Purification Methods for Contaminated Microbial Strains

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News 8 5 月, 2025

During the isolation, preservation, and production of microbial strains, contamination with unwanted microorganisms is common. Therefore, purifying contaminated strains is essential before they can be used in production. Depending on the type and severity of contamination, different purification methods are employed.

1. Removing Bacterial or Yeast Contamination

Contaminated cultures often show sticky colonies on the medium surface. To purify:

Transfer a sample to a slanted agar medium with higher hardness (2.3–2.5% agar) and no condensation.
Lower the incubation temperature to 15–20°C; at lower temperatures, fungal mycelium grows faster than bacteria.
Use a fine inoculation needle to cut the leading edge of the fungal mycelium and transfer it to a fresh slant medium.
Repeat 2–3 times to obtain pure mycelium. Alternatively, break the tube and extract an internal agar block with internal mycelium to transfer onto fresh medium—suitable for cultures contaminated by aerobic bacteria.

2. Removing Mold Contamination

Since molds resemble fungal mycelium, purification focuses on inhibiting mold and promoting the growth difference:

Cut the leading edge of the target fungal colony for transfer to new medium.
Early detection improves purification success.
White mycelium growing away from the inoculation site should be treated as contamination and purified immediately.
If spores have formed (colored), it may be too late as spores spread easily; at early spore formation, edge cutting is still feasible.
For well-established molds:
- Cover the mold colony with sterile filter paper soaked in 0.2% mercuric chloride or 1% carbendazim to inhibit growth and spore spread.
- Scrape off the surface, then hook internal mycelium with a sterile needle for transfer. Repeat 2–3 times if needed.

3. Ring Isolation Method

Place a sterilized glass or metal ring (7–10 mm diameter, 4–6 mm height) onto the center of an agar slant, embedding half of the ring into the medium.
Inoculate contaminated material inside the ring.
Bacteria are restricted inside the ring, while fungal mycelium can grow beyond the ring onto clean medium—transfer this mycelium for purification.

4. Overlay Culture Method

Pour a 2 mm thick layer of fresh medium over the contaminated slant.
After incubation, once fungal mycelium grows through the overlay to form new colonies, cut and transfer it.
A second overlay may improve purification.

5. Substrate Mycelium Purification

For tubes contaminated at the cotton plug:

Break the tube, remove the medium.
Soak in 0.1% mercuric chloride for 2 minutes, rinse with sterile water, dry with sterile filter paper.
Cut 2 cm pieces of medium, slice into small cubes from the middle.
Transfer small cubes onto fresh slants for culture.

6. Chemical Treatment

Add selective antimicrobial agents to the medium for purification:

5–10 mg/L benomyl, thiabendazole, or topsin → prevents fungal mold contamination.
30–40 mg streptomycin, 20–30 mg tetracycline, chloramphenicol, or 50 mg potassium permanganate per mL medium → prevents bacterial contamination.
20 units/mL griseofulvin → inhibits fungal growth.

7. Mycelial Fragmentation Method

Remove contaminated medium, soak in 0.1% mercuric chloride for 2 minutes, rinse, dry.
Place in sterile water with glass beads.
Mechanically disrupt tissue, dilute, plate onto agar, and isolate single colonies for pure culture.