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National food safety standard - Food microbiological examination - Enumeration of moulds and yeasts
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GB 4789.15-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard –
Food Microbiological Examination.
Enumeration of Moulds and Yeasts
ISSUED ON. OCTOBER 19, 2016
IMPLEMENTED ON. APRIL 19, 2017
Issued by. National Health and Family Planning Commission of the PRC
3. No action is required - Full-copy of this standard will be automatically &
immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Culture Medium and Reagents ... 5
4 Examination procedures ... 5
5 Operation Steps ... 6
6 Results and Report... 7
7 Operation Procedures ... 8
Appendix A Culture Medium and Reagents ... 10
Foreword
This Standard replaced GB 4789.15-2010 National Food Safety Standard – Food
Microbiological Examination. Enumeration of Moulds and Yeasts; and SN/T 2552.3-
2010 Microbiological Examination Method for Milk and Milk Products Hygiene – Part
3. Colony-Count Method of Yeast and Moulds.
Compared with GB 4789.15-2010, this Standard has the major changes as follows.
--- Modify the equipment and materials;
--- Modify the culture medium and reagents;
--- Modify the examination procedures and operation steps;
--- Modify the results and report;
--- Modify the Appendix A;
--- Modify the Appendix B into the second method.
National Food Safety Standard –
Food Microbiological Examination.
Enumeration of Moulds and Yeasts
1 Scope
This Standard specifies the enumeration method of moulds and yeasts in the food.
The first method in this Standard is applicable to the enumeration of moulds and yeasts
in various foods; while the second method is applicable to the enumeration of moulds
in the canned tomato sauce and tomato juice.
2 Equipment and materials
In addition to the biological laboratory routine sterilization and cultivating equipment,
other equipment and materials are as follows.
2.1 Incubator. 28°C±1°C.
2.2 Beat-type homogenizer and homogeneous bag.
2.3 Electronic balance. sensitivity of 0.1g.
2.4 Sterile conical flask. capacity of 500mL.
2.5 Sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale).
2.6 Sterile test tube. 18mm × 180mm.
2.7 Vortex mixer.
2.8 Sterile flat plate. diameter of 90mm.
2.9 Constant temperature water batch. 46°C±1°C.
2.10 Microscope. 10× ~ 100×.
2.11 Micro-pipettor and tip. 1.0mL.
2.12 Refractometer.
bottle) containing sterile diluent (distilled water or normal saline or phosphate buffering
solution) or into the sterile homogeneous bag; sufficiently shake, or use beat-type
homogenizer to beat for 1min ~ 2min; and 1.10 sample homogenous solution is
prepared.
5.1.3 Take 1mL of 1.10 sample homogenous solution to inject into the test tube
containing 9mL of sterile diluent; change another 1mL sterile pipette to blow and
absorb repeatedly; or mix evenly on the vortex mixer; such solution is 1.100 sample
homogenous solution.
5.1.4 Operate as per 5.1.3, prepare the 10 times incremental serial sample
homogenous solution. Once incrementally dilute, replace 1 piece of 1mL sterile pipette.
5.1.5 According to the evaluation of the sample pollution, select the sample
homogenous solution (liquid sample can include the stock solution) with 2 ~ 3
appropriate dilution; when performing the 10 times incremental dilution, absorb 1mL of
sample homogenous solution for each dilution, and place into 2 sterile flat plates,
respectively. Meanwhile, take 1mL of sterile diluent and add into 2 sterile flat plates to
make the blank control.
5.1.6 Timely cool off the 20mL ~ 25mL of potato dextrose agar or rose Bengal agar
(can place into a 46°C±1°C constant temperature water bath for thermal insulation) to
46°C, then pour into the flat plate; rotate the flat plate to make it mix evenly. Place on
the horizontal platform till the culture medium is completely solidified.
5.2 Cultivation
After agar solidification, upright the flat plat, and place into 28°C±1°C incubator for
cultivation; observe and record the cultivation results to the first 5d.
5.3 Colony counting
Perform visual examination; if necessary, use magnifier or low power lens to record
dilution factor, and corresponding number of moulds and yeast colonies. It is expressed
by the Colony-Forming Unit (CFU).
Select the flat plate with number of colonies 10CFU ~ 150CFU, count the moulds and
yeasts, respectively according to the colony morphology. When the moulds spreading
and growing over the whole flat plate, it can be recorded as the colony spread.
6 Results and Report
6.1 Results
6.1.1 Calculate the average value of two flat plate colonies at the same dilution, then
multiply the average value by the corresponding dilution factor.
Appendix A
Culture Medium and Reagents
A.1 Normal saline
A.1.1 Components
Sodium chloride 8.5g
Distilled water 1000mL
A.1.2 Preparation
Add sodium chloride into 1000mL of distilled water, mix till it is fully dissolved; after
packaging separately, sterilize for 15min at 121°C, then backup.
A.2 Potato dextrose agar
A.2.1 Components
Potato (peeled and cut into blocks) 300g
Dextrose 20.0g
Agar 20.0g
Chloromycetin 0.1g
Distilled water 1000mL
A.2.2 Preparation
Peel the potato and cut it into blocks; add 1000mL of distilled water, and boil for 10min
~ 20min. Use gauze to filter, add distilled water to 1000mL. Add dextrose and agar,
heating for dissolution; after packaging separately, sterilize for 15min at 121°C, then
backup.
A.3 Rose Bengal agar
Peptone 5.0g
Dextrose 10.0g
Potassium dihydrogen phosphate 1.0g
Magnesium sulfate (anhydrous) 0.5g
......
GB 4789.15-2010
GB
National Standard of the People’s Republic of China
National food safety standard
Food microbiological examination.
Enumeration of moulds and yeasts
ISSUED ON. MARCH 26, 2010
IMPLEMENTED ON. JUNE 01, 2010
Issued by. Ministry of Health of the People’s Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Apparatuses and materials ... 4
3 Culture medium and reagents ... 5
4 Inspection procedure ... 5
5 Operation steps ... 5
6 Results and report ... 7
Appendix A ... 9
Appendix B ... 11
Foreword
This standard will replace GB/T 4789.15-2003 Microbiological examination of food
hygiene-Examination of moulds and yeasts, from the implementation date.
Compared with GB/T 4789.15-2003, the main changes of this standard are as follow.
- Modify the scope;
- Modify the inspection procedure and operation steps;
- Modify the culture medium and reagents;
- Modify the apparatus and materials;
- Modify the appendix.
This standard’s appendix A is normative; appendix B is informative.
The previous versions replaced by this standard are.
- GB 4789.15-1984, GB 4789.15-1994, GB/T 4789.15-2003.
National food safety standard
Food microbiological examination.
Enumeration of moulds and yeasts
1 Scope
This standard specifies the counting methods of moulds and yeasts in foods.
This standard applies to the counting methods of moulds and yeasts in all types of
foods.
2 Apparatuses and materials
In addition to the conventional sterilization and cultivation apparatuses, other
apparatuses and materials are as follows.
2.1 Refrigerator. 2°C~5°C.
2.2 Thermostatic incubator. 28°C±1°C.
2.3 Homogenizer.
2.4 Thermostatic oscillator.
2.5 Microscope. 10x~100x.
2.6 Electronic balance. Sensitivity is 0.1g.
2.7 Sterile Erlenmeyer flasks. 500mL, 250mL capacity.
2.8 Sterile Jars. 500mL.
2.9 Sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale).
2.10 Sterile petri dish. Diameter is 90mm.
2.11 Sterile test tube. 10mmx75mm.
2.12 Sterile Kraft paper bags and plastic bags.
5.2 Cultivation
After the agar is solidified, place the flat-plate upside down. Cultivate it for 5 days at
28°C±1°C. Observe and record.
5.3 Colony counts
Use naked eyes to observe. If necessary, it can use magnifier. Record every
dilution-multiple and corresponding number of moulds and yeasts. Use colony forming
units (CFU) to express.
Select the flat-plate of which the number of colony is at 10 CFU~150 CFU. According
to colony morphology, count the number of moulds and yeasts respectively. For those
that the moulds have spreaded and grown to cover the whole flat-plate, it may be
recorded as “too numerous to count”. The number of colony shall use the average of 2
flat-plates.
6 Results and report
6.1 Calculate the average of colony number for 2 flat-plates. Multiply the average BY
corresponding dilution-multiple.
6.1.2 If all the colony number on the flat-plates are more than 150 CFU, count the
flat-plate which has the highest dilution. Record other flat-plates as “too numerous to
count”. Results shall be calculated by multiplying the highest dilution-multiple BY the
average of colony number.
6.1.3 If all the colony number in the flat-plates are less than 10 CFU, then it shall be
calculated by multiplying the dilution-multiple BY the average of colony number of
which the dilution is lowest.
6.1.4 If all the dilution flat-plates have no colony, multiply the number less than 1 BY
the lowest dilution-multiple; If it is stock solution, count by the number less than 1.
6.2 Report
6.2.1 If the number of colony is less than 100, according to the rounding rules, use the
2 significant figures to report.
6.2.2 If the number of colony is more than or equaling to 100, the first 3 figures adopt
rounding rules, then get the first 2 figures; the last digit is replaced by 0. Otherwise,
use the 10-exponential form to express; under the same rounding rules, get the 2
significant figures.
6.2.3 Weighing sampling shall use CFU/g as the unit for report. Volume sampling shall
use CFU/mL as the unit for report. Report or respectively report the number of moulds
Appendix B
(informative)
Direct microscopic moulds counting method
The common method is Howard moulds measurement method. This method applies
to canned tomato paste.
B.1 Apparatuses and materials
B.1.1 Refractometer.
B.1.2 Microscope.
B.1.3 Howard measuring slide. special slides containing standard measuring
chamber.
B.1.4 Coverslip.
B.1.5 Micrometer. slide containing standard scale.
B.2 Operation steps
B.2.1 Preparation of test sample. Get quantitative test sample. Add distilled water to
dilute to the refractive index of 1.3447~1.3450 (that is, the concentration is
7.9%~8.8%). Keep for spare use.
B.2.2 Calibration of microscope standard vision. Adjust the standard vision of
microscope. According to 90-times~125-times of magnification, make the diameter to
be 1.382mm.
B.2.3 Smear. Clean the Howard measuring slide. Use glass rod to spread out the
prepared standard solution in measuring chamber uniformly, so as to prepare for
observation.
B.2.4 Observation. Put the prepared slide under standard vision of microscope to
observe the moulds. Usually each test sample shall be observed for 50 visions; 2
persons shall observe the same test sample.
B.2.5 Results and calculation. Under the standard vision, if the length of moulds
hypha exceeds 1/6 of standard vision (1.382mm) OR the total length of three hyphae
exceeds 1/6 of standard vision (that is, 1 scale of micrometer), it is positive (+);
otherwise, it is negative (-). Counted according to 100 visions, the number of visions
containing moulds mycelium is the vision percentage of moulds.
......
| Standard ID | GB 4789.15-2016 (GB4789.15-2016) | | Description (Translated English) | National food safety standard - Food microbiological examination - Enumeration of moulds and yeasts | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 8,838 | | Date of Issue | 2016-10-19 | | Date of Implementation | 2017-04-19 | | Older Standard (superseded by this standard) | SN/T 2552.3-2010; GB 4789.15-2010 | | Regulation (derived from) | State Health and Family Planning Commission Notice No.1516 of 2016 | | Standard ID | GB 4789.15-2010 (GB4789.15-2010) | | Description (Translated English) | National food safety standard - Food microbiological examination: Enumeration of moulds and yeasts | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Classification of International Standard | 07.100.30 | | Word Count Estimation | 8,860 | | Date of Issue | 2010-03-26 | | Date of Implementation | 2010-06-01 | | Older Standard (superseded by this standard) | GB/T 4789.15-2003 | | Regulation (derived from) | Circular of the Ministry of Health (2010)7 | | Issuing agency(ies) | Ministry of Health of People's Republic of China | | Summary | This Chinese standard specifies the food molds and yeasts (moulds and yeasts) counting method. This standard applies to all types of food molds and yeasts counts. | | Standard ID | GB/T 4789.15-2003 (GB/T4789.15-2003) | | Description (Translated English) | Microbiological examination of food hygiene. Enumeration of molds and yeasts | | Sector / Industry | National Standard (Recommended) | | Classification of Chinese Standard | C53 | | Classification of International Standard | 07.100.30 | | Word Count Estimation | 6,674 | | Date of Issue | 2003-08-11 | | Date of Implementation | 2004-01-01 | | Older Standard (superseded by this standard) | GB/T 4789.15-1994 | | Quoted Standard | GB/T 4789.2; GB/T 4789.28-2003 | | Drafting Organization | Center for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention | | Administrative Organization | Ministry of Health of the People Republic of China | | Regulation (derived from) | Notice of the Ministry of Health Satcom (2010) on the 7th; Announcement of Newly Approved National Standards No. 5 in 2010 (No. 160 overall) | | Proposing organization | Ministry of Health of the People Republic of China | | Issuing agency(ies) | People Republic of China Ministry of Health China National Standardization Management Committee | | Summary | This standard specifies: Food in molds and yeasts (mould, an, yeasts) count method. This standard applies to: all kinds of molds and yeasts in food count. |
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