Culture media serve as the essential nutrient substrate for microorganisms, animal cells, or plant cells to grow, proliferate, or accumulate metabolites during fermentation processes or large-scale cultivation of animal/plant cells, thereby enabling the synthesis of biochemical products. The preparation and use of culture media constitute a critical step in the testing work of microbiology laboratories.
- Procurement and Acceptance of Culture Media
1.1 Procurement
All chemicals, reagents, and powdered culture media used for microbiological testing must be products manufactured by professional manufacturers, with quality complying with relevant quality standards. Additionally, the manufacturer shall provide information such as the culture medium’s name, product code, batch number, various components and any supplementary ingredients, pH value before use, storage information, and expiration date. This facilitates users in recording and comparing products.
When selecting suppliers, priority should be given to enterprises with a high market reputation, reliable quality assurance capabilities, and sound service support. Whether an enterprise has obtained quality management system certification is a relatively objective evaluation indicator.
1.2 Acceptance
Upon the arrival of purchased culture media, designated personnel shall be responsible for receiving and acceptance. They must carefully verify whether the information provided by the manufacturer, along with the culture medium’s name, specification, quantity, appearance characteristics, batch number, and shelf life, meets the requirements.
Quality testing shall be conducted for each batch of culture media upon arrival. Only after meeting the requirements specified in the testing methods can the culture media be put into use.
- Storage of Culture Media
Powdered culture media should be stored in a cool, dry place, protected from direct sunlight. The shelf life of unopened culture media shall be followed in accordance with the information provided by the manufacturer. Once opened, powdered culture media are prone to moisture absorption; therefore, precautions against moisture should be taken, and the media should be used as soon as possible.
Routine inspections of stored culture media shall be carried out regularly, including rechecking the airtightness of containers, recording the opening date, and conducting sensory inspections of the contents. If the culture media show signs of caking, abnormal color, or other deterioration, they shall no longer be used.
- Preparation of Culture Media
3.1 Water for Culture Medium Preparation
Distilled water with a resistivity of not less than 300,000 Ω·cm or water of equivalent quality shall be used for preparing culture media. It is advisable to avoid using deionized water produced by ion exchangers.
3.2 Weighing
Dedicated spatulas must be used for weighing to prevent cross-contamination that may affect test results. Since powdered culture media are susceptible to moisture absorption, weighing should be conducted in a room with low humidity if conditions permit.
Powdered culture media shall be accurately prepared in strict accordance with the relevant instructions provided by the manufacturer.
3.3 Rehydration
Copper or iron pots shall not be used for rehydration to prevent ion contamination of the culture medium, which may interfere with microbial growth. The volume of the container used must be more than twice the total volume of the rehydrated culture medium to avoid overflow during heating and dissolution.
For culture media containing agar powder, soaking for several minutes before heating and dissolution is recommended. When heating the culture medium, stirring is mandatory—especially for agar-based media. To avoid scorching and boiling overflow, a boiling water bath is preferred for heating small quantities of culture medium.
Scorched culture media have damaged nutrients and may produce toxic substances. If scorching is detected, or if the medium overflows before being uniformly dissolved, the medium shall be discarded and reprepared.
When remelting agar-based culture media, heating via a boiling water bath or flowing steam is required. Remelting shall not be performed more than twice; any unused medium after the second remelting shall be discarded.
3.4 Sterilization
Culture media shall be sterilized promptly under the conditions specified in their formulation—typically 121°C for 15 minutes—to ensure sterilization effectiveness while avoiding damage to the medium’s active components. For sugar-containing or special culture media, sterilization shall be carried out in accordance with national standards or the manufacturer’s instructions.
Prepared culture media must be sterilized immediately to prevent microbial growth and proliferation, which would consume nutrients and alter the medium’s pH. High temperatures can damage the medium’s nutrients and reduce the gel strength of agar; thus, sterilization temperature and time must be strictly controlled, and repeated sterilization is prohibited.
The pressure gauge of the autoclave shall be calibrated regularly. Additionally, the sterilization effect shall be verified periodically using biological indicator bacteria or chemical color-changing paper.
3.5 pH Measurement
Microorganisms can only grow, proliferate normally, or exhibit their biological characteristics within an appropriate pH range. The pH of the medium changes before and after sterilization, so adjustment should be performed before sterilization using 1 mol/L NaOH or 1 mol/L HCl. Note that repeated pH adjustment is not allowed, as it may affect the medium’s osmotic pressure.
3.6 Storage of Prepared Culture Media
After sterilization, the culture medium shall be cooled rapidly to the required temperature to avoid prolonged storage in the autoclave (which causes over-sterilization and impairs the medium’s nutrients or selectivity). Therefore, it is recommended that poured agar plates be used immediately.
For storage, the medium should be kept under conditions that do not alter its composition—i.e., protected from light or sealed and stored in a refrigerator at 4°C to 12°C. If stored for more than 2 days, the medium should be placed in a sealed plastic bag. For prepared broth media stored for more than 2 weeks, they must be kept in test tubes with screw caps or other airtight containers to prevent evaporation.