The antibiotic potency tester is the core equipment for microbial detection of antibiotic activity, and its operational accuracy directly affects the reliability of the detection results. The following is a systematic summary of practical techniques from four aspects: operation process, key skills, daily maintenance, and fault handling, to help improve detection efficiency and data accuracy.
1、 Standardized operating procedures and techniques
Sample preparation and dilution
skill:
Antibiotic standards should be stored by freeze-drying, and special solvents should be used for reconstitution to avoid the use of physiological saline which may cause potency attenuation.
The dilution gradient design should cover 50% -200% of the expected potency. For example, when detecting a 100U/mL sample, three gradients of 50, 100, and 200U/mL should be set to ensure that the linear regression R ² is ≥ 0.99.
Avoiding pitfalls:
It is strictly prohibited to use metal vessels in contact with the sample to prevent catalytic degradation of metal ions.
The diluted sample should be sampled within 30 minutes to avoid loss of potency due to prolonged exposure.
Preparation and sterilization of culture medium
skill:
Select chromatography grade agar to ensure uniform thickness of the bacterial layer.
After high-pressure sterilization, it is necessary to cool to 50 ℃ before pouring the plate to prevent the high temperature from killing the indicator bacteria.
Avoiding pitfalls:
Avoid using water containing calcium/magnesium ions to prepare the culture medium and prevent chelation with antibiotics from affecting activity.
After sterilization, the pH of the culture medium needs to be adjusted to 6.8-7.2. If it exceeds this range, it needs to be reconfigured.
Sampling and incubation
skill:
Use a ceramic sampler with a sampling volume controlled at 0.2mL/hole to avoid edge effects caused by liquid diffusion.
The incubation conditions should be strictly controlled at 35-37 ℃ and humidity ≥ 70%. Saturated sodium chloride solution can be used to maintain humidity.
Avoiding pitfalls:
After sampling, it is necessary to let it stand for 15 minutes for the liquid to absorb, and then transfer it to the incubator to prevent the sample from flowing.
During the incubation period, it is prohibited to open the box for inspection to avoid temperature fluctuations exceeding ± 0.5 ℃.
Measurement and Data Analysis of Antibacterial Zone
skill:
Adopting an automatic image analysis system and setting threshold parameters to improve edge recognition accuracy.
Data processing requires removing outliers and retesting.
Avoiding pitfalls:
Avoid measuring under strong light to prevent reflected light from interfering with image recognition.
When measuring manually, a vernier caliper should be used, and the average value should be taken by repeating the measurement three times.
2、 Key maintenance techniques
Daily cleaning and disinfection
Operation:
Wipe the sampling table and incubator walls with 75% ethanol after daily experiments to prevent cross contamination.
Dismantle the sampling needle every week and clean it with ultrasound for 10 minutes to remove protein residue.
Avoiding pitfalls:
It is strictly prohibited to use chlorine containing disinfectants to prevent corrosion of metal parts.
After cleaning, rinse with pure water three times to avoid residual ethanol affecting subsequent experiments.
Optical system maintenance
Operation:
Wipe the camera lens with a dust-free cloth every month and use compressed air to blow away sensor dust.
Calibrate the light source intensity quarterly to ensure that the image contrast is ≥ 90%.
Avoiding pitfalls:
Avoid direct contact with the lens to prevent fingerprint contamination.
Calibration should be carried out in a dark room to prevent interference from ambient light.
Mechanical component maintenance
Operation:
Lubricate the sampler guide rail every quarter to reduce motion resistance.
Replace the sealing strip of the incubator every year to prevent temperature fluctuations caused by heat leakage.
Avoiding pitfalls:
Do not use engine oil for lubrication to prevent contamination of the culture medium.
After replacing the sealing strip, a 24-hour no-load operation test is required to confirm temperature stability.
3、 Common faults and solutions
| Fault phenomenon |
Possible reasons |
Solution |
| The edge of the antibacterial zone is blurred |
Uneven thickness of culture medium |
Invert the board and calibrate the console using a spirit level |
| Unstable sample size |
Sample needle blockage |
Ultrasonic cleaning needle, replace 0.45 μ m filter membrane |
| Incubation temperature fluctuation>± 1 ℃ |
Temperature control sensor malfunction |
Replace NTC thermistor (accuracy ± 0.1 ℃) and calibrate PID parameters |
| Image recognition error rate>5% |
Light source attenuation |
Replace the LED light board (with a lifespan of ≥ 50000 hours) and adjust the camera focal length |
| Experimental repeatability CV>10% |
Sample dilution error |
Use a pipette calibration kit (accuracy ± 0.5%) to standardize the operating procedures |
4、 Key points of performance verification and quality control
System Applicability Testing
Before each batch of experiments, standard samples need to be run, with a requirement of inhibition zone diameter CV ≤ 5% and linear regression R ² ≥ 0.98.
Mid precision validation is conducted monthly, with 5 repeated tests completed by different operators within 3 days, and the RSD of the results is ≤ 8%.
Data traceability management
The original image needs to be saved in TIFF format and associated with experimental records.
Automatically generate potency reports using the LIMS system to avoid manual input errors.
Regulatory compliance
Meets the requirements of General Rule 1431 of the 2020 edition of the Chinese Pharmacopoeia, and regularly undergoes calibration by the metrology department.
Keep all quality control records for at least 5 years for future reference.
5、 Advanced Optimization Techniques
Automation upgrade
Connect the robot sampling system to shorten the sampling time from 15 minutes per board to 3 minutes, reducing human error.
Using AI image recognition algorithms to automatically remove abnormal antibacterial zones and improve data processing efficiency.
environmental control
Install CO ₂ sensors in the incubator to simulate the optimal growth conditions for microorganisms.
Use a dust-free laboratory to reduce the risk of contamination.
consumables management
Select pre sterilized culture medium plates to reduce errors in on-site preparation and use.
Use disposable sampling needles to avoid cross contamination.