Growing Thoughts: On Propagating Liquid Cultures, Agar Plates, and Grain Spawn

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When I first started growing mushrooms, I tried to just use grain spawn to avoid working with agar plates or liquid cultures. The downside? Its primary contamination vector—the entire mason jar lid.

Unlike liquid cultures—which can transfer between sterilized containers via a small needle (from syringe to mason jar, for example)—grain spawn requires exposing the whole unit to sterile air under a flow hood or still air box. When opening a grain spawn jar, there's very little room for error in sterile technique.

Even with perfect flow hood conditions, contaminants can still hide in the gap between the lid and jar where it screws on. A thorough alcohol spray might miss this spot.

A pro tip here: For extra protection, you can seal this gap with parafilm, grafting tape, or medical tape. I really only take this precaution with liquid culture jars. If you skip this tip, just remember to unscrew the lid slowly and lift it straight up while in a sterile environment, avoiding contact with the jar's rim. Spray the lid down after removing if you want.

My approach evolved to embrace liquid cultures—focusing on liquid culture jars and occasional slurries (transferring agar plates to nutrient broth instead of propagating from existing liquid culture syringes). This worked well until I kept a culture for too long, resulting in contamination across several grain jars within a week.

It’s difficult to tell whether a liquid culture jar is contaminated, especially since I was using agave syrup, which has a brown coloring that hides some of the more apparent growths in a compromised jar.

Each process has their place in growing mushrooms, and I want to share how I approach each methodology and their role in my growing operation.

Using Grain Spawn Propagations

In early-November, I started Badwater Mushrooms back from dormancy, as it had been paused for my work in wildland firefighting during the summer.

The start would have been slow if I was more patient: Starting from scratch, I would have been using old liquid cultures I hadn’t looked at in 6 months, and nothing else. These cultures, having likely eaten all the nutrients in the unrefrigerated syringes I had, would likely be sad and sickly.

But I knew I wanted fruiting bodies by the end of the month, so I decided to get a head start with my usual shortcut: I bought Lion’s Mane grain spawn from North Spore. This is something I hadn’t done for about 2 years! It usually takes 10-15 days to propagate a liquid culture-to-fresh-grain jar, but I wanted to start the substrate fruiting process as soon as possible.

5 days after I ordered the grain spawn bag, I had 8 homemade substrate bags inoculated and propagating.

I also used a small portion of the North Spore grain spawn to mix into my own grain jars, so I would have a plethora of options by the end of the month. In about 2-3 weeks, I had several ready grain spawn jars to mix into my next round of substrate bags.

So let’s do some math: If I had started from my old liquid cultures, it would have taken at least 3-4 weeks to colonize my grain jars, and using them for my grow bags, another 3-4 weeks to arrive at fruiting bodies. 6-8 weeks.

I cut down my “scratch” time down to 3 weeks for fruiting bodies with North Spore’s grain bags.

A conclusion here: When you know you have good quality grain spawn on hand, propagate it with newly sterilized grains. You are turbocharging your grow operation by doing grain-to-grain.

Using Liquid Culture Propagations

The best time to use liquid culture is when you have already started your first round with the grain spawn booster like I did with North Spore, and now you have several weeks to multiply a syringe you might have into a broth jar full of an isolated culture.

It’s been a bit over 2 weeks since I propagated a psilocybin culture in 4 LC jars, and all of them are growing beautifully—to the point that I probably need to refrigerate them so they don’t eat through all the nutrient broth!

This DIY broth propagation will also boost the growing momentum of your culture as nutrient broth accelerates mycelial growth compared to agar plates. This will likely allow the mycelium to colonize your grain spawn jars faster compared to a LC syringe or agar plate you ordered online.

So some new math: If I start from some fresher, healthy LCs propagated in nutrient broth, it would take about 2 weeks to colonize my grain jars, and then 3-4 weeks for fruiting bodies. 5-6 weeks.

Another conclusion: Once you have your first round of substrate bags going, start working with liquid cultures so you have a bulk amount of culture to inoculate jars en masse.

Using Agar Plate Propagations

This is truly the slowest and most advanced method of the bunch, but also provides the most quality assurance. Agar plates are hard to work with when you don’t have a Still Air Box or Flowhood, so I’d stick with Liquid Culture if you don’t want to invest in a truly sterile environment.

The key benefit of agar plates is their ability to help you isolate clean cultures. When you spot contamination on a plate, you can easily transfer a clean section of mycelium to a new plate, effectively creating a pure culture.

However, using agar plates is a large time commitment, and even more so if you’re starting from a multi-spore culture or a spore print.

While grain-to-grain transfers can colonize in 7-10 days and liquid cultures can show visible growth in 3-5 days, agar plates typically take 10-14 days just to show substantial mycelial growth.

I just left several jars inoculated with liquid cultures and agar transfers, and the liquid cultures are fully colonized while the agar transfers are 30-60% done.

So if you started with a spore print or liquid culture on an agar plate, wait 2 weeks for growth, then make a couple more transfers to isolate the strongest growth areas, you're looking at 4-6 weeks before you even begin the process of inoculating grain. Compare this to liquid culture, where you could have multiple generations of fully colonized grain jars in that same timeframe.

But I want to repeat that this really is the best method to work from when you have an established growing operation. The detailed quality control that you get from agar transfers and isolations help you know if they’re carrying contaminants and if they’re quick growers.

Jumping into grain spawn from an unverified liquid culture is a spray-and-pray technique that will sometimes make you wonder if the supplier gave you more than sugar water in that syringe. Some suppliers provide multi-spore syringes, which is essentially a complete guess on the productivity of the spores that succeed in that particular grain jar.

Multi-spore cultures are easy to identify (there will be many gaps between different cultures that can’t fuse together), and to best use your grain jars wisely, you’ll want to isolate one of the possibly thousands of cultures that will grow on an agar plate in the first round of a multi-spore inoculation.

It usually takes me two transfers before I arrive at a quality isolate—but I still have to test this isolate on substrate to see whether even fruits!

Last conclusion: When you have mastered the grain and liquid culture methods of propagation, jump into agar work so you can verify the quality of your cultures and cultures from other suppliers. You’ll start saving a lot of money and materials by isolating to the best genetics that you can find.

Since this episode has a bit more technical information with a lot of numbers, I’ll leave these summary bullet points to end the episode.

• Grain spawn offers quick results but requires careful sterile technique and has higher contamination risks through opening the lid.

• Liquid cultures enable more secure transfers through a needle can and accelerate mycelial growth, though contamination can be harder to detect

• Agar plates provide the best quality control and isolation capabilities but require more time, expertise, and sterile environments

• Starting with quality grain spawn can reduce cultivation time from 6-8 weeks to about 3 weeks. If you're quite new to growing mushrooms, I'd recommend a grain spawn manufacturer like North Spore to start off your grow, and move backwards with DIY techniques to save a lot of money afterward.