This kit adopts the principle of microbial inhibition. The microwell plate is filled with a medium containing Geobacillus stearothermophilus spores and the pH indicator. When cultured at 65℃, the spores germinate, the bacteria grow, and then metabolize to produce acid. The pH value decreases and the indicator changes from the original purple to yellow. If the antibiotics exist in the added milk and dairy products, the antibiotics will inhibit bacterial growth, thus no acid is produced, the indicator does not change the color, and the original purple is maintained; if the antibiotics do not exist in the added milk and dairy products or the concentration of antibiotics is below the detection limit, the spores germinate, metabolize and product acid, and the medium turns yellow.

Reagents and Materials Supplied

Materials required but not supplied

—- Stable 65°C heating equipment (water bath or dry heater)

—- Single channel micropipette (2.5-100µL)

—- Negative control: milk without antibiotics

—- Positive control: milk containing 10mg/L Penicillin G or 10mg/L Ampicillin

Assay Procedure

1. Carefully take the required number of microwells, do not broke the cover film of the remaining microwells. Leave the microwells at room temperature for 15 minutes and store the remaining microwells at 4-8°C immediately.
2. Uncover the cover film of the microwells, then add 50μL of the tested sample to each microwell (Note: do not mix by pipetting). Negative control and positive control are set up at the same time to ensure the accuracy of the kit.
3. Seal the microwells with cover film and place them vertically in a 65°C water bath or dry heater for incubation. Stop culture when the negative control turns yellow (approximately 2h15min to 2h45min).Specific culture time refers to the optimal culture time shown on the outer box.

Note: If precise sensitivity is required, negative control can be used to determine the exact culture time. The specific operation is: incubate until 2h15min to start observing the color of the negative control. It is recommended to observe it every 5 minutes. When the negative control turns yellow, read the results of all other samples immediately.

4. After the reaction, observe the color change from the side of the microwells and determine the result:

Negative result: bright yellow or all yellow

Positive result: purple

Detection limit results: The overall range is between yellow and purple or it does not change color (stay purple), indicating that the antibiotic concentration is within the detection limit range of the kit and should be determined as positive.

Interpretation of Results

Bright yellow         Yellow-purple         Purple

Negative (-)         Detection limit (+)       Positive (++)

Precautions

1. It is recommended to use milk without antibiotics as negative control and milk containing high concentrations of antibiotics, such as 10mg/L Penicillin G or Ampicillin milk as positive control to determine the accuracy of the kit.
2. This product is a detachable microwell plate. When taking the required microwells, first cut the aluminum foil (cover film) along the dividing line with a knife. Do not broke the remaining cover film to prevent contamination and dry cracking of the culture medium. Then the remaining microwell plates should be stored at 4-8°C immediately.
3. This product cannot directly detect acidic substances such as yogurt.
4. This kit is sensitive to antibiotics and other bacteriostatic substances (such as detergents, disinfectants, preservatives, etc.). Avoid contamination with such substances as much as possible during the experiment. Wash and dry hands thoroughly before starting the test, and test on a clean surface.
5. Do not shake the microplate violently, otherwise the solid medium will be loosened, and the final color will be affected.
6. Microplates shall be stored vertically, in a dark place and at a constant temperature, preferably between 4-8°C and protected from freezing. Fluctuations in temperature may soften and loosen the medium. Too high storage temperature will shorten the shelf life.
7. The culture temperature should be strictly controlled at 65±0.5°C, too high or too low temperature and too much temperature fluctuation will affect the reaction time and sensitivity of detection.
8. This reagent may require different test time for different types of dairy products.
9. As the storage time of this kit goes on, the required culture time may gradually increase. Reagents near the end of shelf life need longer incubation time than those just produced, so it is recommended that when the reagents at the end of shelf life are used, the incubation time shall be subject to the time when the negative control turns yellow.
10. The used microplates can only be discarded after being sterilized by high pressure.