Culture medium is a substance in liquid, semi-solid or solid form, containing natural or synthetic components, used to ensure the reproduction of microorganisms or to maintain their vitality. The preparation and use of the culture medium is an important link in the testing work of the microbiological laboratory. The quality of the culture medium itself, whether it is properly stored, and whether it is prepared and used will directly affect the accuracy of the test results. This article discusses the quality control measures that microbiology laboratories should take in the purchase, storage, preparation, and use of culture media, and talks about some opinions and opinions. Hope to help colleagues in microbiological testing work better.

1. Purchase and acceptance of medium

1.1 Purchase

From the perspective of the quality of the medium itself, there will be differences between products from different manufacturers, and even products from different batches of the same manufacturer[1]. According to ISO/IEC17025 "General Requirements for the Capability of Testing and Calibration Laboratories", it is an important step in the medium purchase process to select qualified suppliers after evaluating the medium supplier. Enterprises with high product market reputation, quality assurance capabilities and service assurance capabilities should be selected; whether the enterprise has passed the certification of the quality management system is a more objective evaluation index. Manufacturers are required to provide: medium component name and product number, batch number, pH before use, storage information and expiration date, performance evaluation and test strains used, technical data list, quality control certificate, and necessary safety/hazard data and other materials.

1.2 Acceptance

After the purchased culture medium arrives, special personnel should do a good job of acceptance and inspection, and carefully check whether the information provided by the manufacturer, culture medium name, specification quantity, appearance characteristics, batch number, and shelf life meet the requirements. The quality of each batch of medium should be tested after it arrives at the goods, and it can be put into use only after meeting the requirements specified in the test method.

2. Storage of medium

Dry powder medium should be stored in a cool and dry place, avoiding direct sunlight; the unopened medium can be stored at room temperature for up to 2 years, and the dry powder medium after opening the bottle is easy to absorb moisture, so it should be protected from moisture and stored at 6 Use within one month. Routine inspections of media in storage should be performed regularly, such as rechecking container tightness, date of first opening, sensory inspection of contents, and media should not be used if clumping, abnormal color and other signs of deterioration occur.

3. Preparation of medium

3.1 Medium water

The water for medium preparation should use distilled water with a resistivity of not less than 300,000Ω cm or water of the same quality, and it is best not to use deionized water produced by an ion exchanger.

3.2 Weighing

When weighing, use a special angle spoon to avoid cross-contamination and affect the test results; dry powder medium is easy to absorb moisture, if possible, try to weigh the medium in a room with low humidity.

3.3 Rehydration

Do not use copper or iron pots when rehydrating, to prevent ions from mixing into the medium and affecting the growth of microorganisms; the size of the container should be more than twice the total volume of the medium after rehydration, and avoid heating and dissolving Medium, the medium overflows; the medium containing agar powder can be soaked for several minutes before heating to dissolve; be sure to stir when heating the medium, especially the agar medium. In order to avoid scorching and boiling over, it is best to use a boiling water bath for heating a small amount of medium. The nutrients of the burnt medium are destroyed and may produce toxic substances. If it is found to be coked, or the medium overflows before it is evenly dissolved, the medium cannot be used and should be prepared again. When re-melting agar medium, use a boiling water bath or flowing steam to heat it, and it can only be heated twice at most, and the unused medium after the second re-melting should be discarded.

3.4 Sterilization

The general medium is sterilized by moist heat, that is, high-pressure steam sterilization, usually at 121°C for 15 minutes, and sugar-containing or special medium is sterilized according to the national standard or the instructions provided by the manufacturer. The prepared medium must be sterilized immediately to avoid the consumption of nutrients due to the growth and reproduction of microorganisms and change the pH of the medium. High temperature can destroy the nutrient content of the medium and also reduce the gel strength of the agar, so the sterilization temperature and time must be strictly controlled, and the sterilization cannot be repeated. The pressure gauge of the high-pressure steam sterilizer should be calibrated regularly, and the sterilization effect should be verified by the biological indicator bacteria method or chemical discoloration paper regularly.

3.5 pH measurement

Microorganisms must be in a suitable pH range to grow and reproduce normally or to reflect their biological characteristics. The pH required by the medium formula is the pH value after sterilization or disinfection, that is, the pH before use of the medium, and should be measured with a precision acidity meter at a temperature of 25°C. The solid agar medium should be measured with a special colloidal surface electrode Take measurements. During use, if the pH of the culture medium needs to be adjusted, it should be adjusted with 1mol/L NaOH or 1mol/LHCL. Be careful not to adjust the pH repeatedly, otherwise the osmotic pressure of the culture medium will be affected.

3.6 Preservation of the prepared medium

After sterilization, the culture medium should be cooled to the required temperature quickly to avoid long-term storage in the sterilizer, which will cause excessive sterilization and affect the nutritional content or selective effect of the culture medium. The amount of the prepared medium should be just used up within the longest storage period. The dye-containing medium should be stored in closed light; if the poured agar plate is not used up on the day of preparation, it should be stored in cold storage. If the storage time exceeds 2 days, it should be stored in a sealed plastic bag; Broth media if stored for longer than 2 weeks should be placed in a screw cap test tube or other airtight test tube or container to prevent evaporation.

4. Performance test of medium

4.1 Appearance Control

The prepared medium should have a corresponding color, and the general medium should be clear, without turbidity, and without precipitation. The medium should be checked for color change and evaporation/dehydration before use.

4.2 Pollution Control

Select a part of each batch of prepared medium for contamination test (sterility test) to confirm that there is no microbial growth before use.

4.3 Performance test

4.3.1 Selection of test strains The test strains are a set of strains that have the stable characteristics of their representative species and can effectively prove the best performance of the specific medium in the laboratory. They should come from the ATCC standard strains of the International/National Standard Culture Collection Center. The selection of bacterial strains refers to the health industry standard WS/T232-2002 "Quality Inspection Regulations for Commercial Microbial Culture Media".

4.3.2 Quantitative test method Improved Miles-Misra method The overnight culture of the test strain was diluted 10 times; the test plate and the reference plate were divided into 4 areas and marked; starting from the highest dilution, drop a drop of dilution solution on the The marked area of the test plate and the control plate; apply the diluent to the entire 1/4 area, and incubate at 37°C for 18 hours; count the easy-to-count areas, and calculate the growth rate according to the formula (growth rate = on the medium plate to be tested) The total number of colonies obtained/the total number of colonies obtained on the reference medium plate). The growth rate of the target bacteria on the non-selective medium should not be lower than 0.7, and this type of medium should be easy for the growth of the target bacteria; the growth rate of the target bacteria on the selective medium should not be lower than 0.1.

4.3.3 Semi-quantitative test method Improved streaking method Inoculation method The plate is divided into four areas, ABCD, and a total of 16 lines are drawn. The parallel lines are about 0.5cm apart. Only half of the line with colony growth was recorded as 0.5 points, no colony growth or the growth amount was less than half of the line was recorded as 0 points, and the scores were added up to obtain the growth index G. The target bacteria should show typical growth on the medium, while the growth of non-target bacteria should be partially or completely inhibited. When the growth index G of the target bacteria is greater than 6, the medium is acceptable.

4.3.4 Qualitative test method Plate inoculation observation method Take the test bacteria culture with an inoculation loop, and draw a parallel line on the surface of the test medium. Cultivate the inoculated plate according to the culture time and temperature specified in the standard. The target bacteria should grow well and have typical colony appearance, size and shape, and the non-target bacteria should grow weakly or have no growth.

5. Discussion

The quality of culture medium is the basis of microbiology laboratory testing work, which is related to the accuracy and reliability of testing work, and plays an important role in microbiology laboratory testing work. If quality problems in any link are ignored in the work, it will lead to the deviation of the scientific and objective test results. Therefore, only by strengthening the laboratory quality control of the culture medium, earnestly doing a good job in the quality control of the culture medium, and constantly improving and improving the quality control level of the culture medium can we provide high-quality culture medium for the microbiology testing laboratory.