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GB 4789.28-2024: National Food Safety Standards--Food Microbiology Testing--Quality Requirements for Medium and Reagents
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....