Food hygiene microbiological examination
National Standard Food Hygiene Microbiological Examination of the People's Republic of China GB 4789.2-94
Determination of the total number of colonies ──────────────────────────────────────
1 Subject content and scope of application This standard specifies the method for determining the total number of colonies in food.
This standard applies to the determination of the total number of colonies in food.
2 The term total number of colonies refers to the total number of colonies contained in the 1 mL (g) sample obtained after the food sample has been treated and cultured under certain conditions (such as culture composition, culture temperature and time, pH, aerobic properties, etc.). . The results obtained under the culture conditions specified in the method include only a total number of mesophilic aerobic colonies grown on nutrient agar.
The total number of colonies is mainly used as a marker to determine the degree of contamination of food. This method can also be used to observe the dynamics of bacterial propagation in foods in order to provide a basis for hygienic evaluation of the samples to be tested.
3 Reference standards
GB 4789.28 Food hygiene microbiological test staining method, medium and reagents
4 Equipment and materials
4.1 Thermostat: 36 ± 1 °C.
4.2 Refrigerator: 0 ~ 4 ° C.
4.3 Constant temperature water bath: 46 ± 1 °C.
4.4 Balance.
4.5 Electric furnace.
4.6 Straw.
4.7 Jar or flask: The capacity is 500mL.
4.8 Glass beads: about 5mm in diameter.
4.9 Plate: 90 mm in diameter.
4.10 test tube.
4.11 Magnifying glass.
4.12 Colony counter.
4.13 Alcohol lamp.
4.14 Homogenizer or mortar.
4.15 test tube rack.
4.16 Sterilizer or scissors.
4.17 Sterilize the tweezers.
5 media and reagents
5.1 Nutrient agar medium: According to 4.7 of GB 4789.28.
5.2 Phosphate buffer dilution: According to 3.22 of GB 4789.28.
5.3 Saline.
5.4 75% ethanol.
6 Inspection procedures The total number of colonies tested is as follows.
┌─────â”
│ Sample │
└─────┘
↓
┌──────────────â”
│ Make a few suitable dilutions │
└──────────────┘
↓
┌──────────────â”
│ Select 2~3 suitable dilutions│
│ Each 1mL is added to the sterilized plate │
└──────────────┘
↓
┌──────────────â”
│ Add appropriate amount of nutrient agar to each dish │
└──────────────┘
36±1°C ↓ 48±2h
┌─────â”
│ Colony Count │
└─────┘
↓
┌──────â”
│ Report │
└──────┘
7 Operating steps
7.1 Sample dilution and culture
7.1.1 Aseptically, smash 25g (or mL) of the sample into a sterilized glass bottle containing 225mL of sterile saline or other diluent (pre-set the appropriate number of glass beads in the bottle) or sterilize In the mortar, fully shaken or ground to make 1:10
Uniform dilution.
After the solid sample is added to the diluent, it is best to treat it in the homogenizer at a rate of 8,000 to 10 000 r/min for 1 min.
Make a 1:10 uniform dilution.
7.1.2 Pipette 1 mL of 1:10 dilution solution with a 1 mL sterile pipette, and slowly inject the test tube containing 9 mL of sterile saline or other diluent along the tube wall (note that the tip of the pipette does not touch the dilution in the tube), shake the tube, mix Evenly, made 1:10
0 dilution.
7.1.3 Take another 1mL sterile pipette, do the 10-fold incremental dilution according to the above operation sequence, so every incremental dilution,
That is to replace one 1mL sterile straw.
7.1.4 According to the requirements of food hygiene standards or the estimation of the contamination of the specimen, select 2 to 3 suitable dilutions, and at the same time as the 10-fold incremental dilution, remove the 1 mL dilution solution from the pipette to absorb the dilution for sterilization. In the dish, make two plates for each dilution.
7.1.5 After the diluent is transferred into the plate, it should be cooled to 46 °C nutrient agar medium (can be placed in a 46±1 ° C water bath) to inject about 15 mL into the plate, and turn the plate to mix evenly. At the same time, the nutrient agar medium was poured into a sterilized dish to which 1 mL of the diluted solution was added as a blank control.
7.1.6 After the agar is solidified, turn the plate and place it in a 36±1°C incubator for 48±2h.
7.2 Colony counting method When the plate colony count is made, it can be observed with the naked eye, and if necessary, check with a magnifying glass to prevent omission. After the number of colonies of each plate was recorded, the average number of colonies of each plate of the same dilution was determined.
7.3 Report on colony count
7.3.1 Selection of plate colony number The plate with the number of colonies between 30 and 300 was selected as the standard for the total number of colonies. Two plates should be used for one dilution. The average number of two plates should be used. When one plate has larger flaky colonies, it should not be used. Instead, the plate with no flaky colonies should be used as the number of colonies of the dilution. If the flaky colony is less than half of the plate and the colony distribution is very uniform in the remaining half, then half of the plate can be calculated and multiplied by 2 to represent the number of colonies. If there are chain colonies growing in the plate (there is no obvious boundary between the colonies), if there is only one chain, it can be regarded as a colony; if there are several chains from different sources, each chain should be counted as one colony.
7.3.2 Selection of dilution
7.3.2.1 The dilution of the average number of colonies between 30 and 300 should be selected and multiplied by the dilution factor (see Example 1 in the table).
7.3.2.2 If there are two dilutions, the number of colonies growing is between 30 and 300, depending on the ratio of the two.
If the ratio is less than or equal to 2, the average number should be reported; if it is greater than 2, the smaller number is reported (see Example 2 in the table).
3).
7.3.2.3 If the average number of colonies for all dilutions is greater than 300, the average number of colonies with the highest dilution should be multiplied by the dilution factor (see Example 4 in the table).
7.3.2.4 If the average number of colonies for all dilutions is less than 30, the average number of colonies with the lowest dilution should be multiplied by the dilution factor (see Example 5 in the table).
7.3.2.5 If all dilutions are grown aseptically, multiply by less than 1 by the lowest dilution factor (see Example 6 in the table).
7.3.2.6 If the average number of colonies in all dilutions is not between 30 and 300, some of them are greater than 300 or less than 30.
When multiplied by the average number of colonies closest to 30 or 300, the dilution factor is reported (see Example 7 in the table).
7.3.3 Reporting the number of colonies When the number of colonies is less than 100, the actual number is reported. When the number of colonies is greater than 100, two significant figures are used, and the values ​​after the two significant digits are rounded off. In order to shorten the number of zeros after the number, it can also be expressed by an index of 10 (see the "Report Mode" column in the table).
Dilution selection and colony number reporting method ─┬─────────────┬──────┬────┬─────────
│ Diluent and colony number │ │ Total number of colonies │ Reporting method ├ ─ ─ ─ ─ ─ ─ ─ ─ ─ ┤ two dilution ratio │ / g or mL │ / g or mL
│ 10-1 │ 10-2 │ 10-3 │ │ │
───┼────┼────┼───┼──────┼────┼─────────
1 │Multiple │ 164 │ 20 │ - │ 16 400 │16 000 or 1.6×10**4
2 │Multiple │ 295 │ 46 │ 1.6 │ 37 750 │3 8000 or 3.8×10**4
3 │Multiple │ 271 │ 60 │ 2.2 │ 27 100 │27 000 or 2.7×10**4
4 │More than │Multiple │ 313 │ - │ 313 000│31 000 or 3.1×10**5
5 │ 27 │ 11 │ 5 │ - │ 270 │270 or 2.7×10**2
6 │ 0 │ 0 │ 0 │ - │ <1×10│ <10
7 │Multiple │ 305 │ 12 │ - │ 30 500│31 000 or 3.1×10**4
───┴────┴────┴───┴──────┴────┴─────────
──────────
Additional information:
This standard was proposed by the Health Supervision Department of the Ministry of Health.
This standard is drafted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health.
The main drafter of this standard is Liu Hongdao.
This standard is interpreted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health, which is entrusted by the Ministry of Health.
National Standard Food Hygiene Microbiological Examination of the People's Republic of China GB 4789.2-94
Determination of the total number of colonies ──────────────────────────────────────
1 Subject content and scope of application This standard specifies the method for determining the total number of colonies in food.
This standard applies to the determination of the total number of colonies in food.
2 The term total number of colonies refers to the total number of colonies contained in the 1 mL (g) sample obtained after the food sample has been treated and cultured under certain conditions (such as culture composition, culture temperature and time, pH, aerobic properties, etc.). . The results obtained under the culture conditions specified in the method include only a total number of mesophilic aerobic colonies grown on nutrient agar.
The total number of colonies is mainly used as a marker to determine the degree of contamination of food. This method can also be used to observe the dynamics of bacterial propagation in foods in order to provide a basis for hygienic evaluation of the samples to be tested.
3 Reference standards
GB 4789.28 Food hygiene microbiological test staining method, medium and reagents
4 Equipment and materials
4.1 Thermostat: 36 ± 1 °C.
4.2 Refrigerator: 0 ~ 4 ° C.
4.3 Constant temperature water bath: 46 ± 1 °C.
4.4 Balance.
4.5 Electric furnace.
4.6 Straw.
4.7 Jar or flask: The capacity is 500mL.
4.8 Glass beads: about 5mm in diameter.
4.9 Plate: 90 mm in diameter.
4.10 test tube.
4.11 Magnifying glass.
4.12 Colony counter.
4.13 Alcohol lamp.
4.14 Homogenizer or mortar.
4.15 test tube rack.
4.16 Sterilizer or scissors.
4.17 Sterilize the tweezers.
5 media and reagents
5.1 Nutrient agar medium: According to 4.7 of GB 4789.28.
5.2 Phosphate buffer dilution: According to 3.22 of GB 4789.28.
5.3 Saline.
5.4 75% ethanol.
6 Inspection procedures The total number of colonies tested is as follows.
┌─────â”
│ Sample │
└─────┘
↓
┌──────────────â”
│ Make a few suitable dilutions │
└──────────────┘
↓
┌──────────────â”
│ Select 2~3 suitable dilutions│
│ Each 1mL is added to the sterilized plate │
└──────────────┘
↓
┌──────────────â”
│ Add appropriate amount of nutrient agar to each dish │
└──────────────┘
36±1°C ↓ 48±2h
┌─────â”
│ Colony Count │
└─────┘
↓
┌──────â”
│ Report │
└──────┘
7 Operating steps
7.1 Sample dilution and culture
7.1.1 Aseptically, smash 25g (or mL) of the sample into a sterilized glass bottle containing 225mL of sterile saline or other diluent (pre-set the appropriate number of glass beads in the bottle) or sterilize In the mortar, fully shaken or ground to make 1:10
Uniform dilution.
After the solid sample is added to the diluent, it is best to treat it in the homogenizer at a rate of 8,000 to 10 000 r/min for 1 min.
Make a 1:10 uniform dilution.
7.1.2 Pipette 1 mL of 1:10 dilution solution with a 1 mL sterile pipette, and slowly inject the test tube containing 9 mL of sterile saline or other diluent along the tube wall (note that the tip of the pipette does not touch the dilution in the tube), shake the tube, mix Evenly, made 1:10
0 dilution.
7.1.3 Take another 1mL sterile pipette, do the 10-fold incremental dilution according to the above operation sequence, so every incremental dilution,
That is to replace one 1mL sterile straw.
7.1.4 According to the requirements of food hygiene standards or the estimation of the contamination of the specimen, select 2 to 3 suitable dilutions, and at the same time as the 10-fold incremental dilution, remove the 1 mL dilution solution from the pipette to absorb the dilution for sterilization. In the dish, make two plates for each dilution.
7.1.5 After the diluent is transferred into the plate, it should be cooled to 46 °C nutrient agar medium (can be placed in a 46±1 ° C water bath) to inject about 15 mL into the plate, and turn the plate to mix evenly. At the same time, the nutrient agar medium was poured into a sterilized dish to which 1 mL of the diluted solution was added as a blank control.
7.1.6 After the agar is solidified, turn the plate and place it in a 36±1°C incubator for 48±2h.
7.2 Colony counting method When the plate colony count is made, it can be observed with the naked eye, and if necessary, check with a magnifying glass to prevent omission. After the number of colonies of each plate was recorded, the average number of colonies of each plate of the same dilution was determined.
7.3 Report on colony count
7.3.1 Selection of plate colony number The plate with the number of colonies between 30 and 300 was selected as the standard for the total number of colonies. Two plates should be used for one dilution. The average number of two plates should be used. When one plate has larger flaky colonies, it should not be used. Instead, the plate with no flaky colonies should be used as the number of colonies of the dilution. If the flaky colony is less than half of the plate and the colony distribution is very uniform in the remaining half, then half of the plate can be calculated and multiplied by 2 to represent the number of colonies. If there are chain colonies growing in the plate (there is no obvious boundary between the colonies), if there is only one chain, it can be regarded as a colony; if there are several chains from different sources, each chain should be counted as one colony.
7.3.2 Selection of dilution
7.3.2.1 The dilution of the average number of colonies between 30 and 300 should be selected and multiplied by the dilution factor (see Example 1 in the table).
7.3.2.2 If there are two dilutions, the number of colonies growing is between 30 and 300, depending on the ratio of the two.
If the ratio is less than or equal to 2, the average number should be reported; if it is greater than 2, the smaller number is reported (see Example 2 in the table).
3).
7.3.2.3 If the average number of colonies for all dilutions is greater than 300, the average number of colonies with the highest dilution should be multiplied by the dilution factor (see Example 4 in the table).
7.3.2.4 If the average number of colonies for all dilutions is less than 30, the average number of colonies with the lowest dilution should be multiplied by the dilution factor (see Example 5 in the table).
7.3.2.5 If all dilutions are grown aseptically, multiply by less than 1 by the lowest dilution factor (see Example 6 in the table).
7.3.2.6 If the average number of colonies in all dilutions is not between 30 and 300, some of them are greater than 300 or less than 30.
When multiplied by the average number of colonies closest to 30 or 300, the dilution factor is reported (see Example 7 in the table).
7.3.3 Reporting the number of colonies When the number of colonies is less than 100, the actual number is reported. When the number of colonies is greater than 100, two significant figures are used, and the values ​​after the two significant digits are rounded off. In order to shorten the number of zeros after the number, it can also be expressed by an index of 10 (see the "Report Mode" column in the table).
Dilution selection and colony number reporting method ─┬─────────────┬──────┬────┬─────────
│ Diluent and colony number │ │ Total number of colonies │ Reporting method ├ ─ ─ ─ ─ ─ ─ ─ ─ ─ ┤ two dilution ratio │ / g or mL │ / g or mL
│ 10-1 │ 10-2 │ 10-3 │ │ │
───┼────┼────┼───┼──────┼────┼─────────
1 │Multiple │ 164 │ 20 │ - │ 16 400 │16 000 or 1.6×10**4
2 │Multiple │ 295 │ 46 │ 1.6 │ 37 750 │3 8000 or 3.8×10**4
3 │Multiple │ 271 │ 60 │ 2.2 │ 27 100 │27 000 or 2.7×10**4
4 │More than │Multiple │ 313 │ - │ 313 000│31 000 or 3.1×10**5
5 │ 27 │ 11 │ 5 │ - │ 270 │270 or 2.7×10**2
6 │ 0 │ 0 │ 0 │ - │ <1×10│ <10
7 │Multiple │ 305 │ 12 │ - │ 30 500│31 000 or 3.1×10**4
───┴────┴────┴───┴──────┴────┴─────────
──────────
Additional information:
This standard was proposed by the Health Supervision Department of the Ministry of Health.
This standard is drafted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health.
The main drafter of this standard is Liu Hongdao.
This standard is interpreted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health, which is entrusted by the Ministry of Health.
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