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GB 4789.28-2024 English PDF (GB4789.28-2024)

GB 4789.28-2024 English PDF (GB4789.28-2024)

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GB 4789.28-2024: National Food Safety Standards--Food Microbiology Testing--Quality Requirements for Medium and Reagents
This standard specifies the quality requirements for culture media and reagents used in food microbiology testing. This standard applies to the quality control of culture media and reagents for food microbiology testing. 2 Terms and definitions 2.1 Quality control Technical operations and activities undertaken to meet quality requirements. 2.2 Lots of media and reagents The complete traceability unit of culture media and reagents, which refers to a certain number of products produced in a specific production cycle and meeting production quality requirements. These products have the same batch number. 2.3 Performance of media and reagents The reaction of culture media and reagents to the test strain under certain conditions. 2.4 Culture medium Substances in liquid, semi-solid or solid form, containing natural or synthetic ingredients, used to ensure the reproduction, identification or maintenance of vitality of microorganisms. 2.5 Liquid culture medium A culture medium consisting of an aqueous solution containing one or more GB 4789.28-2024 www.ChineseStandard.net ? Buy True-PDF ? Auto-delivery. Page 6 of 100 components (e.g. buffered peptone water). 2.6 Solid media A culture media, which is formed by adding certain amount of solidified material (such as agar) to the liquid culture medium, after boiling or moist heat sterilization, cooled to room temperature and solidified. Solid culture medium poured into a petri dish is generally called a and quot;plate and quot;; solid culture medium poured into a test tube and placed at an angle is often called a and quot;slant and quot;, when the culture medium solidifies. 2.7 Semi-solid medium A culture media, which is formed by adding a small amount of solidified matter (such as agar) to the liquid culture medium, after boiling or moist heat sterilization, semi-solidified after cooling. 2.8 Transport media A culture medium, that protects and maintains microbial activity after sampling and before laboratory processing, AND does not allow obvious proliferation ...
GB 4789.28-2024
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standards - Food microbiology testing -
Quality requirements for medium and reagents
ISSUED ON: FEBRUARY 08, 2024
IMPLEMENTED ON: AUGUST 08, 2024
Issued by: National Health Commission of PRC;
State Administration for market Regulation.
Table of Contents
Foreword ... 4
1 Scope ... 5
2 Terms and definitions ... 5
2.1 Quality control ... 5
2.2 Lots of media and reagents ... 5
2.3 Performance of media and reagents ... 5
2.4 Culture medium ... 5
2.5 Liquid culture medium ... 5
2.6 Solid media ... 6
2.7 Semi-solid medium ... 6
2.8 Transport media ... 6
2.9 Preservation medium ... 6
2.10 Suspension medium ... 6
2.11 Recovery medium ... 7
2.12 Enrichment medium ... 7
2.13 Selective enrichment medium ... 7
2.14 Non-selective enrichment medium ... 7
2.15 Isolation medium ... 7
2.16 Selective isolation medium ... 7
2.17 Non-selective isolation medium ... 7
2.18 Identification medium (specific medium) ... 7
2.19 Qualification media ... 8
2.20 Enumeration medium ... 8
2.21 Reference medium ... 8
2.22 Commercial ready-to-use media ... 8
2.23 Commercial dehydrated synthetic medium ... 9
2.24 Homemade culture medium ... 9
2.25 Reagents ... 9
2.26 Test strains ... 9
2.27 Standard strains ... 9
2.28 Standard reserve strains ... 9
2.29 Reserve strains ... 10
2.30 Working strains ... 10
3 Quality assurance of culture media and reagents ... 10
3.1 Supporting documents ... 10
3.2 Storage ... 11
3.3 Laboratory preparation of culture media ... 12
3.4 Use of culture medium ... 13
3.5 Disposal of culture medium ... 14
4 Preservation and use of test strains ... 15
4.1 General requirements ... 15
4.2 Commercially sourced test strains ... 15
4.3 Standard stock strains prepared in the laboratory ... 15
4.4 Reserve strains ... 15
4.5 Working strains ... 16
5 Quality requirements for culture media and reagents ... 16
5.1 Basic requirements ... 16
5.2 Microbiological requirements ... 17
6 Test methods of media and reagent performance ... 18
6.1 Quantitative test method for non-selective isolation and enumeration of target bacterial growth rate in solid culture media ... 18
6.2 Test methods for selective isolation and enumeration of solid media ... 19 6.3 Semi-quantitative test method for non-selective enrichment media ... 21 6.4 Semi-quantitative testing method for selective enrichment media ... 22 6.5 Semi-quantitative test method for selective liquid enumeration media ... 23 6.6 Quantitative test methods for suspension media and transport media ... 24 6.7 Qualification media test methods ... 25
6.8 Test methods for experimental reagent ... 27
Appendix A Flow chart for media performance testing method ... 28
Appendix B Analysis of quality problems and reasons caused by incorrect preparation of culture medium ... 32
Appendix C Preparation of standard reserve strains and working strains ... 34 Appendix D Medium components specified in food safety microbiological inspection standards ... 36
Appendix E Quality control criteria for reference media in food safety microbiological inspection standards ... 38
Appendix F Quality control criteria for culture media and reagents used in food microbiology laboratories ... 44
Appendix G Test strain ... 96
Appendix H Spiral plate method ... 99
National food safety standards - Food microbiology testing -
Quality requirements for medium and reagents
1 Scope
This standard specifies the quality requirements for culture media and reagents used in food microbiology testing.
This standard applies to the quality control of culture media and reagents for food microbiology testing.
2 Terms and definitions
2.1 Quality control
Technical operations and activities undertaken to meet quality requirements. 2.2 Lots of media and reagents
The complete traceability unit of culture media and reagents, which refers to a certain number of products produced in a specific production cycle and meeting production quality requirements. These products have the same batch number. 2.3 Performance of media and reagents
The reaction of culture media and reagents to the test strain under certain conditions. 2.4 Culture medium
Substances in liquid, semi-solid or solid form, containing natural or synthetic ingredients, used to ensure the reproduction, identification or maintenance of vitality of microorganisms.
2.5 Liquid culture medium
A culture medium consisting of an aqueous solution containing one or more Note: The identification medium that can be used for isolation and culture is called isolation (identification) medium (such as: XLD agar).
2.19 Qualification media
A culture medium capable of producing one or more specific identification reactions that usually does not require further confirmatory testing (e.g. lactose fermentation tubes).
Note: The qualification medium used for isolation is called isolation (qualification) medium. 2.20 Enumeration medium
Selective (e.g., MYP agar) or non-selective media (e.g., plate enumeration agar) capable of quantitatively enumeration microorganisms.
Note: Enumeration media may contain the characteristics of recovery and/or enrichment media.
2.21 Reference medium
The high-quality culture medium which is specially prepared according to the culture medium formula and used for quality control of the culture medium, to ensure the quality of the culture medium for food microbiological testing. For the reference culture medium, a nationally certified and accredited testing agency with corresponding testing capabilities shall issue a testing report. 2.22 Commercial ready-to-use media
Liquid, solid or semi-solid culture media supplied in containers (e.g. dishes, test tubes or other containers) or carriers in ready-to-use or molten form:
- Completely ready-to-use media (e.g. test strips, etc.);
- Culture media that need to be remelted (e.g. for plate pouring techniques); - Culture media (such as preparation culture media) that need to be re-melted and aliquoted (such as poured into plates) before use;
- Media that need to be re-melted, added, aliquoted before use (such as TSC media and Baird Parker agar).
Note: Please refer to Appendix F for the test methods and evaluation criteria of the test pieces. If it cannot refer to the parts of Appendix F (such as colony morphology), it can refer to the operating instructions provided by the manufacturer.
2.23 Commercial dehydrated synthetic medium
Dry culture medium that needs to be added with water and processed before use (such as powder, small particles, freeze-dried, etc.):
- Complete culture medium, which has no need to add other ingredients when using; - Incomplete culture medium, which needs to add additives when using.
2.24 Homemade culture medium
According to the specific ingredients of the complete formula of the culture medium, the culture medium as prepared by the laboratory.
2.25 Reagents
Reagents for food microbiological testing and supporting reagents for culture media preparation.
2.26 Test strains
Microorganisms commonly used for media performance testing.
Note: Test strains can be further defined depending on their origin (see 2.27 ~ 2.30). 2.27 Standard strains
Strains which obtained directly from professional culture preservation agency and defined at least to the genus or species level. It is classified and described according to strain characteristics, preferably strains originating from food, water or their production environment.
2.28 Standard reserve strains
A set of identical independent strains obtained by transferring a standard strain in the laboratory for one generation.
- The expiry date of the product;
- Other necessary documentation.
3.2 Storage
3.2.1 General requirements
Culture media and reagents shall be stored and used, in strict accordance with the storage conditions, expiration date, usage methods provided by the supplier. 3.2.2 Commercial dehydrated synthetic culture medium and its additives
Acceptance shall include the following:
- Container tightness inspection;
- Sensory examination of contents.
The quality of the dehydrated synthetic medium after opening depends on the storage conditions. By observing the fluidity, uniformity, agglomeration, color changes of the powder or granules, the quality changes of the dehydrated culture medium can be judged. If the culture medium is found to be damp or has obvious changes in physical properties, it shall no longer be used.
3.2.3 Commercial ready-to-use media and reagents
It shall be stored and used, in strict accordance with the storage conditions, validity period, usage methods provided by the supplier.
3.2.4 Laboratory-made media
Store it under conditions that ensure that its ingredients will not change, that is, avoid light and dry storage. If necessary, store it in a refrigerator at 2 °C ~ 8 °C. It is usually recommended that the storage period of plates shall not exceed 4 weeks; the storage period of bottled and test tube media shall not exceed 6 months.
If it needs to add unstable additives to the culture medium, they shall be prepared and used immediately. The solid culture media containing active chemicals or unstable components shall also be prepared and used immediately, which shall not be melted twice.
Storage of media shall establish a validated expiry date. Observe the culture medium for color changes, evaporation (dehydration) or microbial growth. When such changes occur in the culture medium, use shall be prohibited.
The culture medium shall be taken out and equilibrated to room temperature before use or reheating.
3.3 Laboratory preparation of culture media
3.3.1 General requirements
The correct preparation of culture media is one of the most basic steps in microbiological testing. When using dehydrated culture media and other ingredients, especially those containing toxic and harmful substances (such as bile salts or other selective agents), good laboratory practices and instructions for use provided by manufacturers shall be followed. Improper preparation of culture medium can lead to quality problems in the culture medium (see Appendix B).
When using commercial dehydrated synthetic medium to prepare culture medium, the preparation shall be strictly in accordance with the instructions provided by the manufacturer, such as mass (volume), pH, preparation date, sterilization conditions, operating procedures, etc.
When the laboratory uses various basic components to prepare culture media, it shall be accurately prepared according to the formula, meanwhile relevant information shall be recorded, such as the name and type of culture medium and reagent level, content of each component, manufacturer, batch number, pH, culture medium volume (division volume), aseptic measures (including implementation method, temperature and time), preparation date, personnel, etc. for traceability.
3.3.2 Water
The conductivity of the test water shall not exceed 25 µS/cm (equivalent to resistivity ≥ 0.04 MΩ/cm) at 25 °C, unless otherwise specified.
The microbial contamination of test water shall not exceed 103 CFU/mL. Microbial contamination levels shall be checked regularly according to GB 4789.2. 3.3.3 Weighing and dissolving
Accurately weigh the required amount of dehydrated synthetic culture medium (if necessary, wear a mask or operate under a fume hood or exhaust hood to prevent inhalation of harmful substances such as culture medium powder). First add an appropriate amount of water. Mix thoroughly (be careful to avoid medium agglomeration). Then add water to the required amount. Heat appropriately. Stir repeatedly or continuously to disperse quickly. The culture medium containing agar shall be soaked for 3 ~ 5 minutes before heating and dissolving.
3.3.4 pH measurement and adjustment
Unless otherwise specified, the pH after sterilization shall be within the standard pH of the culture medium. The melted culture medium shall be used as soon as possible and shall not be left for more than 4 hours. Unused culture medium cannot be re- solidified for next use.
3.4.2 Deoxygenation of culture medium
Place the culture medium in a boiling water bath or steam bath, to heat it for 15 minutes before use. Loosen the lid of the container during heating. Close it tightly after heating. Quickly cool it to the use temperature (such as liquid thioglycolate culture medium). 3.4.3 Addition of additional ingredients
When the culture medium cools to 47 °C to 50 °C, add thermally unstable ingredients that have been equilibrated to room temperature, to avoid agar condensation or flake formation. Slowly mix the medium with added ingredients and mix thoroughly. Divide it into containers to be used as soon as possible.
3.4.4 Plates preparation and storage
Pour the melted culture medium into a flat dish (for a flat dish with a diameter of 90 mm, usually add 15 mL ~ 20 mL of agar medium), so that the thickness in the flat dish is at least 3 mm. Place the lid on the plate and place it on a flat surface, to allow the agar to cool and solidify. If the plate needs to be stored, or the culture time exceeds 48 hours or the culture temperature is higher than 40 °C, more culture medium needs to be poured. The solidified culture medium shall be used immediately or stored in a dark place and/or in a sealed bag in a refrigerator at 2 °C ~ 8 °C, to prevent changes in the composition of the culture medium. Mark the bottom or side of the plate, with the name, preparation date and/or expiration date. Marking can also be done using an appropriate media coding system.
For solid media cultured using surface inoculation, the agar surface shall be dried first in a biological safety cabinet or clean workbench. Be careful not to over-dry. Commercial plate agar media shall be used, according to the instructions provided by the manufacturer.
3.5 Disposal of culture medium
All contaminated and unused media shall be disposed of in a safe manner, in compliance with relevant laws and regulations.
4 Preservation and use of test strains
4.1 General requirements
In order to successfully preserve and use strains, different preservation methods shall be used for different strains. It can choose to use freeze-drying preservation, ultra-low temperature (not higher than -70 °C) freezing preservation, liquid nitrogen preservation or other effective preservation methods.
4.2 Commercially sourced test strains
For original packaged test strains obtained from a standard strain preservation center or other validly certified commercial agency, recovery and use shall be carried out in accordance with the instructions provided by the manufacturer.
4.3 Standard stock strains prepared in the laboratory
When storing and using the standard reserve strain, which is used for performance testing (see Figure C.1 in Appendix C), care shall be taken to avoid cross-contamination and reduce strain mutations or typical characteristic changes. It shall prepare multiple copies of the standard reserve strain; it shall be stored at ultra-low temperature (not higher than -70 °C) or in freeze-dried form. Storage times at higher temperatures shall be shortened.
Standard stock strains used as test strains for culture media shall have their growth characteristics fully described in the documentation.
Standard stock strains cannot be used to prepare standard strains.
4.4 Reserve strains
Reserve strains are usually prepared from standard reserve strains that are freeze-dried or cryopreserved (see Figure C.2).
The preparation of stock strains shall avoid cross-contamination and/or degradation of standard stock strains. When preparing reserve strains, the standard reserve strain shall be made into a bacterial suspension and cultured in non-selective medium, to obtain strains with stable characteristics.
For commercially sourced strains, the manufacturer's instructions shall be strictly followed.
- Change the dosage of peptone and dipped powder according to nutritional needs; - Change the dosage of agar according to the desired gelling effect;
- Determine the amount of buffer substance according to buffering requirements; - Determine the dosage of bile salts, bile extracts, deoxycholates, antibacterial dyes based on selectivity requirements;
- Determine the dosage of the antibiotic based on its potency.
5.2 Microbiological requirements
5.2.1 Introduction
The culture medium and reagents shall meet the requirements of quality control criteria in Appendix F; their performance testing methods shall be implemented in accordance with Chapter 6. When the laboratory uses commercial culture media and reagents, the information provided by the manufacturer in accordance with 3.1.1 shall be retained and acceptance procedures shall be established. If the manufacturer can provide corresponding inspection capabilities and obtain an inspection report for the same batch of products issued by the relevant national certification and accreditation qualification inspection agency, the laboratory can retain the relevant certification documents and conduct repeated tests on the performance of the batch of culture media, based on actual needs. (Medium and reagents not included in Appendix F can be tested based on the performance of the culture medium and reagents, by referring to the method established in Appendix F).
5.2.2 Growth characteristics
5.2.2.1 General requirements
Select one of the following methods to evaluate each batch of finished media or reagents: - Quantitative methods;
- Semi-quantitative methods;
- Qualitative methods.
When using quantitative methods, the reference medium (see Appendix E) shall be used as a control; when using semi-quantitative and qualitative methods, using a medium that can obtain "positive" results as a control will help in the interpretation of the results. 5.2.2.2 Growth rate
Inoculate an appropriate amount of working bacterial suspension of the test strain into solid, semi-solid and liquid culture media, using appropriate methods as specified. The growth rate of strains on each medium shall reach the specified minimum limit (see Appendix F).
5.2.2.3 Selectivity
In order to quantitatively evaluate the selectivity of the culture medium, an appropriate amount of the working culture of the test strain shall be inoculated into the selective medium and reference medium, using an appropriate method in accordance with requirements; the selectivity of the culture medium shall reach the specified value (see Appendix F).
5.2.2.4 Physiological and biochemical characteristics (specificity)
Determine the colony morphological characteristics, identification characteristics of the culture medium, or the identification characteristics of the reagents, to obtain the basic characteristics of the culture medium or reagents (see Appendix F).
5.2.2.5 Performance evaluation and result interpretation
If all specified test strains are used and the performance test of the culture medium or reagents meets the standard requirements, then the performance test results of this batch of culture media or reagents meet the requirements. If the basic requirements and microbiological requirements are met, the batch of media or reagents can be accepted. 6 Test m...
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