Summary and determination of the total number of colonies
1. Total number of colonies
1. Definition
Colony refers to the growth of bacteria that grows on a solid medium and can be recognized by the naked eye. It is a collection of tens of thousands of the same bacteria. When the sample is diluted to a certain degree and mixed with the culture medium, under certain culture conditions, each bacterial cell that can grow and reproduce can form a visible colony on the plate.
The total number of colonies refers to the total number of bacterial colonies grown per gram (per milliliter) of the sample under certain conditions (such as aerobic conditions, nutritional conditions, pH, culture temperature and time, etc.).
2. The significance of the total number of colonies
The determination of the total number of colonies is used to determine the degree of food contamination by bacteria and the hygienic quality. It reflects whether the food meets the hygienic requirements during the production process, so as to make an appropriate hygienic evaluation of the tested sample. To a certain extent, the total number of colonies marks the quality of food hygiene.
Second, the inspection method
For the determination of the total number of colonies, the test sample is generally made into several different 10-fold increasing dilutions, and then 1 mL is taken from each dilution and placed in a sterilized dish and mixed with nutrient agar medium at a certain temperature. After incubation for a certain period of time (generally 48 hours), record the number of colonies formed in each dish, and calculate the total number of bacterial colonies contained in each gram (or per ml) of the original sample based on the dilution factor.
Basic operations generally include: dilution of the sample-pouring into the dish-incubation for 48 hours-counting report.
3. Instruments used in the test
HBM series flapping homogenizer (sterile homogenizer): Tianjin Hengao Technology Development Co., Ltd .;
HMS series oscillator: Tianjin Hengao Technology Development Co., Ltd .;
Glass bottle
test tube;
Flat dish
Straws, etc.
Fourth, the test process
(1) Sample processing and dilution:
1. Aseptic operation: The glassware used must be completely sterilized, and no bacteria or bacteriostatic substances should remain. The scissors and tweezers used must also be sterilized. If the sample is packaged, wipe it with 75% ethanol at the opening of the package and take a sample.
The operation should be carried out on an ultra-clean workbench or a sterilized sterile room. The agar plate is exposed to the workbench for 15 minutes, and each plate must not exceed 15 colonies.
2. Operation method: Take 25g (or 25ml) of the sample aseptically and put it in a sterilized glass bottle with 225mL sterilized physiological saline or other diluent (pre-set appropriate amount of glass beads in the bottle) or sterilized mortar , Shake well with a shaker to make a 1:10 uniform dilution.
After adding the diluent to the solid sample, it is best to put it in a sterilizing homogenizer and process it at a speed of 8000-10000r / min for 1min to make a 1:10 uniform diluent.
Use a 1ml sterilized pipette to absorb 1ml of 1:10 dilution solution, slowly inject it into a test tube containing 9ml of sterilized physiological saline or other dilution solution along the wall of the tube, shake the test tube to mix evenly, and make a 1: 100 dilution solution.
Take another 1ml sterilized pipette and make a 10-fold incremental dilution according to the above operation sequence. In this way, each incremental dilution is replaced with a 1ml sterilized pipette.
3. Representativeness of sampling: If it is a solid sample, it should not be concentrated when sampling, and it is advisable to collect more parts. Solid samples must be homogenized (available with a homogenizer, only for reference) or ground, and liquid samples must be oscillated (available with an oscillator, only for reference) to obtain a uniform dilution.
4. Sample dilution error: In order to reduce the sample dilution error, during successive dilutions, each dilution should be fully shaken to make it uniform, and at the same time, each dilution should be replaced with a straw.
During continuous dilution, the liquid in the pipette should flow along the wall of the pipe, and the tip of the pipette should not extend into the diluent, so as to prevent the test liquid adhering to the outside of the pipette from dissolving in it.
To reduce the dilution error, the SN standard uses 10mL of diluent and injects it into 90mL of buffer.
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 the damaged bacterial cells of the food. Sterilized distilled water can be used to dilute foods with high salt content (such as soy sauce).
(Two) pouring cultivation
1. Operation method: According to the standard requirements or the estimation of the pollution situation, select 2-3 suitable dilutions, respectively, while making a 10-fold incremental dilution, and pipette 1ml of the dilution liquid into the sterilization plate with the pipette sucking the dilution. Make two dishes for each dilution.
Put the nutrient agar medium cooled to 46 ° C into the dish for about 15ml, and rotate the dish to mix evenly. At the same time, the nutrient agar medium was poured into a sterilized plate with 1 ml of diluent (without sample) as a blank control.
After the agar solidifies, flip the plate, place it in a 36 ± 1 ℃ incubator for 48 ± 2h, take out the number of colonies in the plate, and multiply by the dilution factor to obtain the total number of colonies per gram (per milliliter) of sample.
2. The culture medium for pouring should be kept in a 46 ° C water bath. Too high a temperature will affect the growth of bacteria. Too low agar is easy to congeal and cannot be mixed well with the bacterial solution. If there is no water bath, the skin should be hot rather than hot.
The amount of medium poured is different, ranging from 12-20ml, generally 15ml is more suitable, too thick plate can affect the observation, too thin and easy to crack. When pouring, if there is sediment at the bottom of the base, 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 from the start of the sample dilution to the last plate should not exceed 20min. To prevent the bacteria from dying or multiplying. .
4. The cultivation temperature is generally 37 ° C (the cultivation temperature of aquatic products is 30 ° C because of the low water temperature in its living environment). The cultivation time is generally 48h, some methods only require 24h cultivation 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 the confusion between the tiny particles in the food or the impurities in the base and the bacterial colony, it is not easy to distinguish, and it can be used as a plate mixed with agar and base at the same time. Make control observation.
In some cases, in order to prevent food particles from being confused with colonies, triphenyltetrazolium chloride (TTC) can be added to nutrient agar. After cultivation, the colonies are red and easy to separate.
(3) Counting and reporting
1. Operation method: After the cultivation time, count the number of colonies on each plate. It can be observed with the naked eye and checked with a magnifying glass when necessary to prevent omissions. After recording the total number of colonies on each plate, find the average number of colonies on each plate at the same dilution, calculate the number of colonies per gram (or per ml) in the original sample, and report.
2. When the specified cultivation time is reached, it should be counted immediately. If it cannot be counted immediately, the plate should be placed at 0-4 ℃, but not more than 24h.
3. When counting, the plate with a colony number of 30-300 should be selected (SN standard requires 25-250 colonies). If there are two dilutions between 30-300, the two should be based on the national standard method. The ratio determines that the ratio is less than or equal to 2 to take the average, and the ratio is greater than 2 to the smaller number (some provisions do not consider the size of the ratio, and all are reported as averages).
4. If all dilutions are not within the counting interval. If both are greater than 300, the average number of colonies at the highest dilution is multiplied by the dilution factor to report. If both are less than 30, report the average number of colonies at the lowest dilution times the dilution factor. If the number of colonies is more than 300, and some is less than 30, but not between 30-300, the average colony number closest to 300 or 30 should be multiplied by the dilution factor to report. If all dilutions are aseptically grown, they should be reported as less than 1 times the lowest dilution multiple. According to some regulations, the number of colonies calculated for the above situations is reported as an estimate.
5. The number of colonies at different dilutions should be inversely proportional to the dilution factor (the number of colonies of two plates at the same dilution should be basically close), that is, the higher the dilution factor, the fewer 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 inspection (some foods may sometimes have a reverse phenomenon, such as acidic beverages, 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 are no obvious boundaries between the chain-growing colonies, it should be counted as a colony. If there are several chains from different sources, each chain should be a colony. For calculation, do not count each colony growing on the chain separately. If there are flaky colonies growing, the plate is generally not suitable for use. If the flaky colonies are less than half of the plate, and the other half is evenly distributed, the number of colonies on a half plate can be multiplied by 2 to represent the number of colonies on the whole plate.
7. When the number of colonies in the counting plate is too large (that is, when all dilutions are greater than 300), but the distribution is very uniform, half or 1/4 of the plate can be taken for counting. Then multiply the corresponding dilution factor as the number of colonies on the plate.
8. The number of colonies is reported according to the national standard method. When the number of colonies is 1-100, the actual number is reported. If it is greater than 100, the first two significant digits are reported, and the third digit is calculated by rounding. Solid samples are reported in grams (g), liquid samples are reported in milliliters (ml), and surface smears are reported in square centimeters (cm2).
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