Mold Sourcing

Part of Antibiotics

Finding, isolating, and maintaining Penicillium chrysogenum for antibiotic production when pure laboratory cultures are unavailable.

Why This Matters

Penicillin production requires the right mold. Penicillium chrysogenum (formerly called P. notatum) is a blue-green mold found naturally in the environment — it is not rare or exotic. Alexander Fleming’s famous discovery happened because P. notatum contaminated one of his culture plates. This means the mold you need is likely already present in your environment.

However, not all Penicillium strains produce equal amounts of penicillin. Wild strains vary enormously in productivity. Some produce almost none. The NRRL 1951.B25 strain used in WWII industrial production produced 1,000 times more penicillin than Fleming’s original isolate — found on a moldy cantaloupe in Peoria, Illinois.

Your goal is to find, isolate, identify, test, and maintain the most productive strains available in your local environment. This is achievable with simple equipment and systematic methodology.

Identifying Penicillium

Visual Identification

Penicillium appears as:

• Color: Blue-green, grey-green, or turquoise — the classic “bread mold” color
• Texture: Powdery or velvety surface (the powdery appearance comes from spores)
• Growth pattern: Circular colonies spreading outward from a center point
• Back of colony: Typically white, cream, or pale yellow when viewed from below

Common confusion organisms:

• Aspergillus: Also blue-green but colonies tend to be darker, more olive; avoid — some species are toxic
• Trichoderma: Bright green, typically faster-growing; not used for penicillin
• Cladosporium: Dark green to black; avoid
• Mucor: White, fluffy, grows very fast (often 5–10 cm in 24 hours); not Penicillium

If you have access to a magnifying glass or basic microscope, Penicillium shows characteristic brush-like structures (penicilli) when spores are examined — hence the name. The spore chains emerge from branching stalks resembling a small paintbrush.

Common Locations for Wild Penicillium

High-probability locations:

• Citrus fruits (oranges, lemons) that have begun to go moldy — blue-green mold on citrus is frequently Penicillium digitatum or P. italicum (relatives with some penicillin production)
• Bread and grain products left in cool, damp locations
• Aged cheeses — Brie, Camembert, Roquefort all use Penicillium; taking a scraping from commercial cheese is a legitimate starting point
• Damp soil beneath leaf litter in temperate forests
• Old wooden structures in shaded, damp conditions

Season and conditions: Penicillium thrives in cool (15–25°C) and moderately damp conditions. Spring and autumn in temperate climates are best for finding environmental isolates.

Isolation Procedure

From Environmental Sample

1. Prepare isolation plates: Pour thin layer of cooled sterile nutrient broth into flat plates or shallow containers; allow to solidify if agar is available, or use liquid broth for surface growth

2. Sample collection: Using a sterile implement (flamed metal probe, cooled before touching), take a small amount of mold material from your source

3. Streak or inoculate: Touch the probe to the center of your prepared medium and allow the spores to settle naturally if in liquid medium, or streak across solid medium

4. Incubate: 20–24°C for 5–7 days

5. Select: Identify Penicillium-looking colonies (blue-green, powdery). Multiple colonies will likely appear — each may be a different organism or different Penicillium strain

From Moldy Citrus

This is perhaps the most reliable environmental source:

1. Allow a citrus fruit to mold naturally in a cool, slightly damp environment
2. The blue-green mold that appears within 7–14 days is frequently Penicillium species
3. Take a spore sample with a sterile implement
4. Transfer to growth medium
5. Allow to grow 5 days at room temperature (20–24°C)
6. Assess colony appearance

Testing Strains for Penicillin Production

Once you have isolated Penicillium strains, you must test which produce usable amounts of penicillin:

Simple Agar Diffusion Test

1. Prepare plates with solid medium (boiled broth plus agar or gelatin cooled to gel)
2. Inoculate surface with a known pathogen — a throat swab or wound swab from a current infection works; alternatively, maintain a standard test organism like a wound infection culture
3. Incubate to produce a lawn of bacteria across the plate
4. Place a small piece of cloth soaked in each candidate Penicillium’s culture broth in the center
5. Re-incubate 24 hours
6. Measure the clear zone around each cloth piece

Strains producing larger clear zones are producing more penicillin. Select these for continued cultivation.

Cross-Contamination Prevention

When working with multiple strains:

• Label all containers clearly
• Work with only one strain at a time before cleaning surfaces
• Keep strains physically separated during incubation

Maintaining a Mold Bank

Once you have productive strains, protect them:

Short-Term Storage (Weeks)

Transfer mold culture to fresh medium every 3–4 weeks. Label with strain ID, transfer number, and date. Keep at 4°C (cold room, cellar, or refrigerator) to slow metabolism.

Long-Term Storage (Months to Years)

Soil-based storage:

1. Mix healthy mold growth with sterile dry soil or sand
2. Dry slowly at room temperature
3. Store in sealed container at cool temperature
4. Revive by adding small amount to fresh growth medium

Silica gel storage:

1. Grow mold on agar slope in sealed container
2. Add sterile silica gel granules to desiccate the surface
3. Seal tightly; store at 4°C
4. Viable for 1–2 years

Freeze-drying (if conditions allow): Mix mold spores with skim milk or sucrose solution; freeze; allow to desiccate in cold, dry conditions. Seal; store frozen. Viable for many years.

Protecting Your Best Strains

Maintain at least two physically separate stocks of each productive strain. A production failure should never risk losing your entire culture collection. Keep one stock as primary production; keep one as archive that is only opened to revive from catastrophic loss.

Document every strain: source location and date, productivity assessment (inhibition zone measurement), any observed characteristics. This record becomes foundational to your community’s pharmaceutical capability.