Methods for Purifying Contaminated Microbial Strains Under Different Conditions

News 11 9 月, 2024

Microbial strains are prone to contamination by unwanted bacteria, yeast, or molds during isolation, preservation, and production processes. It is crucial to purify these contaminated strains to ensure their suitability for production. Depending on the type and degree of contamination, different purification methods must be employed.

1. Eliminating Bacterial or Yeast Contamination

In microbial cultures, bacterial or yeast contamination often appears as sticky colonies on the surface of the culture medium. To purify, transfer the contaminated culture onto a slant medium with a high agar concentration (2.3%–2.5%) without condensation water, and lower the incubation temperature to 15–20°C. At these lower temperatures, the growth rate of fungal mycelium often exceeds that of bacteria. Using a fine inoculation needle, cut the leading edge of the fungal mycelium and transfer it to a new slant medium. Repeating this process 2-3 times usually yields a pure fungal strain.

Another method involves breaking the test tube and picking the internal agar block with base mycelium, transferring it to a new medium without condensation water. This technique is effective for aerobic bacterial contamination.

2. Eliminating Mold Contamination

Molds, similar to edible fungal mycelium, possess both aerial and substrate mycelia. The primary approach to eliminating mold contamination involves suppressing mold growth while promoting the edible fungal mycelium. Cutting the leading edge of the edible fungal colony and transferring it to new media increases the success rate of separation, especially when detected early. Mold contamination, indicated by white mycelium at non-inoculated sections of slant media, should be immediately purified.

If colored spores have already formed, they may spread easily, mingling with edible fungal mycelium. For immature, uncolored spores, the leading-edge mycelium cutting method may still be effective. If the mold colony is already dark, spore spread is inevitable. A piece of sterile filter paper treated with 0.2% mercuric chloride or 1% carbendazim can suppress mold growth, preventing spore spread. The surface is then scraped off, and the base mycelium is transferred to new media for further purification.

3. Restricted Culture Method

Place a 7–10 mm diameter and 4–6 mm high glass or stainless-steel ring, sterilized with an alcohol burner flame, onto the center of the slant medium while still hot, embedding half of the ring into the medium. Place the contaminated inoculum inside the ring. This restricts bacterial growth within the ring, while edible fungal mycelium can grow beyond the ring onto the surrounding medium, allowing for the isolation of purified mycelium after transfer.

4. Overlay Culture

Pour a 2 mm layer of medium over contaminated fungal mycelium on slant media. After some time, edible fungal mycelium will grow through the new medium layer, forming a new colony, which can then be cut and transferred. For best results, perform a second overlay.

5. Substrate Mycelium Purification

For slant media contaminated with mold, break the test tube, retrieve the medium, and immerse it in 0.1% mercuric chloride for 2 minutes. Rinse with sterile water, absorb excess moisture with sterile filter paper, and cut the medium into rice-sized blocks with a sterile blade. Transfer these blocks to new slant media for cultivation.

6. Antimicrobial Treatment

To purify strains, add selective antimicrobial agents to the medium. For instance, incorporating 5–10 mg/L of fungicides like carbendazim (MBC), thiabendazole (TBZ), or benomyl can prevent mold contamination. Adding 30–40 mg of streptomycin, 20–30 mg of tetracycline or aureomycin, or 50 mg of potassium permanganate per milliliter of medium effectively prevents bacterial contamination. Griseofulvin (20 units/mL) can inhibit fungal growth.

7. Fragmenting Mycelium

Remove contaminated medium from the test tube and immerse it in 0.1% mercuric chloride solution for 2 minutes. Rinse with sterile water, dry with sterile paper, and place in sterile water containing glass beads. Fragment the mycelium by agitation, dilute, and inoculate onto agar plates. Isolate single colonies for purification.