GB 4789.28-2013 English PDF

GB 4789.28-2013: National food safety standard - Food Microbiological Examination - Quality requirements of culture media and reagents
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GB 4789.28-2013
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Food Microbiological Examination -
Quality requirements of culture media and reagents
ISSUED ON: NOVEMBER 29, 2013
IMPLEMENTED ON: JUNE 01, 2014
Issued by: National Health and Family Planning Commission of the
People's Republic of China
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions ... 4 
3 Quality assurance of culture medium and reagent ... 8 
4 Preservation and use of quality control strains... 14 
5 Quality requirements for culture medium and reagents ... 15 
6 Test methods for culture medium and reagent performance ... 18 
7 Record of test results ... 32 
Annex A Analysis of quality problems and causes of incorrect preparation of
culture medium ... 33 
Annex B Preparation of standard storage strains and working strains ... 35 
Annex C Medium composition specified in the Food Safety Microbiological Test
Standard ... 37 
Annex D Manufacturer and laboratory quality control standards for homemade
medium and reagents ... 38 
Annex E Quality control standards for commercially available medium and
reagents in laboratory ... 62 
Annex F Spiral plate method ... 73 
Annex G Record of test results for laboratory self-made or commercial medium
... 75 
National Food Safety Standard -
Food Microbiological Examination -
Quality requirements of culture media and reagents
1 Scope
This Standard specifies the quality requirements of culture medium and
reagents used for food microbiological examination.
This Standard is applicable to the quality control of culture medium and
reagents used for food microbiological examination.
2 Terms and definitions
2.1 quality control
technical operations and activities to meet quality requirements
2.2 batch of medium or reagent
complete traceable unit of medium or reagent, referring to a certain amount of
semi-finished or finished product with stable product model and quality that
meet product requirement (internal control) and performance testing; these
products are produced in specific production cycles and are numbered the
same
2.3 performance of culture medium and reagent
the response of the medium to the test strain under specific conditions
2.4 culture medium
a substance in liquid, semi-solid or solid form containing natural or synthetic
ingredients used to ensure microbial growth (bacteriostatic agents with or
without certain microorganisms), to identify or maintain their viability
2.5 pure chemical medium
medium prepared from chemical components of known molecular structure and
purity
2.6 undefined and partially defined chemical medium
a medium consisting wholly or partly of natural substances, processed
substances or other impure chemicals
2.7 solid medium
a medium that a certain amount of solidified material (such as agar, gelatin, etc.)
are added to the liquid medium, heated to 100°C to dissolve, solidified into a
solid state after cooling
the solid medium poured into the dish is generally called "plate"; pour into a test
tube and place it in a solid medium with a bevel; when the medium solidifies, it
is often called a "bevel"
2.8 semi-solid medium
a medium that a small amount of solidified material (such as agar, gelatin, etc.)
are added to the liquid medium, heated to 100°C to dissolve, and solidified into
a semi-solid state after cooling
2.9 transport medium
a medium that protects and maintains microbial activity and does not allow
significant proliferation after sampling and prior to laboratory treatment
substances that promote the proliferation of microorganisms are generally not
allowed in the transport medium, but the medium should protect the strain (e.g.
buffered glycerol-sodium chloride solution transport medium)
2.10 preservation medium
a medium for protecting and maintaining the vitality of microorganisms for a
certain period of time, preventing the adverse effects of long-term storage, or
making them easy to resuscitate after long-term storage (e.g. nutrient agar
slope)
2.11 suspension medium
the microorganisms of the test sample are dispersed into the liquid phase, and
no proliferation or inhibition occurs during the entire contact (e.g., phosphate
buffer)
2.12 recovery medium
a medium that repairs damaged or stressed microorganisms and restores
normal growth, but does not necessarily promote microbial growth
2.13 enrichment medium
- product expiration date;
- provision of documents.
3.2 Storage
3.2.1 General requirements
Storage and use of medium and reagents shall be carried out in strict
accordance with the storage conditions, expiration dates and methods of use
provided by the supplier.
3.2.2 Quality management and quality control of dehydrated synthetic
medium and its added ingredient
Dehydrated synthetic medium is usually packaged in a closed container in
powder or granular form. The added ingredient for microbial selection or
identification is usually a lyophilizate or liquid. The purchase of the medium shall
be planned to facilitate the turnover of the stock (that is, the principle of using
the first-come first-served). The laboratory shall maintain a list of valid medium
catalogues. The list shall include the following:
- container tightness check;
- record of the date of the first opening;
- sensory examination of contents.
For dehydrated synthetic medium after opening, the quality depends on storage
conditions. Determine the change of the quality of the dehydrated medium by
observing the fluidity, uniformity, agglomeration and color change of the powder.
If the medium is found to be damp or the physical properties change significantly,
it shall not be used.
3.2.3 Commercial ready-to-use medium and reagents
It shall be stored and used in strict accordance with the storage conditions,
expiration date and method of use provided by the supplier.
3.2.4 Laboratory-made medium
Preserve under conditions that ensure that its composition does not change, i.e.
protect from light, dry preserved. Store in a refrigerator at 5°C ± 3°C if necessary.
It is generally recommended that the tablet does not exceed 2 weeks to 4 weeks.
Bottled and test tube culture mediums do not exceed 3 months to 6 months,
unless some standard or experimental results indicate a longer shelf life than
the above.
Carefully weigh the required amount of dehydrated synthetic medium (if
necessary, wear a mask or operate in a fume hood to prevent inhalation of
media powder containing toxic substances). Add the right amount of water first,
mix well (be careful to avoid agglomeration of the medium). Then add water to
the required amount and heat it properly. Repeatedly or continuously stir to
disperse rapidly and shall be completely dissolved if necessary. The agar-
containing medium shall be soaked for a few minutes before heating.
3.3.4 pH measurement and adjustment
Use a pH meter for pH measurement. Adjust before sterilization if necessary.
Unless otherwise specified, the pH shall be within the standard pH±0.2 when
the medium is sterilized and cooled to 25°C. The pH of the medium is generally
adjusted using a sodium hydroxide solution having a concentration of about 40
g/L (about 1 mol/L) or a hydrochloric acid solution having a concentration of
about 36.5 g/L (about 1 mol/L). If it is necessary to adjust after sterilization, use
a sterilized solution.
3.3.5 Sub-packing
Dispense the prepared medium into a suitable container. The volume of the
container shall be at least 20% larger than the volume of the medium.
3.3.6 Sterilization
3.3.6.1 General requirements
The medium shall be hygrothermally sterilized (3.3.6.2) or filtered and sterilized
(see 3.3.6.3).
Some medium cannot or do not require autoclaving. It may use boiling for
sterilization. If the specific medium such as SC broth contains light and heat
sensitive substances, it shall be cooled quickly after boiling, and stored in the
dark. Some reagents do not require sterilization and can be used directly (see
relevant standards or supplier instructions).
3.3.6.2 Wet heat sterilization
Perform hygrothermal sterilization in an autoclave or media preparer.
Autoclaving is generally sterilized at 121°C ± 3°C for 15 min. The specific
medium is sterilized according to the provisions of the food microbiological test
standards. The volume of the medium shall not exceed 1000mL, otherwise it
may cause excessive heating during sterilization. All operations shall be carried
out in accordance with the standards or instructions for use.
Control of sterilization effect is a key issue. Cool in an appropriate manner after
heating to prevent overheating. This is important for large volumes and
are specific requirements, please refer to the specified standards.
The temperature of the medium poured into the sample shall be controlled at
about 45°C.
3.4.2 Deoxygenation of culture medium
If necessary, put the medium in a boiling water bath or steam bath for 15min
before use. Loosen the lid of the container while heating. After heating, cover
tightly and quickly cool to the use temperature (e.g. FT medium).
3.4.3 Addition of added ingredient
Addition of heat-labile added ingredient shall be added when the medium is
cooled to 47°C ~ 50°C. Sterile added ingredient shall be placed at room
temperature prior to addition to prevent agar from condensing or forming a
sheet. Slowly mix the medium to which the added ingredients are added.
Dispense as soon as possible into the container to be used.
3.4.4 Preparation and storage of plate
Pour the melted medium into a dish to form a plate with a thickness of at least
3 mm (90 mm diameter plate, usually 18 mL to 20 mL agar medium). Cover the
plate and put it on the horizontal plane to cool and solidify the agar. If the plate
needs to be stored, or if the culture time exceeds 48h or the culture temperature
is higher than 40°C, more medium needs to be poured. The solidified medium
shall be used immediately or stored in a sealed bag in a dark place and/or 5°C
± 3°C refrigerator to prevent changes in the composition of the medium. Mark
the bottom or side of the plate. The content of the mark includes the name, date
of preparation, and/or expiration date. It can also use a suitable media coding
system to mark.
Store the poured plate in a sealed bag for refrigerated storage to extend shelf
life. In order to avoid the generation of condensed water, the plate shall be
cooled and then placed in the bag. Do not dry the surface of the medium before
storage.
For solid media cultured in a surface inoculated form, the agar surface shall be
dried first. Uncover the plate and pry the plate into the oven or incubator
(temperature set at 25°C ~ 50°C). Or place in a sterilized clean bench with
convection until the water droplets on the surface of the medium disappear. Be
careful not to over dry. Commercialized plate agar medium shall be used
according to the manufacturer's instructions.
3.5 Disposal of culture medium
Disposal of all contaminated and unused medium shall be carried out in a safe
- qualitative method.
When using the quantitative method, the reference medium (see Annex D) shall
be used for comparison. When using semi-quantitative and qualitative methods,
the use of reference medium or medium that yield "positive" results can be
useful for interpretation of results. The reference medium shall be selected from
good-quality medium in recent batches or batch medium or ready-to-use
medium with long-term stability from other suppliers.
5.2.3.2 Test strain
The test strain is a set of strains that have stable characteristics of their
representative species and can effectively demonstrate the best performance
of the laboratory-specific medium. The test strains are mainly purchased in the
standard strain collection center, or they can be isolated strains of the laboratory
with good characteristics. The laboratory shall test and record the
characteristics of standard stock strains. Or choose a new strain with typical
characteristics that shall be noted when used. It is best to use strains isolated
from food or water.
For medium without indicator or selection agent, only one positive strain shall
be tested. For culture medium or reagents containing indicators or selection
agents, tests shall be carried out using strains that demonstrate their indication
or selection. The complex medium (such as the medium to which the additive
is added) needs to be verified by the following strains:
- well-growth positive strain with typical reaction characteristics;
- weakly positive strain (strain sensitive to agents such as selection agents
in the medium);
- negative strain without this characteristic;
- partially or completely inhibited strain.
5.2.3.3 Growth rate
Inoculate working cultures of appropriate test strains into solid, semi-solid and
liquid medium by appropriate methods.
The growth rate of the strain on each medium shall reach the specified minimum
limit (see Annex D, Annex E).
5.2.3.4 Selectivity
In order to quantitatively evaluate the selectivity of the medium, the working
culture of the appropriate amount of the test strain shall be inoculated into the
selective medium and the reference medium in an appropriate manner as
In accordance with the requirements of 6.1.1.3.
6.1.2.1.4 Calculation
In accordance with the requirements of 6.1.1.4.
6.1.2.1.5 Explanation of results
The target bacteria shall exhibit typical growth on the medium. The growth rate
of the target bacteria on the selective separation solid medium is generally not
less than 0.5, and the minimum shall be 0.1. Selective counting of the growth
rate of the target bacteria on the solid medium is generally not less than 0.7.
Refer to Annex F for medium quality control standards.
6.1.2.2 Semi-quantitative test method for non-target bacteria (selective)
6.1.2.2.1 Plate preparation and st...