Summary from the front line! A compilation of microbial sampling, sample preparation techniques, as well as dilution, inoculation, and cultivation methods!

1. Sample Pre-treatment

(1) Regardless of the sampling scheme, the samples must be representative. The sampling process follows aseptic operating procedures to prevent any possible external contamination.

If it is a frozen sample, it should be stored frozen as required. Dried food can be placed in a cool, dark place at room temperature, while perishable and refrigerated samples should be kept in an environment of 10°C. If frozen samples cannot be tested in time, they should be placed in a freezer below -15°C, and the storage time for samples awaiting testing should not exceed 36 hours.

(2) Sample Thawing

If it is a frozen sample, after taking it out, thaw it in a refrigerator at 0-8°C for no more than 18 hours; it can also be thawed in an environment not exceeding 45°C for no more than 15 minutes.

(3) Preparation Before Testing

Before the experiment, move the tools to the sterile area, and it is best to sterilize them with ultraviolet light for 20 minutes to achieve a sterile state.

Check that the preparation items for the operation experiment are complete, including homogenization cups, alcohol and alcohol cotton, pipettes, sterilized tips, petri dishes, diluents, scissors, tweezers, sterile basins or baskets, etc.

2. Testing

1. Sample Identification

Label the samples accordingly, with the total number of colonies according to operation 4789.2, making two parallels for each dilution, generally making three dilutions for each sample in a stack.

The detection of coliforms and Staphylococcus aureus is performed according to the MPN method in 4789.3 and 4789.10, with three tubes for each dilution, generally also making three dilutions.

2. Sampling

First, spray the outer packaging of the sample on both sides with alcohol (if there is condensation on the sample surface, wipe it clean before spraying with alcohol).

(1) Solid Samples

① Disinfect the opening and surrounding area with 75% alcohol cotton, then use flame-sterilized scissors to cut it open.

② Place the homogenization cup on the electronic scale and tare it to zero.

③ Use flame-sterilized scissors and tweezers to take a sample, weighing 25g of a representative sample.

④ Add 225ml of sterilized phosphate buffer or sterile saline (when opening the bottle, the mouth and stopper should be passed through the flame 1-2 times to kill any bacteria that may have fallen from the air).

⑤ After cleaning the scissors and tweezers, soak them in a 75% alcohol container.

⑥ Cover the homogenization cup and homogenize at 8000 rpm for 1-2 minutes to obtain the sample solution.

(2) Liquid Samples

① Use the sample only after it has completely thawed.

② Disinfect the opening and surrounding area with 75% alcohol cotton, then use flame-sterilized scissors to open it, and use a sterilized pipette to take 25ml of the well-mixed sample.

③ Add 225ml of sterilized phosphate buffer or sterile saline and mix as the sample solution.

(3) Powdered Samples and Semi-solid Samples

① Mix the sample evenly.

② Disinfect the opening and surrounding area with 75% alcohol cotton, then use flame-sterilized scissors to open it, and use a sterile spoon to take 10g of the mixed sample.

③ Add 90ml of sterilized phosphate buffer, and when opening the bottle, the mouth and stopper should be passed through the flame 1-2 times to kill any bacteria that may have fallen from the air.

④ The same as above.

3. Dilution Method

① Accurately aspirate 1mL of the homogenized sample solution, then slowly inoculate it along the wall of the tube into 9ml of phosphate buffer, cap the test tube, and shake well to mix, creating a 1:100 dilution. (Make sure the tip of the pipette extends into the dilution liquid by 2-3mL to avoid sucking in air, which could contaminate the sample and cause the experiment to fail.)

②Repeat this operation to prepare 1:1000, 1:10000... dilutions as needed.

③ To reduce dilution errors, when performing consecutive dilutions (original solution first, dilution liquid later), each dilution should be shaken thoroughly to ensure uniformity, and a new pipette tip should be used for each dilution.

④ Do not blow out the pipette in the diluent.

4. Sample Inoculation Method

① In the sterile room, take out the sterilized pipette tip or sterilized pipette.

② Accurately aspirate the sample solution, injecting 1mL, 1mL, 1mL into the clearly labeled petri dishes for total colony count, coliforms, and Staphylococcus aureus culture within 2-4 seconds.

③ If any sample solution has been left for more than 3 minutes before inoculation, it should be re-homogenized.

④ To verify the sterility of the diluent, culture medium, petri dishes, pipette tips, etc., a blank control should be performed.

5. Pouring Culture Medium

Hold the culture medium triangular flask (which has been kept in a 45°C water bath) with the right hand next to the flame, and use the left thumb and index or middle finger to open the petri dish no more than 30°, quickly pour in about 15-20ml of culture medium, cover it, and gently shake the petri dish, making three circles to the left and three to the right to evenly distribute the culture medium at the bottom of the petri dish, then lay it flat on the table.

6. Cultivation

• Invert the petri dishes and place them in the constant temperature incubator (stacking a maximum of 6 petri dishes per stack, leaving gaps between the dishes for air circulation, allowing the temperature of the culture to quickly match the temperature of the incubator), and cultivate at the specified time and temperature.
• Slanted surfaces, high-level slanted surfaces, and liquid culture media are placed in a test tube rack and put into a constant temperature incubator for cultivation at the specified time and temperature.

7. Counting Determination and Precautions

• Due to the wide variety of bacteria, there are significant differences. During counting, a colony counter is generally used for careful observation. If necessary, a magnifying glass can be used to check for omissions, especially for colonies growing at the edges of the plates.
• The counting method refers to GB 4789.2-2016.
• In culture dishes with low dilution, it is difficult to distinguish between microbial colonies and food debris. Generally, the diluted liquid after filtering through a filter mesh is taken to reduce the impact of food debris.
• To avoid confusion between small particles in food or impurities in the culture medium and bacterial colonies, which are difficult to distinguish, a diluted liquid can be prepared and mixed with agar medium on a plate, without cultivation, and placed in a 4°C environment for control observation during counting.
• If necessary, to prevent confusion between food particles and colonies, tri phenyl tetrazolium chloride (TTC) can be added to the agar in the plate counting (1 ml of 0.5% TTC added to 100 ml). After cultivation, the colonies will appear red, making them easy to distinguish.
• In fermentation experiments, the gas production in the fermentation tube can fill the entire tube with gas in large amounts, while smaller amounts can produce bubbles smaller than millet grains, or during fermentation, acid production or small bubbles slowly rising along the tube wall should be further tested. If there are doubts about fermentation that produces acid but no gas, gently shaking the test tube can help; if bubbles rise along the tube wall, similar to carbon dioxide bubbles in beer, it should be considered that gas may be produced, and further testing should be conducted.