Microbiological testing in food is based on the detection of living microorganisms, contaminated live bacteria in the sample distribution is uneven, and dynamic changes, its number may change over time. Microbiological testing activities mainly rely on people rather than instruments, the results of traceability fully rely on the observation, recording and analysis of the process of human experiments. Food microbiological testing needs to accumulate a wealth of practical experience. However, first of all, do a good job of key impact factors of risk assessment and quality control of the inspection process. Especially in the case that the microbial project cannot be re-examined, we should pay more attention to the control of process elements.
I. Equipment and materials
Microbiological testing equipment has electronic balance, thermostatic culture box, sterilization equipment, biosecurity cabinet, automatic microbial biochemical identification system. Electronic balance should be checked regularly, check qualified before it can be used. The thermostatic culture box should be calibrated regularly and the calibration results should meet the requirements of temperature accuracy and accuracy, using temperature correction factors if necessary. Sterilization pressure vessels should regularly use biological indicators to check the effectiveness of sterilization equipment and record to ensure the sterility of items after sterilization. Biosaturation cabinets should regularly calibrate parameters such as airflow mode, cleanliness, high efficiency/ultra-efficient filter detection to maintain their performance in protecting samples, personnel and the environment.
The materials of microbiological testing are mainly sterile conical bottles, sterile straws or nosuction heads, sterile flat dishes, etc. Relevant materials should meet the requirements of sterility, sterile straw should meet the accuracy required by the method.
II. Cultures and reagents
Culture and reagents are the most basic and important items of microbiological testing, and their performance indicators such as growth-promoting, inhibitory and indicative determine whether the target microorganism can be accurately detected and/or identified. At present, there are more culture base manufacturers on the market, but because of the differences in the quality and production process of raw materials such as peptone and agar, resulting in uneven quality of finished culture meth, so the laboratory should have an impact on the test results of the culture and reagents in accordance with GB 4789.28 for technical acceptance. The laboratory should have a record of the batch number, storage date, opening date, etc. of the key culture (reagent).
For ready-to-use mediums, commercialized dehydrated synthetic mediums, test acceptance of each batch of medium removal standard strains, if necessary, with artificial contamination of actual samples to test to better verify the suitability of the medium.
Test water for use such as culture kits should also be monitored for conductivity and microbial contamination as required by GB 4789.28. Too high conductivity indicates that too many metal ions in the water may lead to the precipitation of the mediums, and high microbial contamination may lead to the failure of the mediums’ sterilization.
III. Quality control strains
The laboratory must keep a standard strain/strain (standard culture) that meets the test needs. In addition to the strains specified in the testing method, it should also include strains required for medium (reagent) acceptance/quality control, method /confirm/iation//confirm/iation, positive control, negative control, personnel training assessment and assurance of the quality of results. Standard strains must be obtained from approved strains or specimen collection methods, and laboratories should have documented procedures to manage standard strains (original standard strains, standard reserve strains and working strains), covering the purchase, storage, use, transmission, storage of strains, etc., to ensure traceability and stability.
Isolated strains (wild strains) obtained from routine laboratory inspections or environments are identified and preserved as reference strains for quality control within the laboratory.
IV. Test the environment
Food microbiological inspection clean area should meet the requirements of GB 50687 "Food industry clean room building technical specifications". Clean area before and after use should be in accordance with GB/T 27405 "laboratory quality control norms" food microbiological testing. The cleanliness, planktonic bacteria, sedimentary bacteria, pressure difference and other indicators of clean areas should be monitored regularly, and the use should be stopped immediately if they do not meet the requirements;
The identification of pathogenic microbial isolation should be carried out in a biosecurity laboratory of level 2 or above. The pressure difference of the secondary biosathic laboratory should be monitored on a regular basis, and the clean-level secondary biosansy chamber should be set up and the cleanliness should be monitored on a regular basis.
The microbiological testing of the PCR method shall be carried out in the molecular biology laboratory, the laboratory partition and quality control shall meet gb/T 27403 Laboratory Quality Control Specification Food Molecular Biology Testing, and the pressure difference shall be monitored regularly, and the molecular biology laboratory with the clean level shall also monitor the cleanliness on a regular basis.
V. The inspection process
1. Avoid cross-contamination
The accuracy of microbial test results comes from two aspects: first, to avoid microbial cross-contamination, mainly to test the environment of aerosols and testing equipment cross-contamination, and second, the cross-contamination of antibiotics contained in the culture, such as E. coli O157:H7 test used in improved EC broth (mEC-n) containing new mycomycin sodium salt, It can kill gram-positive bacteria such as Staphylococcus aureus, resulting in false-negative results for gram-positive bacteria. Therefore, laboratories should strengthen the management of culture meth waste disposal and vessel washing.
2. Regular internal quality control
It is recommended to refer to the "Quality Assurance ofORA-LAB.5.9 Inspection Results" in the LABORATORY Manual of the U.S. FDA: Internal quality control at 5% per batch or group of samples or 1 frequency per 20 samples. Internal quality control methods are positive control, negative control and blank control, such as Staphylococcus amethyst test using Staphylococcus aureus as a positive control, Staphylococcus epidermidis as a negative control, using culture meth, reagents, straws, etc. as a blank control. Internal quality control also includes functional verification of the use of standard or quality control substances, measurement and testing equipment, period verification of measuring equipment, repeated testing using the same or different methods, in-laboratory pairing, blind sample testing, etc. Items recommended for quantitative testing, such as total number of places, mold and yeast counts, are quality-controlled or blind samples purchased from institutions with the ability to verify the qualifications of the provider, and quality-controlled or blind samples of items for qualitative testing can be purchased or prepared by the laboratory itself.
3. Regular external quality control
Laboratories should compare the level of monitoring capability with the results of other laboratories in two main ways: first, to participate in capacity verification, to participate in capacity verification areas and frequency recommendations to refer to the CNAS-RL02 "capability verification rules" requirements;
4. Pay attention to the results of non-conformity
In the case of microbial non-conformity, inspectors and laboratory management must attach great importance to: review the entire test process, if necessary, the replacement count or identification of cultures, excluding all factors that may cause microbial contamination in the laboratory, before the sample can be determined to be unqualified, while retaining isolated wild strains.
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