plates for each test strain in parallel according to step S2.
4. The method of claim 1, wherein the test solutions are poured into plates and agar medium is poured immediately to prepare 2 plates in parallel according to step S4.
5. The method of claim 1, wherein the step S5 is performed by diluting a test sample with a diluent of calcium chloride-containing polyethylene glycol solution, and adding a diluent control group to the dilution to examine the degree of influence of microorganisms on the test sample during the preparation process.
6. The method of claim 1, wherein the diluent is injected into the dish during the preparation process, the agar medium is poured immediately, and 2 dishes are prepared in parallel according to the step S6.
7. The method of claim 1, wherein the preparation process comprises sealing the sample solution of oxytetracycline of step S2 with a sealing membrane, rotating the sealed sample solution on a vortex mixer to form a suspension, dispersing the microorganisms contaminated in oxytetracycline into the solution, centrifuging the suspension in a centrifuge, collecting the supernatant, adding polyethylene glycol containing calcium chloride, mixing the mixture, performing membrane filtration on the whole mixture, rinsing the mixture with polyethylene glycol containing calcium chloride for 5 times, wherein the total rinsing amount is 500ml, removing the filter membrane, attaching the filter membrane to a TSA plate, culturing the filter membrane in an inverted state for 72 hours, and counting.
8. The method of claim 1, wherein a small amount of polyethylene glycol containing calcium chloride is added during the preparation according to step S10, after the filter membrane is wetted, suspensions of pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, candida albicans and aspergillus niger are added respectively, and then the filter membrane is washed with the polyethylene glycol containing calcium chloride, removed, stuck upwards on an agar plate, and counted after inverted culture.
9. The method of claim 1, wherein the preparation process comprises the steps of S11, adding Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus niger bacteria solution into a sterilized centrifuge tube, adding sterilized polyethylene glycol containing calcium chloride, sealing with a sealing film, rotating on a vortex mixer to uniformly distribute the bacteria, centrifuging, collecting the supernatant, filtering with a membrane, washing with a polyethylene glycol solution containing calcium chloride for 5 times, collecting the total washing amount of 500ml, removing the filter membrane, attaching the filter membrane to an agar plate, culturing in an inverted state for 72 hours, and counting.
10. The method of claim 1, wherein the preparation process comprises sealing the polyethylene glycol solution containing calcium chloride with a sealing film, rotating the solution on a vortex mixer, centrifuging the solution in a centrifuge, sucking the upper solution, adding the polyethylene glycol containing calcium chloride, mixing the solution uniformly, performing membrane filtration on the mixed solution, washing the mixed solution with the polyethylene glycol containing calcium chloride as a washing solution for 5 times, washing the solution with 500ml in total, removing the filter membrane, attaching the filter membrane to a TSA plate, performing inverted culture for 72 hours, and counting.
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