Mycology 102: Mastering Sterile Technique & Culture Work

Building on the fundamentals from Mycology 101, this guide takes a deep dive into the critical skills that separate successful cultivators from those constantly battling contamination. Master these techniques and you'll have the foundation for consistently clean cultures and reliable results.

Why Sterile Technique Matters

Contamination is the number one cause of failed grows. Mold spores and bacteria are everywhere—in the air you breathe, on your skin, and on every surface in your home. A single spore landing on your agar plate can outcompete mushroom mycelium within days.

The good news? With proper technique and equipment, you can create conditions clean enough for successful culture work. It's not about achieving hospital-grade sterility—it's about reducing contamination pressure to manageable levels.

The 80/20 Rule

About 80% of contamination comes from just a few sources: poor air quality during transfers, inadequate substrate sterilization, and contaminated source material. Focus on these areas first.

Building Your Clean Workspace

You have two main options for creating a clean work environment: a Still Air Box (SAB) or a Laminar Flow Hood. Each has its place in a cultivator's toolkit.

Still Air Box (SAB)

A SAB is simply a clear plastic tub with arm holes cut into the sides. Air movement carries contaminants, so by working in still air, you dramatically reduce exposure.

Advantages:

Low cost (under $30)
No electricity required
Portable and easy to store
Surprisingly effective when used correctly

Limitations:

Arm holes create air disturbance
Limited working time before air quality degrades
Requires careful technique
Not suitable for large-scale work

Build Your SAB

Use a 60-70 quart clear plastic tub. Cut two 4-5 inch holes on one long side, positioned shoulder-width apart. Flame-polish the edges to remove sharp plastic.

Prepare the Workspace

Wipe down the inside with 70% isopropyl alcohol. Let it evaporate completely—alcohol vapor is flammable. Place your tools and materials inside before starting work.

Settle the Air

After placing your arms through the holes, wait 30-60 seconds for air currents to settle before opening any cultures.

Work Methodically

Move slowly and deliberately. Rapid movements create air turbulence. Keep open containers pointed away from you—you're the biggest source of contamination.

Laminar Flow Hood

A flow hood pushes HEPA-filtered air across your work surface, creating a clean zone. Air flows in one direction (usually horizontal toward you), carrying any contaminants away from your work.

Advantages:

Consistent clean air supply
Longer working sessions possible
Faster workflow (no settling time)
Essential for commercial operations

Limitations:

Significant investment ($300-1500+)
Requires electricity
Filter replacement costs
Takes up permanent space

DIY vs Commercial

Building your own flow hood can save money, but requires careful attention to CFM (cubic feet per minute) airflow and filter sealing. A poorly built hood is worse than a well-used SAB.

Which Should You Choose?

Situation	Recommendation
Just starting out	SAB
Budget under $100	SAB
Fewer than 20 transfers/month	SAB
Commercial or scaling up	Flow Hood
High contamination environment	Flow Hood
Working with valuable genetics	Flow Hood

Agar: Your First Line of Defense

Agar plates are the cornerstone of culture work. Working on agar lets you verify culture health, isolate clean tissue, store genetics, and catch contamination before it spreads to expensive grain spawn.

Understanding Agar Media

Agar is a gelatinous substance derived from seaweed. By adding nutrients and pouring it into petri dishes, you create a surface where mycelium can grow. Different recipes favor different growth characteristics.

Media Type	Full Name	Best For	Notes
MEA	Malt Extract Agar	General purpose	Good balance of growth speed and visibility
PDA	Potato Dextrose Agar	Most species	Widely available, reliable results
MYPA	Malt Yeast Peptone Agar	Fast colonization	Rich nutrients accelerate growth
DFA	Dog Food Agar	Aggressive species	High nitrogen for vigorous growers
WA	Water Agar	Germination	Low nutrient forces rhizomorphic growth

Basic MEA Recipe

This reliable recipe works for most common species:

500ml distilled water
10g light malt extract (LME)
10g agar powder
Optional: 1g nutritional yeast for faster growth

Mix Ingredients

Combine water and malt extract in a heat-resistant container. Stir to dissolve. Add agar powder and stir again—it won't fully dissolve until heated.

Sterilize

Pressure cook at 15 PSI for 20 minutes. Use a jar with a modified lid (micropore tape over a hole) or cover with foil.

Cool and Pour

Let the jar cool until you can comfortably hold it (around 120-140°F). Work in front of your flow hood or inside your SAB. Pour approximately 15-20ml per plate, just enough to cover the bottom.

Let Plates Set

Leave plates stacked with lids on until the agar solidifies (30-60 minutes). Store upside down to prevent condensation from dripping onto the agar surface.

Common Mistake

Don't pour plates while the agar is too hot—excess condensation promotes bacterial growth. If plates are dripping wet inside, your agar was too hot when poured.

Tissue Cloning: Capturing Genetics

Tissue cloning lets you capture the genetics of a mushroom you want to reproduce. Found a particularly large fruit, interesting mutation, or early fruiter? Clone it.

The Cloning Process

The goal is to transfer a small piece of interior tissue from a mushroom to an agar plate, where it will grow out into mycelium.

Select Your Specimen

Choose a healthy, mature (but not old) mushroom. Look for characteristics you want to preserve—size, shape, early fruiting, or disease resistance.

Prepare Your Workspace

Flame-sterilize your scalpel. Have your agar plates ready. Work in your SAB or in front of your flow hood.

Split the Mushroom

Tear the mushroom in half lengthwise with clean hands. The interior tissue is sterile—you're exposing it for sampling.

Harvest Tissue

Using your sterile scalpel, cut a small piece (2-3mm) from the interior tissue where the cap meets the stem. This junction often has vigorous mycelium.

Transfer to Agar

Place the tissue piece in the center of your agar plate. Close the lid immediately. Label with strain, date, and source.

Expect to see mycelium growing out from the tissue within 3-7 days. If you see colored growth (green, black, orange) before white mycelium appears, the clone is contaminated.

Isolation Transfers

Clones often carry contamination—not because you did something wrong, but because no mushroom is truly sterile throughout. The solution is isolation transfers.

When mycelium grows out from your clone, you'll often see the leading edge running ahead of any contamination. By transferring a piece from this clean edge to a fresh plate, you can "outrun" the contaminants.

Repeat this process 2-3 times until you have plates that grow out completely clean. These isolated cultures become your master stock.

Liquid Culture: Scaling Your Inoculum

Liquid culture (LC) is mycelium grown in nutrient-rich broth. It allows you to rapidly multiply your inoculum and inoculate grain spawn much faster than agar-to-grain transfers.

Why Use Liquid Culture?

Speed: LC can colonize grain in 7-10 days vs 14-21 days from agar
Volume: One LC jar can inoculate dozens of grain bags
Verification: Healthy mycelium is visible in the liquid
Storage: Refrigerated LC remains viable for months

Simple LC Recipe

500ml distilled water
20g light malt extract (4% solution)

That's it. Some cultivators add honey or corn syrup, but simple malt extract works reliably.

Prepare the Jar

Use a quart jar with a modified lid—drill a hole and cover with a self-healing injection port and micropore tape for gas exchange.

Mix and Sterilize

Dissolve malt extract in water. Pour into jar, leaving headspace. Pressure cook at 15 PSI for 20 minutes.

Inoculate

Once cooled, inoculate with a small piece of agar culture or a few drops of existing LC. Work in your clean workspace.

Incubate with Agitation

Place on a magnetic stir plate or shake vigorously once daily. Agitation breaks up mycelium and encourages growth.

Reading LC Health

Healthy liquid culture should be:

Clear broth with white mycelial clouds
Wispy appearance—mycelium forms delicate strands
Sweet smell (or no smell) when opened

Discard If You See

Cloudy or milky broth (bacterial contamination)
Sour or foul smell
Colored blobs (green, black, yellow)
Slimy or thick consistency

Long-Term Culture Storage

Clean cultures represent significant investment. Protect that investment with proper storage.

Refrigerated Agar

Fully colonized agar plates can be stored in the refrigerator for 6-12 months. Wrap plates in parafilm to prevent drying and contamination. When ready to use, transfer to fresh plates and allow a few days for the culture to "wake up."

Agar Slants

Slants are test tubes with agar poured at an angle, creating more surface area. They're more compact than plates and can store for 1-2 years refrigerated.

Refrigerated LC

Liquid culture stores well at refrigerator temperatures (35-40°F). Before use, allow it to return to room temperature and confirm health by looking for new wispy growth after a few days.

Long-Term: Cryopreservation

For truly long-term storage, cultures can be frozen in glycerol solution at -80°C. This is typically only available to commercial operations or those with access to laboratory freezers.

Putting It All Together: A Sample Workflow

Here's how these techniques fit into a practical cultivation workflow:

Acquire genetics via spore syringe, tissue clone, or culture trade
Grow out on agar to verify health and isolate if needed
Transfer to LC for rapid multiplication
Inoculate grain spawn from LC
Store backup cultures on agar slants in the fridge

When starting a new strain, log each culture plate and LC jar as a separate item. Note the source material, transfer dates, and any observations about growth characteristics. This lineage tracking becomes invaluable when you need to trace back a contamination issue or identify your best-performing genetics.

Next Steps

With solid sterile technique and culture work skills, you're ready to move on to Mycology 103: Substrate Science & Spawn Production, where we'll dive deep into grain preparation and bulk substrate formulation.

Quick Reference: Sterile Technique Checklist

Before every transfer session:

Clean work surface with 70% isopropyl alcohol
Flame-sterilize tools until red hot
Let alcohol evaporate before using flame
Move slowly and deliberately
Keep open containers angled away from you
Work from cleanest to least clean items
Label everything immediately after transfer
Log new cultures with source and date