Method for detecting the total number of colonies
2021-06-03 15:04:25
Method for detecting the total number of colonies <br> First, the total number of colonies:
Colonies are growth-producing organisms formed by the growth and reproduction of bacteria on solid media. They are made up of tens of thousands of identical bacteria. When the sample is diluted to a certain extent and mixed with the medium, each of the bacterial cells capable of growing and reproducing can form a visible colony on the plate under certain culture conditions.
The total number of colonies refers to the total number of bacterial colonies grown per gram (per milliliter) of samples under certain conditions (such as aerobic conditions, nutrient conditions, pH, culture temperature and time). According to the national standard method, the total number of bacterial colonies that can grow on common nutrient agar plates under aerobic conditions at 37 ° C for 48 h, so anaerobic or microaerobic bacteria, special nutritional requirements and non-mesophilic Bacteria, because the existing conditions can not meet their physiological needs, it is difficult to breed and grow. Therefore, the total number of colonies does not indicate the total number of bacteria in the actual, and the total number of colonies cannot distinguish the types of bacteria therein, so it is sometimes referred to as the number of bacteria, the number of aerobic bacteria, and the like.
The total number of colonies is used to determine the degree of contamination of the food by the bacteria and the quality of the hygiene. It reflects whether the food meets the hygiene requirements in the production process in order to make an appropriate hygienic evaluation of the sample to be tested. The total number of colonies indicates the quality of food hygiene to a certain extent.
Second, the test method for the determination of the total number of colonies, generally the test sample is made into several different 10-fold incremental dilutions, and then each sample is taken out of 1mL in a separate dish and mixed with nutrient agar medium, After a certain period of time (usually 48 hours) at a certain temperature, the number of colonies formed in each dish was recorded, and the total number of bacterial colonies contained per gram (or per ml) of the original sample was calculated based on the dilution factor.
The basic operations generally include: dilution of the sample - pouring the plate - culturing for 48 hours - counting report.
The methods for determining the total number of colonies at home and abroad are basically the same. There is no significant difference in the sample processing, dilution, and pouring of the tablets to the counting report. However, there are slight differences in some specific requirements. For example, in some countries, sample dilution and pouring culture are carried out. The flow rate of the liquid in the pipette, the amplitude of the oscillation of the diluent, the time and the number of times, and the time of placement are all specified.
For the test method, see:
GB4789.2-94 "National Standards for Food Hygiene Microbiological Examination of Total Number of Colonies"
SN0168-92 "Country Import and Export Commodity Inspection Industry Standard Export Food Colony Count"
Third, explain (a) sample processing and dilution:
1. Method of operation: Take 25g (or 25ml) of the sample aseptically, place it in a sterilized glass bottle of 225mL sterile saline or other diluent (pre-positioned with the appropriate number of glass beads in the bottle) or in a sterile chyle Fully shaken or ground to make a 1:10 uniform dilution.
After adding the diluted solution, it is best to treat it in a sterilizing homogenizer at a speed of 8000~10000r/min for 1min to prepare a 1:10 uniform dilution.
Pipette 1 ml of a 1:10 dilution with a 1 ml sterile pipette, and slowly inject a tube containing 9 ml of sterile physiological saline or other diluent along the tube wall, shake the tube and mix well to make a 1:100 dilution.
Another 1ml sterile pipette was taken, and the 10-fold incremental dilution was prepared according to the above operation sequence. Thus, each incrementing dilution was replaced with a 1 ml sterilization pipette.
2. Sterile operation: The concept of “aseptic operation†must be used in the operation. The glassware used must be completely sterilized and no bacteria or bacteriostatic substances remain. The scissors, tweezers and other utensils used must also be disinfected. If the sample is packaged, use 75% ethanol to wipe the package opening and sample.
The operation should be carried out in an ultra-clean work station or in a sterile, sterile room. The agar plates were exposed to the bench for 15 minutes and each plate should not exceed 15 colonies.
3. Representativeness of sampling: If it is a solid sample, it should not be concentrated when sampling. It is advisable to take several parts. Solid samples must be homogenized or ground and the liquid sample must be shaken to obtain a uniform dilution.
4. Sample dilution error: To reduce sample dilution error, each dilution should be shaken to make it uniform during continuous dilution, and a pipette should be replaced for each dilution.
When performing serial dilution, the liquid in the straw should flow along the wall of the tube. Do not allow the tip of the straw to protrude into the diluent to prevent the test solution adhering to the outside of the straw from being dissolved.
In order to reduce the dilution error, the SN standard uses 10 mL of the dilution solution and is injected into 90 mL of the buffer solution.
5. Diluent: The sample diluent is mainly sterilized physiological saline, and some use phosphate buffer (or 0.1% peptone water), which has a certain protective effect on bacterial cells whose food has been damaged. For dilution of foods with high salt content (such as soy sauce), sterile distilled water can be used.
(2) pouring culture 1. Method of operation: According to the standard requirements or the estimation of the pollution situation, select 2~3 suitable dilutions, and then take 1ml dilution liquid into the sterilization dish by taking the dilution pipette while making the 10-fold incremental dilution. Make two plates for each dilution.
Inject the nutrient agar medium cooled to 46 ° C into a plate of about 15 ml, and rotate the plate and mix well. At the same time, the nutrient agar medium was poured into a sterilized plate to which 1 ml of the diluent (without the sample) was added as a blank control.
After the agar solidified, the plate was inverted and placed in a 36±1 °C incubator for 48±2 h. The number of colonies in the plate was counted and multiplied by the dilution factor to obtain the total number of colonies per gram (per ml) of the sample.
2. The culture medium for pouring should be kept in a water bath at 46 ° C. If the temperature is too high, the growth of the bacteria will be affected. If the agar is too low, the agar will be easily condensed and thus cannot be thoroughly mixed with the bacterial liquid. If there is no water bath, it should be better to feel hot and not hot.
The amount of the pouring medium is different, ranging from 12 to 20 ml, generally 15 ml is more suitable, the plate is too thick to affect the observation, too thin and easy to dry. When pouring, if there is sediment in the base of the culture, the bottom should be discarded to avoid confusion with the colony and affect the counting observation.
3. In order to make the colonies evenly distributed on the plate, after the test solution is added to the plate, the medium should be poured as soon as possible and rotated and mixed, which can be rotated in both directions. The time taken for the sample to be diluted from the beginning to the last plate should not exceed 20 minutes. To prevent bacteria from dying or breeding. .
4. The culture temperature is generally 37 ° C (the culture temperature of aquatic products, due to the low temperature of the living environment, 30 ° C is often used). The culture time is generally 48 hours, and some methods only require 24 hours of culture to count. The incubator should maintain a certain humidity. After 48 hours of agar plate culture, the weight loss of the medium should not exceed 15%.
5. In order to avoid confusion between the tiny particles or the impurities in the food and the bacterial colonies in the food, it is difficult to distinguish, and the flat plate mixed with the agar-based base can be simultaneously prepared without being cultured, and placed in an environment of 4 ° C for counting. Take a control observation.
In some cases, in order to prevent food particles and colonies from being confused, triphenyltetrazolium chloride (TTC) can be added to the nutrient agar. After the culture, the colonies are red and easy to separate.
(3) Counting and reporting 1. Method of operation: After incubation for a period of time, count the number of colonies on each plate. It can be observed with the naked eye and checked with a magnifying glass if necessary to prevent omission. After the total number of colonies of each plate was recorded, the average number of colonies of each plate of the same dilution was determined, and the number of colonies per gram (or per ml) in the original sample was calculated and reported.
2. When the prescribed culture time is reached, it should be counted immediately. If it is not possible to count immediately, the plate should be placed at 0-4 ° C, but not more than 24 h.
3. When counting, the plate with the number of colonies between 30 and 300 should be selected (the SN standard requires 25 to 250 colonies). If the two dilutions are between 30 and 300, the requirements should be based on the national standard method. The ratio determines that the ratio is less than or equal to 2, and the ratio is greater than 2, and the smaller number (some regulations do not consider the ratio of the ratio, are reported as an average).
4. If all dilutions are not in the counting range. If both are greater than 300, the average number of colonies at the highest dilution is multiplied by the dilution factor. If less than 30, the average number of colonies in the lowest dilution is multiplied by the dilution factor. If the number of colonies is more than 300, and some are less than 30, but none of them are between 30 and 300, the average number of colonies closest to 300 or 30 should be multiplied by the dilution factor. If all dilutions are aseptically grown, they should be reported as less than 1 times the lowest dilution factor. Some regulations report the number of colonies calculated for the above several cases as estimated.
5. The number of colonies of different dilutions should be inversely proportional to the dilution factor (the number of colonies of the two plates of the same dilution should be close to each other), that is, the higher the dilution factor, the smaller the number of colonies, and the lower the dilution factor, the more colonies. If there is a reverse phenomenon, it should be regarded as an error in the test (some foods may sometimes have reverse phenomena, such as acidic drinks, etc.), and should not be used as the basis for the sample count report.
6. When there are chain colonies growing on the plate, if there is no obvious boundary between the colonies growing in chains, it should be counted as a colony. If there are several chains of different origin, each chain should be colonized by one colony. Calculate, do not count each colony growing on the chain separately. If there is flaky colony growth, the plate is generally not suitable. If the flaky colony is less than half of the plate and the other half is evenly distributed, the colony number of the half plate can be multiplied by 2 to represent the number of colonies of the whole plate.
7. When counting too many colonies in the plate (ie, all dilutions are greater than 300), but the distribution is very uniform, half or 1/4 of the plate can be counted. Multiply by the corresponding dilution factor as the number of colonies of the plate.
8. The number of colonies is reported according to the national standard method. When the number of colonies is between 1 and 100, the actual number is reported. If it is greater than 100, the first two significant figures are reported, and the third digit is rounded off. Solid samples are reported in grams (g), liquid samples are reported in milliliters (ml), and surface rubs are reported in square centimeters (cm2).
Colonies are growth-producing organisms formed by the growth and reproduction of bacteria on solid media. They are made up of tens of thousands of identical bacteria. When the sample is diluted to a certain extent and mixed with the medium, each of the bacterial cells capable of growing and reproducing can form a visible colony on the plate under certain culture conditions.
The total number of colonies refers to the total number of bacterial colonies grown per gram (per milliliter) of samples under certain conditions (such as aerobic conditions, nutrient conditions, pH, culture temperature and time). According to the national standard method, the total number of bacterial colonies that can grow on common nutrient agar plates under aerobic conditions at 37 ° C for 48 h, so anaerobic or microaerobic bacteria, special nutritional requirements and non-mesophilic Bacteria, because the existing conditions can not meet their physiological needs, it is difficult to breed and grow. Therefore, the total number of colonies does not indicate the total number of bacteria in the actual, and the total number of colonies cannot distinguish the types of bacteria therein, so it is sometimes referred to as the number of bacteria, the number of aerobic bacteria, and the like.
The total number of colonies is used to determine the degree of contamination of the food by the bacteria and the quality of the hygiene. It reflects whether the food meets the hygiene requirements in the production process in order to make an appropriate hygienic evaluation of the sample to be tested. The total number of colonies indicates the quality of food hygiene to a certain extent.
Second, the test method for the determination of the total number of colonies, generally the test sample is made into several different 10-fold incremental dilutions, and then each sample is taken out of 1mL in a separate dish and mixed with nutrient agar medium, After a certain period of time (usually 48 hours) at a certain temperature, the number of colonies formed in each dish was recorded, and the total number of bacterial colonies contained per gram (or per ml) of the original sample was calculated based on the dilution factor.
The basic operations generally include: dilution of the sample - pouring the plate - culturing for 48 hours - counting report.
The methods for determining the total number of colonies at home and abroad are basically the same. There is no significant difference in the sample processing, dilution, and pouring of the tablets to the counting report. However, there are slight differences in some specific requirements. For example, in some countries, sample dilution and pouring culture are carried out. The flow rate of the liquid in the pipette, the amplitude of the oscillation of the diluent, the time and the number of times, and the time of placement are all specified.
For the test method, see:
GB4789.2-94 "National Standards for Food Hygiene Microbiological Examination of Total Number of Colonies"
SN0168-92 "Country Import and Export Commodity Inspection Industry Standard Export Food Colony Count"
Third, explain (a) sample processing and dilution:
1. Method of operation: Take 25g (or 25ml) of the sample aseptically, place it in a sterilized glass bottle of 225mL sterile saline or other diluent (pre-positioned with the appropriate number of glass beads in the bottle) or in a sterile chyle Fully shaken or ground to make a 1:10 uniform dilution.
After adding the diluted solution, it is best to treat it in a sterilizing homogenizer at a speed of 8000~10000r/min for 1min to prepare a 1:10 uniform dilution.
Pipette 1 ml of a 1:10 dilution with a 1 ml sterile pipette, and slowly inject a tube containing 9 ml of sterile physiological saline or other diluent along the tube wall, shake the tube and mix well to make a 1:100 dilution.
Another 1ml sterile pipette was taken, and the 10-fold incremental dilution was prepared according to the above operation sequence. Thus, each incrementing dilution was replaced with a 1 ml sterilization pipette.
2. Sterile operation: The concept of “aseptic operation†must be used in the operation. The glassware used must be completely sterilized and no bacteria or bacteriostatic substances remain. The scissors, tweezers and other utensils used must also be disinfected. If the sample is packaged, use 75% ethanol to wipe the package opening and sample.
The operation should be carried out in an ultra-clean work station or in a sterile, sterile room. The agar plates were exposed to the bench for 15 minutes and each plate should not exceed 15 colonies.
3. Representativeness of sampling: If it is a solid sample, it should not be concentrated when sampling. It is advisable to take several parts. Solid samples must be homogenized or ground and the liquid sample must be shaken to obtain a uniform dilution.
4. Sample dilution error: To reduce sample dilution error, each dilution should be shaken to make it uniform during continuous dilution, and a pipette should be replaced for each dilution.
When performing serial dilution, the liquid in the straw should flow along the wall of the tube. Do not allow the tip of the straw to protrude into the diluent to prevent the test solution adhering to the outside of the straw from being dissolved.
In order to reduce the dilution error, the SN standard uses 10 mL of the dilution solution and is injected into 90 mL of the buffer solution.
5. Diluent: The sample diluent is mainly sterilized physiological saline, and some use phosphate buffer (or 0.1% peptone water), which has a certain protective effect on bacterial cells whose food has been damaged. For dilution of foods with high salt content (such as soy sauce), sterile distilled water can be used.
(2) pouring culture 1. Method of operation: According to the standard requirements or the estimation of the pollution situation, select 2~3 suitable dilutions, and then take 1ml dilution liquid into the sterilization dish by taking the dilution pipette while making the 10-fold incremental dilution. Make two plates for each dilution.
Inject the nutrient agar medium cooled to 46 ° C into a plate of about 15 ml, and rotate the plate and mix well. At the same time, the nutrient agar medium was poured into a sterilized plate to which 1 ml of the diluent (without the sample) was added as a blank control.
After the agar solidified, the plate was inverted and placed in a 36±1 °C incubator for 48±2 h. The number of colonies in the plate was counted and multiplied by the dilution factor to obtain the total number of colonies per gram (per ml) of the sample.
2. The culture medium for pouring should be kept in a water bath at 46 ° C. If the temperature is too high, the growth of the bacteria will be affected. If the agar is too low, the agar will be easily condensed and thus cannot be thoroughly mixed with the bacterial liquid. If there is no water bath, it should be better to feel hot and not hot.
The amount of the pouring medium is different, ranging from 12 to 20 ml, generally 15 ml is more suitable, the plate is too thick to affect the observation, too thin and easy to dry. When pouring, if there is sediment in the base of the culture, the bottom should be discarded to avoid confusion with the colony and affect the counting observation.
3. In order to make the colonies evenly distributed on the plate, after the test solution is added to the plate, the medium should be poured as soon as possible and rotated and mixed, which can be rotated in both directions. The time taken for the sample to be diluted from the beginning to the last plate should not exceed 20 minutes. To prevent bacteria from dying or breeding. .
4. The culture temperature is generally 37 ° C (the culture temperature of aquatic products, due to the low temperature of the living environment, 30 ° C is often used). The culture time is generally 48 hours, and some methods only require 24 hours of culture to count. The incubator should maintain a certain humidity. After 48 hours of agar plate culture, the weight loss of the medium should not exceed 15%.
5. In order to avoid confusion between the tiny particles or the impurities in the food and the bacterial colonies in the food, it is difficult to distinguish, and the flat plate mixed with the agar-based base can be simultaneously prepared without being cultured, and placed in an environment of 4 ° C for counting. Take a control observation.
In some cases, in order to prevent food particles and colonies from being confused, triphenyltetrazolium chloride (TTC) can be added to the nutrient agar. After the culture, the colonies are red and easy to separate.
(3) Counting and reporting 1. Method of operation: After incubation for a period of time, count the number of colonies on each plate. It can be observed with the naked eye and checked with a magnifying glass if necessary to prevent omission. After the total number of colonies of each plate was recorded, the average number of colonies of each plate of the same dilution was determined, and the number of colonies per gram (or per ml) in the original sample was calculated and reported.
2. When the prescribed culture time is reached, it should be counted immediately. If it is not possible to count immediately, the plate should be placed at 0-4 ° C, but not more than 24 h.
3. When counting, the plate with the number of colonies between 30 and 300 should be selected (the SN standard requires 25 to 250 colonies). If the two dilutions are between 30 and 300, the requirements should be based on the national standard method. The ratio determines that the ratio is less than or equal to 2, and the ratio is greater than 2, and the smaller number (some regulations do not consider the ratio of the ratio, are reported as an average).
4. If all dilutions are not in the counting range. If both are greater than 300, the average number of colonies at the highest dilution is multiplied by the dilution factor. If less than 30, the average number of colonies in the lowest dilution is multiplied by the dilution factor. If the number of colonies is more than 300, and some are less than 30, but none of them are between 30 and 300, the average number of colonies closest to 300 or 30 should be multiplied by the dilution factor. If all dilutions are aseptically grown, they should be reported as less than 1 times the lowest dilution factor. Some regulations report the number of colonies calculated for the above several cases as estimated.
5. The number of colonies of different dilutions should be inversely proportional to the dilution factor (the number of colonies of the two plates of the same dilution should be close to each other), that is, the higher the dilution factor, the smaller the number of colonies, and the lower the dilution factor, the more colonies. If there is a reverse phenomenon, it should be regarded as an error in the test (some foods may sometimes have reverse phenomena, such as acidic drinks, etc.), and should not be used as the basis for the sample count report.
6. When there are chain colonies growing on the plate, if there is no obvious boundary between the colonies growing in chains, it should be counted as a colony. If there are several chains of different origin, each chain should be colonized by one colony. Calculate, do not count each colony growing on the chain separately. If there is flaky colony growth, the plate is generally not suitable. If the flaky colony is less than half of the plate and the other half is evenly distributed, the colony number of the half plate can be multiplied by 2 to represent the number of colonies of the whole plate.
7. When counting too many colonies in the plate (ie, all dilutions are greater than 300), but the distribution is very uniform, half or 1/4 of the plate can be counted. Multiply by the corresponding dilution factor as the number of colonies of the plate.
8. The number of colonies is reported according to the national standard method. When the number of colonies is between 1 and 100, the actual number is reported. If it is greater than 100, the first two significant figures are reported, and the third digit is rounded off. Solid samples are reported in grams (g), liquid samples are reported in milliliters (ml), and surface rubs are reported in square centimeters (cm2).
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