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Blog Synthetic Biology

My Experience in Learning PCR (Polymerase Chain Reaction)

When starting your journey in learning synthetic biology, you will inevitably run into something in your reading called PCR (polymerase chain reaction).

I found it a little confusing at first because in some contexts, PCR refers to the process of amplifying DNA, while at other times, it refers to the physical hardware used to perform the amplification.

Now that I’ve gained some experience in running PCR, I thought I’d share a little more about how I learned it using internet resources alone.

What is PCR?

The short-version is that PCR is a technique that is used to make a lot of copies of DNA. Why? DNA is incredibly small and so we must make a lot of copies of it in order to study it in more detail. Sequencing, for example, requires a certain concentration of DNA to provide a reliable reading of nucleotide sequences.

How PCR works is more involved but the short version is that you heat and cool a sample of DNA in a series of cycles. In each cycle, a special enzyme (DNA polymerase) mixed into your tube of extracted templated DNA works to double the amount of DNA with each heat / cool cycle.

The heat / cool cycles are also referred to as denaturation and annealing in the literature.

In days of old, temperature cycling was achieved by moving the tube of DNA back and forth between a hot and cold bath of specific temperatures for specific periods of time. As you might imagine, this process required much attention and energy over several hours and is prone to error.

Nowadays, the process is automated via the use of a thermocycler machine.

How I Learned PCR Fundamentals

One of my favorite options for learning just about anything is to use Khan Academy. Khan has an Introduction to Genetic Engineering section that covers PCR, Gel electrophoresis, sequencing and more.

I spent some time watching the videos, reading the articles and making notes – really making sure I understood what was being said to the extent that I could explain it by heart to someone else.

In addition, I found it helpful to search for images, like the one below, that could help me develop a mental model of what was happening at the molecular level.

Personally, I learn my saturating myself with information to the point that certain terms and methods begin to repeat themselves across sources and eventually come into focus. To saturate myself with knowledge and develop a clear understanding, I have to be exposed to the same information in a variety of formats and contexts. This is quite different than how most folks are taught in school where most people are given a single source (usually a textbook) and the learning typically revolves around memorizing, not internalizing, the information.

diyBio Thermocycler Options

There are a few options available to diybio folks who want to run PCR reactions at home. Keeping in mind that all thermocyclers really do is heat and cool DNA samples over a series of cycles, the difference in options comes down to price, size and degree of automation.

Here are some of the options.

MiniPCR mini8 Cost: $650, Samples: 8

MiniPCR mini8

PocketPCR Cost: ~$120 Samples: 5

PocketPCR

Bento Lab Cost: $1600 Samples 32

BentoLab

There was also OpenPCR but it looks like the project has been discontinued and replaced with a qPCR machine that starts at around $5000. I’m going to skip that one since that is out of the range for most diy biologists.

So which thermocycler did I end up choosing?

I weighed all of the options and went in another direction: buying a used thermocycler on eBay!

Buying on eBay is inherently risky, but it can be done. You absolutely need to make sure the listing states the the unit has been tested and works. You absolutely need to make sure it comes with the heat block (the big chunk of metal that the tubes sit in); otherwise, buying a new block can cost more than the machine itself.

I’ve heard some folks get lucky with repairing broken units, but I wouldn’t recommend this approach if you are new to diybio as you’re already going to be overwhelmed with things to do and learn.

I ended up buying an Applied Biosystems GeneAmp PCR 9700 for about $300 ($150 for the unit + $150 for shipping).

This option was the cheapest for me out of all the options mentioned above, even with the high shipping costs. Although it’s not portable and it cannot be controlled via an app, it works. It has a 96-well heat block which is more than I’ll ever need for my home lab. It allows me to easily program and store my PCR routines. And surprisingly, many of the disposables and peripherals are still sold by ThermoFisher.

Top-View of Thermocycler in Shipped Packaging
View of the Thermocycler Display

Additional Components Necessary for Running PCR

A thermocycler is only one piece of the puzzle in amplifying DNA. There are other procedures and required tools + reagents you’ll need to successfully run a PCR reaction.

My experience is in extracting and amplifying the ITS gene from fungal samples, but the same general requirements will be needed for any gene(s) you want to amplify:

  • DNA Extraction – you’ll need a sample, protocol for extracting the dna, reagents to break the DNA out of your cultures’ cells, centrifuge, and 1.5ml or .2ml Eppendorf tubes.
  • Oligonucleotides (Primers) primers are little strands of DNA that prefix and suffix the gene(s) you wish to amplify. Once you extract your DNA, you’ll mix in the primers per your protocol prior to running PCR. During PCR, the primers will attach to the gene of interest where the next reagent below will work to double the gene of interest.
  • TAQ Polymerase – this reagent is added with the primers and it’s a special heat-resistant enzyme that facilitates the doubling of the gene(s) of interest between each denaturation / annealing cycle.
  • Buffer – buffer is usually just water and Tris-HCL pH 8.0 (sometimes with EDTA) but it’s role it’s to balance and stabilize the extracted DNA and/or create primer dilutions.

After running PCR, gel electrophoresis can be used to test whether or not your PCR reaction was successful. This is an important step because it ensures you’ve amplified the correct gene before proceeding with costly next steps like sequencing.

In closing, I’ll say that learning PCR is an incredibly useful and versatile tool. Many of the folks I’ve spoken with reflect on their time spent learning and running PCR reactions fondly. Much of modern-day genetic work would not be possible without PCR. So go forth and learn it! It’ll be worth your time.

Categories
Blog

Current Legal Status of Psilocybin Therapy (as of September 2020)

I am of the belief that a wave of support for novel treatments of depression and PTSD are coming to the United States, particularly from the therapeutic use of psychedelics such as psilocybin and LSD.

After many years of an expensive drug war that has ruined lives, filled our prisons and cost taxpayers an exorbitant amount of money, Americans are beginning to question to the long-term effects of such obtuse and short-sighted policy making.

In 2015, an antidepressant was the top-selling prescription drug in the US and today, multiple antidepressants rank in the top 25 most prescribed drugs. Meanwhile, rates of depression and mental illness continue to rise, especially among the young, around the globe. Suicidality and self-harm that arrise from depression and PTSD, are only part of the long-term harm to our society. Mental illness is a significant contributor to cormorbidities such as hyptertension, heart attack, heart disease, obesity, drug addiction and more.

It doesn’t take a rocket scientists to see that simply relying on FDA-approved antidepressants to address the mental health crisis alone isn’t working. We need alternative treatments and psychedelics, especially under the care of a trained professional, offer a new option that many who have experienced a significant trip will attest not only aids in their mental health but offers a transcendent mindshift in their role in the world and connectivity within it.

I’ll be using this post to track the legal status and ongoing efforts to legalize psilocybin use in the United States.

StateSpores LegalLegal StatusEfforts to Decriminalize
AlabamaYesProhibited for Any Use-
AlaskaYesProhibited for Any Use-
ArizonaYesProhibited for Any Use-
ArkansasYesProhibited for Any Use-
CaliforniaProhibitedProhibited for Any UseDecriminalized in Oakland and Santa Cruz
ColoradoYesProhibited for Any Use-
ConnecticutYesProhibited for Any Use-
DelawareYesProhibited for Any Use-
FloridaYesProhibited for Any Use-
GeorgiaProhibitedProhibited for Any Use-
HawaiiYesProhibited for Any Use-
IdahoProhibitedProhibited for Any Use-
IllinoisYesProhibited for Any Use-
IndianaYesProhibited for Any Use-
IowaYesProhibited for Any Use-
KansasYesProhibited for Any Use-
KentuckyYesProhibited for Any Use-
LouisianaYesProhibited for Any Use-
MaineYesProhibited for Any Use-
MarylandYesProhibited for Any Use-
MassachusettsYesProhibited for Any Use-
MichiganYesProhibited for Any Use-
MinnesotaYesProhibited for Any Use-
MississippiYesProhibited for Any Use-
MissouriYesProhibited for Any Use-
MontanaYesProhibited for Any Use-
NebraskaYesProhibited for Any Use-
NevadaYesProhibited for Any Use-
New HampshireYesProhibited for Any Use-
New JerseyYesProhibited for Any Use-
New MexicoYesProhibited for Any Use-
New YorkYesProhibited for Any UseBill to decriminalize introduced in May 2020
North CarolinaYesProhibited for Any Use-
North DakotaYesProhibited for Any Use-
OhioYesProhibited for Any Use-
OklahomaYesProhibited for Any Use-
OregonYesProhibited for Any UseLegalization (Measure 109) to appear on November 2020 ballot
PennsylvaniaYesProhibited for Any Use-
Rhode IslandYesProhibited for Any Use-
South CarolinaYesProhibited for Any Use-
South DakotaYesProhibited for Any Use-
TennesseeYesProhibited for Any Use-
TexasYesProhibited for Any Use-
UtahYesProhibited for Any Use-
VermontYesProhibited for Any Use-
VirginiaYesProhibited for Any Use-
WashingtonYesProhibited for Any Use-
West VirginiaYesProhibited for Any Use-
WisconsinYesProhibited for Any Use-
WyomingYesProhibited for Any Use-
District of ColumbiaYesProhibited for Any UseDecriminalization on ballot for November 2020

City and Statewide Ballot Initiatives

Ann Arbor, Michigan

Ann Arbor City Council to consider decriminalization of entheogenic substances on September 21, 2020.

Supported by Decriminalize Nature Ann Arbor (DNA2).

Oregon

Measure 109 will create a psilocybin therapy program so Oregonians have the best therapeutic options available.”

Categories
Blog

EverymanBio YouTube Channel Launched!

The EverymanBio YouTube channel has launched!

Please support me by subscribing, watching and liking any videos that you find interesting.

Personal Challenges in Starting a New Channel

For months, I’ve been thinking about, obsessing and, quite frankly, stressing about starting this channel.

I have this vision of what I want in a channel that includes a lot of cool shots, plenty of B-roll with inspirational voice-overs and self-authored music all edited down into a nice cohesive narrative that makes an emotional appeal to the audience while simultaneously teaching them something new about genetic engineering.

Couple my grand vision with watching other great YouTubers (e.g. Thought Emporium, Fresh From the Farm Fungi, David Ishee) continually crank out quality content, it really got me down. Why would anyone ever want to watch my crappy videos? How will I ever get enough followers to make this venture a sustainable business?

In order to move forward, I had to come to terms and admit that I am but just one man with only so many hours and energy in a day. I could continue to put up roadblocks and watch another day pass with no progress on my channel, or I could embrace the suck and take a lesson from the software development world: iterate.

So I’ve been doing just that. I’ve been hosting regular live streams on Instagram, gradually getting comfortable on camera and making small adjustments to how I speak, framing the short and engaging my audience.

I am optimistic that in time, my videos will get better and eventually will evolve into a different vision; one that emerges from the cumulative experiences accrued on the journey. After all, sharing the journey with you all is why I started EverymanBio in the first place.

Categories
Blog Well-Being

How Studying Fungi Can Help Navigate a World in Chaos

If you’ll be so kind as to oblige me. I’d like to try something new and share with you a few thoughts I’ve had as of late as a sort of experiment in personal self-expression. My brother-in-law recently commented on Sam Harris’s Mushroom Trip monologue and after listening to it closely one afternoon, I couldn’t help but to feel inspired to write down some of my own thoughts on the matter and record this incantation.

Where this goes, the medium through which it will be published and the reaction to be received in consequence, well – I can’t say for sure . But I do know, like so many of you, I yearn to be heard, for some of my thoughts to be explained and perhaps most importantly, to be understood.

I’m often reminded of Sir Francis Bacon’s declaration that “reading makes a full man; conference a ready man; and writing an exact man.”

Indeed; writing as a form allows for one’s thoughts to be carefully constructed, poured over and conveyed to the limits of one’s ability. 

I suppose if I am going to talk about my first psychedelic experience and the impact it had on me, some background information and a lens through which I entered into the experience would be useful.

How Mushrooms Can Help Navigate a World in Chaos

My journey with mushrooms began over ten years ago when I discovered that mushrooms are part animal, part plant and that human-beings share more DNA in common with mushrooms than plants. Fungi provide medicine, food, clothing, and a wealth of other benefits to mankind. This initial discovery led me down a path to dig into the kingdom of fungi and the incredible symbiotic relationship that we share with these often misunderstood and feared organisms.

I recently had a conversation with a close friend and he was asking me how I was fairing with the epidemic. Personally, I haven’t been too affected by it. I certainly have my opinions and have my moments but by and large, I’ve been navigating through this time like any other. My friend was apt to point out to me that I would often steer the conversation towards one of my current projects involving the study and cultivation of a variety of medicinal mushrooms. It dawned on us both that there is something special about the process of mushroom growing that is actually helping me during this time and that it might actually be something worth sharing.

I can talk and share all kinds of really fascinating research and information about fungi but for now, I’d like to just talk at a meta-level about getting involved in learning about and cultivating mushrooms and how we might apply that to our lives and our workplaces to improve our overall well-being.

Learning and Attending to Fungi is a Great Place to Occupy a Worried Mind

Prolonged social distancing and isolation are precisely the opposite of what human-beings need to develop a sense of well-being, belonging and connectedness in this world. Many of the coping mechanisms we are accustomed to leaning on during times of distress are no longer accessible to us.

For the intellectually minded, those who are on a constant chase for new information and learning, fungi offer an outlet unlike any other. For example, studying the slime mold Physarum polycephalum  offers software engineers a chance to peek into how nature has solved NP-hard problems – that is a problem that is non-deterministic and cannot be easily approached with a predetermined algorithm. This same slime mold was also recently used to simulate the dark matter that connects all matter in the universe. 

There are many experiments you can do from home to learn algorithms that nature has laid at our footsteps.

For the layperson, you might start by looking up an antibiotic that you have laying around the house such as a prescription or from a tube of antiseptic cream. You’ll quickly discover that the key ingredient is derived from a fungus that was discovered by accident. This might take you down a path of learning how to grow your own antibiotic mold in some Tupperware laying around the house. While others might be interested in knowing more about how fungus generates enzymes that “plug into” our body or harmful pathogens and soon, the world of microbiology is at your fingertips in a way you would never experience in a classroom setting.

Learning about fungi and mushrooms from all angles provides you with knowledge, a better understanding of our world and a sense of empowerment in realizing the information has applications to medicines, including the current epidemic, when your mind might otherwise be sitting in fear, anxiety and a sense of helplessness.

Learning About Mushrooms Increases a Sense of Connectedness

Research suggests that one’s subjective sense of well-being is highly correlated with how interconnected the mind is with itself and that one of the biological mechanisms of trauma and its corollary effects is that it shuts down parts of the brain (or you might say self). The research suggests that it’s a series of deeply connected interpersonal experiences that shape how we view the world and our capacity for healing and growth.

When you dig into the world of fungi, you will begin to see the parallels in how connectedness, in a variety of micro and macro contexts, is not just healthy for humans, but has been employed by nature for millennia.

Fungi facilitates nutrient exchange between just about every type of plant there is. Underneath our feet is a massive network of mycelium, resembling the topology of the internet, connecting living organisms and acting as message pathways that alert life in the local ecosystem of potential dangers.

In my learning, I have discovered a plethora of parallels in how fungi grows, travels, reproduces to that of human well-being. I would postulate that the more we can emulate the degree of complex interconnectedness we have with our own minds and in larger social contexts, the more our well-being is improved – offering up a vast amount of benefits to individuals and society at-large.

In short, if you can get over the hump and make a connection to mushrooms, it won’t be long before you begin to draw parallels between other organisms and systems you interact with. The outcomes, in my experience, offer a counter-balance to the ever disconnected and isolated world in which we live in and are increasingly moving towards.

There are many ways to get started with growing and studying mushrooms. In my case, I’ve built my own clean room, poured agar plates, germinated fungal colonies, transferred isolated strains to bulk and fruited whole mushroom bodies. I’ve filmed and grown bioluminescent mushrooms, cultured yeast that naturally lives on our bodies and now am I on a journey to use the awe-inspiring natural biosynthetic power of a common yeast to produce valuable and life-saving compounds. And I’m doing it as an average person with no fancy degree, no formal training. It’s me. Books. The internet. And voracious appetite for knowledge and to manifest into the present that which has lived in the corners of my mind, often neglected and attended to, for far too long.

Other folks might get started with a $20 oyster mushroom grow kit from Home Depot.

Whatever methodology you choose, there is definitely something to be said about the process and it’s healing, regulative effect on the psyche. Growing mushrooms takes patience and each step is fraught with the possibility of failure. The process often means that you do a lot of prep work and then you wait to see the results.

I’ll find myself in my lab for hours working on a project or inspecting growths on my Petri dishes. As each stage of the process advances, I find myself checking on my experiments and ensuring they have the right humidity and temperature to colonize without introducing contamination.

In a sense, I’m a modern farmer of fungi and I’ve developed a strong sense that the act of farming – the journey from seed to harvest – is itself a healing process that instills a sense of regulation and calm. With each step of the process, I can only move as fast as the mushrooms will allow. Developing a trust in this serves as a metronome for the parts of me that are seeking instant hits of gratification that I might otherwise lean on to avoid being with myself or feeling whatever lurks deeper within me.

The satisfaction of having paced myself, attuning myself to my observations, listening to what the growths are telling me, the daily ritual of measuring and logging my observations and then finally seeing the finished product in my hands is indescribable. It’s probably akin to how farmers of old felt by having a successful crop they invested everything into, knowing that they were able to create food and resources through a beautifully coordinated dance with the ultimate incubator of life: nature.

Closing Remarks

There is much we can learn about how to adapt to these challenging times by looking to nature and emulating how it has solved problems that we are only now understanding apply to us. Mycology, or the study of fungi, has been an incredible gateway to increased connectedness, meaningful learning, and obtaining the satisfaction of the slow and steady progression of tending to a crop before feasting on a glorious harvest.

To those of you who find yourself, like so many of us, in a perpetual struggle for meaning and balance in life – for those of you seek to transcend the flawed constructs of modern day society and the division it sews amongst our fellow man, I offer up this anecdotal experience and words of hope that there are ways you can navigate chaos, fear, anxiety, and other emotional challenges. There is much we can learn about ourselves from the fungi world and I hope this post will offer up a new pathway – a new connection – that you might explore to help improve your personal and work life as it has for me.

Categories
Blog Experiments Organic Chemistry

A Brief Introduction to Thin Layer Chromatography from an Amateur Scientist

I was hanging around some mycology forums one day and a fellow diyBio scientist suggested that I look into learning something called thin-layer chromatography. The idea came as a response to my question to the community: how does one quantify the amount of a particular compound in a mushroom?

This question has been on my mind for some time, especially as the popularity and the science behind the medicinal benefits of consuming mushrooms becomes more mainstream. It’s been my experience that the majority of amateur mushroom growers have no means to detect and quantify the presence of these medicinal compounds. As a result, mushroom growers which are interested in creating unique strains are restricted to using physical phenotypes alone in their selective breeding process.

Imagine if one could detect the amount of cordycepin or terpines in two separate strains. That information could then be used to select for the strains which express the highest yields of these compounds and immediately give themselves a competitive advantage over other breeders focused on gourmet and medicinal mushrooms.

So I started to look into thin-layer chromatography (TLC) in a quest to explore if it would be possible for me to develop this technique to benefit the amateur mycology community.

What’s in a Name?

Let’s look at the word chromatography. Chroma means color and –ography is the study of something. From this, we can infer that TLC has something to do with the study of color…and that isn’t far off.

The general idea behind chromatography is to take a substance, separate out the compounds using a solvent and then interpret the color-based result in order to determine what compounds are present in the analyte (the substance being analyzed).

Here, let’s take a look a little drawing I made of the components.

Thin-Layer Chromatography Drawing
Thin-Layer Chromatography Drawing

The components of TLC are:

  • developing chamber: a glass jar with a lid to hold the solvent and stationary phase. I found using an empty seasoning bottle or a glass salsa jar worked just fine. Preferably, the jar has a flat bottom.
  • mobile phase: this is a solvent like acetone or methylene chloride, both of which can be obtained off Amazon or eBay for minimal cost. The mobile phase is what is drawn up the stationary phase, separating out the analyte on its way up.
  • origin: the origin indicates where on stationary phase the analyte was deposited. It’s used in the calculation for the Rf factor and so usually is drawn with a carbon-based pencil onto the TLC strip a couple of cm from the bottom.
  • stationary phase: this component is typically known as a TLC plate, a silica dioxide coated strip of glass or clear plastic. The solvent moves up this plate via capillary action.
  • solvent front: this is where the solvent stopped at the end of the procedure. It needs to be marked soon after the plate is removed from the chamber as its used in the Rf calculation and will eventually disappear as the solvent evaporates completely from the plate.

Here’s a video explainer of the process with a demo.

As mentioned in the video, the less polar a compound is, the higher the compound will travel up the plate and the more polar compound is, the lower on the plate will stay.

Here is what my very first setup looked like and the results under a UV-A lamp.

TLC Setup
First TLC Setup
Thin-Layer Chromatography with Paper, Acetone and Highlighters
Thin-Layer Chromatography with Paper, Acetone and Highlighters

On the left, we see the results of TLC run using a couple of dabs of yellow highlighter. Note how the acetone was drawn up by the chromatography paper, separating out the highlighter dye as it went up the paper, revealing the blueish hue seen near the solvent front.

On the right, we see several trials using various highlighters, including the yellow one on the left.

Note that I used a UV-A light. UV lamps come in various wavelengths and important to note that only some compounds fluoresce and those that do may only fluoresce under specific wavelengths.

Chart Showing The Different Wavelength Bands of UV Light

And as I learned, some compounds require that you spray the plate afterwards with an additional reagent in order to induce fluorescence. This is the case with psilocybin, for example, which requires Ehrlich’s reagent to properly develop the plate in order to make it fluoresce. And in the case of psilocybin, a short wave uv (UV-C) light is required.

When you do your own TLC experiments, you will be faced with a few questions that I’m still trying to work through myself. These questions include:

  • Which solvent or solvent mixture should I use for my mobile phase?
  • Is there an additional development step required to make the compounds visible?

Obtaining the answers to these questions is still a challenge. A biologist friend of mine told me most folks can find the right solvents to use but must go through a series of trials and errors to narrow-in on the right parameters that best optimize the experiments for compound visibility.

How to find TLC methods and reagents for your subject compound

Here’s my current approach to finding out what solvents, development steps and techniques are used for a compound I wish to analyze:

  1. Head over to scholar.google.com
  2. Search for: your_compound thin layer chromatography (ex: cordycepin thin layer chromatography)
  3. Look for a research paper and read through it.

That’s pretty much of it. The only caveat here is that you might only find a paper that was written over 30 years ago and uses an antiquated procedure or reagent that is impossible for the average person to obtain.

For example, I was unable to develop my psilocybin TLC plate correctly because I am currently unable to obtain p-dimethylaminobenzaldehyde (DMAB).

TLC Setup Outside
My TLC Setup Performed Outside

How I setup TLC for Mushroom and Common Analgesic Extract Experiment

Note: Although my test results were inconclusive due to not having the proper developing reagents, the analyte preparation and testing procedures don’t change much.

Here was my general procedure:

  • Use a mortar and pestle to grind ibuprofen, aspirin and 1.5g dried mushroom fruit body
  • Mix each analyte with 2ml acetone in individual air-tight containers and leave for 24 hours
  • Pipette 1mL of each into eppendorf tubes and centrifuge at 10K rpm for 1 hour
  • Measure and mark the point of origin on the test plates
  • Use seperate capillary tubes to drop some of the supertanant onto each of the test plates (real silica plates I cut myself), being sure to mark the top of each plate with the name of the analyte
  • Develop the plate using methylene chloride in a glass developing chamber
  • Once the solvent front has reached a few cm from the top of the plate, remove from the chamber and mark the location of the solvent front
  • Let the plate dry and check plate under shortwave UV lamp
EverymanBio Josh TLC
Pipetting Extracts for Placement on TLC Plates

What is Rf factor and how to calculate it?

Rf means retardation factor. It is the ratio of how far the compound traveled up the plate over the total migration distance of the solvent front.

Most compounds have a published Rf factor which can be used as a reference to determine whether the spot on your plate is indeed the compound in question.

But in a real-world scenario, a scientist would be running TLC alongside a control containing a pure sample of the compound being analyzed. So if the control had an Rf factor of 0.4, you can be reasonably sure that the compound you are looking for will have the same Rf value all things being equal.

Closing Remarks on Thin-Layer Chromatography

Most folks I’ve spoken to who have taken an organic chemistry lab at a college or university speak fondly of their experience in using TLC. It’s really fun to run an experiment where you can watch a physical observable result happen in near real-time.

Aside from the fun, it’s also an incredible useful technique for identifying the presence or absence of a compound. Learning TLC has opened up some new connections to other organic chemistry concepts like polarity, solubility, solvents and qualitative experimentation in a way that I would not have learned had I been presented the information in abstract as is often the case in formal education systems.

Although my final tests (shown in the big green image above) did not yield the clear definitive results I was hoping for, I am confident that once I obtain the correct arrangement of solvents and developing reagents, I’ll be a TLC pro in no time.

So there you have it. There’s TLC from an amateur scientist who went from never having heard of it to running experiments in just two weeks time. Give it a shot – it’s really a lot of fun.

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Quick procedural test of the fungal chromatography kit I’m developing to help mushroom farmers improve production of medicinal compounds in their proprietary strains. Different compounds travel up and settle on the plate at various heights and colors based on their respective polarity and solubility. A cheap and easy TLC kit will give growers a competitive advantage over breeders who do selective breeding using physical phenotypes alone. #chromatography #thinlayerchromatography #labiotech #diybio #scienceexperiments #diyscience #mycology #mushroomgrowers #medicine #biotechnology #startupcompany #startup #lifescience #biohacking #amateurchemist #organicchemistry #selectivebreeding #phenotypes #geneticengineering

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Blog Interviews

Interview with Experienced Dutch Mushroom Grower and Researcher Jop Mens

I’m pleased to present a special post this week featuring an interview of a new friend I’ve made in the amateur mushroom growing community: Jop Mens.

Jop is a vocal and respected voice in the world of mushroom growing and dedicates much of his time towards helping to educate newcomers to the expansive fields of diy biology, mycology, botany and more.

I think you’ll enjoy learning more about Jop, his background and his general perspective. Enjoy!


How did you get started with growing mushrooms?

When I was studying chemistry and some philosophy in college I grew magic mushrooms (psychedelics), starting out with a kit. It was still legal here at the time, before the government banned those kinds of mushrooms in response to incidents with tourists.

I got some results with the kit but unfortunately it contaminated prematurely. After that I grew more of them with my own materials, using a classic method (PF tek). And some “monotubs” as well. At the time I did not focus on fungi much, the real passion came later.

I’ve always loved DIY with many things, but particularly fermentation. I brewed cider when I was in high school.

The sheer make-ability of all that, it is like sorcery to me. Chemistry also fascinates me because there is both an inquiry into what the world is made of fundamentally as well as an exploration of what is possible and what can be created or transformed.

How did your interest in growing develop over time?

I went through a hard time after college and having a job in IT for a while, I had to move around a couple of times. I live in the south of the Netherlands again now. When I got an apartment and could stop moving, I built a cabinet for growing plants. 

Jop’s Plant & Cacti Grow Cabinet

Basically “I came for the mescaline containing cacti (and other ethnobotanicals) and stayed for the beauty”. Those were never for consumption anyway, but actually most I have now are sister species and varieties and virtually contain no mescaline at all.

Then the same sort of thing happened with growing mushrooms. I started trying to grow gourmet edible mushrooms and soon enough looked into other applications and other species and it really took off.
My penchant for experimenting and a tendency towards novelty became evident in my projects and I also started to collect different species like a madman. There are dozens upon dozens and they were basically all traded from all over the world.

I was helped poorly in my search for work and basically wasted a year of my life checking out 2 traineeships, just waiting on bureaucracy until neither worked out. This made me frustrated and angry, but that anger gave me a drive to make my own destiny.

And then in one moment I realized how starting my own business focused on novel applications of fungi could combine my interest in nature, my aptitude for science and experimentation (and background in chemistry), my budding passion for fungi, all into one.

I know Holland has one of the more liberal drug policies that attracts people from all over the world, with psychedelic sclerotia legally sold in “smart shops”. How do you think that impacts the amateur mushroom community?

Despite being legal, mushrooms then and sclerotia now may still be misunderstood and feared, just as psychedelics in general. This has started to shift significantly only since relatively recently. The US, Paraguay and Portugal for example have all surpassed the Netherlands with laws for cannabis, which is not legal here but only condoned in an inelegant and sometimes messy way.

Psilocybin-containing sclerotia are often called magic truffles, which is technically incorrect. Actual truffles are underground reproductive fruiting bodies and they spread spores which are similar to seeds. It’s fascinating really: they have evolved to become so aromatic to attract certain kinds of critters even from underground, so that they would get covered in spore mass and take it away with them to spread it around.

However sclerotia are differentiated hardened tissues which act more like the tubers of plants (such as a potato). They are not just a storage vehicle for nutrients but also protect the fungus from the elements until conditions become suitable once again.

The amateur mushroom community which revolves for a significant part around psychedelic mushrooms remains mostly underground (just as growlights immediately make people think about growing cannabis, and if I mention growing mushrooms a lot of people also assume psychedelic ones).

To be fair, it is how I started originally of course!

My country had progressive ideas at one time, but managed the issue of drugs terribly so now there is a lot of organized crime and the European Union also made it very hard for the Netherlands to really manifest those liberal ideas about drugs.

It must be a big difference though, for there to not be a ‘war on drugs’ in this country. You are generally left alone as a user and judged by the actual problems that may arise and not on principle.

As far as I know the mushroom community was never a very well represented and outspoken one, probably because in absolute terms mycology (the study of fungi) is very lol populated but in relative terms it seems those growing psychedelic mushrooms are overrepresented among amateur mushroom growers. I think it is because psychedelic mushrooms are illegal so they force more people into growing their own and a part of them discover how much they like the growing in general. It wouldn’t happen that way with gourmet mushrooms.

Wild Clone of a Stinkhorn Mushroom Egg (Phallus impudicus)

There are apparently very few people working on fungi as I have learned becoming introduced to the world of mycologists. (I go on excursions with a group of – mostly elderly – mushroom enthusiasts with a quite different angle at them).

Most of them just love the fungi and are not that distracted nor attracted by issues of legality or applications even as food. This really surprised me, enjoying eating wild mushrooms just seemed like a very convenient byproduct of hunting them but it seemed like it didn’t enter their minds for the most part.

What do you do professionally now, and what will you be doing in the future?

I have an agreement with a world renowned art academy (it is not really a school, but basically hosts post-doc level artists for a year long residency) to grow fungi for them. A grow room (dubbed the Mush Room) was already designed and built by me. But due to covid I cannot continue my project there until september and I am using the time to finish building other facilities elsewhere.

Their restaurant is one of the “labs” there and it is integrated in a bio & eco focused mother department. I will produce mushrooms for a large part but can also experiment with growing more unusual or difficult species for eating as well as fungi to use for bio-materials and bio-art. The methods will be taught to other staff members to free up time for me to develop more projects methods and experiments, repeat.

I also sell and trade cultures of fungi but not yet in a professional capacity, as I cannot and will not prioritize it over work for the academy.

Other than that I am working on multiple new concepts for utilizing fungi in novel ways, and I study/explore crossing and hybridizing them.

“Learning to grow is much less bewildering if you try to enjoy the process…”

Jop Mens

What are some misconceptions about growing mushrooms?

There are a number of myths, for example that mushrooms require darkness to grow yet paradoxically when lighting is used people may forget that they are not plants that perform photosynthesis so it does not have to be intense light (preferably not overly bright actually).

But also among growers certain myths can become very persistent because it is kind of an obscure and closed off world and much work still needs to be done. There isn’t quite as much work being done on fungi as we might like and we have barely gotten started from a historical perspective, shockingly.

And so it seems that many of us have an inborn fear and distrust of fungi which are ours to shake, for our benefit at least. As with many primordial instincts, purely from an evolutionary survival point of view it pays to be apprehensive, but in many subtle ways we hold ourselves back if we cannot dispel the irrational parts.

Mycophobia (the fear of fungi) may originate from our associations with disease, death and decay whether it is our bodies or plants/trees or our food that may become infected… it is not the whole picture. If that is where we stop to think about it, I feel we are hung up on our egocentricity, how it affects us personally.

The other part of the story is that fungi can complete the picture of recycling. Hardly never do we appreciate that earth would be uninhabitable if dead materials would just pile up instead of returning them to nature for new life to flourish.

If there was one thing you wish you knew when you started learning mycology, what is it?

Hericium coralloides “Coral Tooth” fungus

There isn’t one very best way to do things, not necessarily. Choices depend on someone’s own unique situation, even if we try to standardize as much as possible. Sometimes this is forgotten and someone’s success or failure is assumed to be universal for the method. Yes, at first it is good to just read a lot, assimilate and take teks (written out methods) at face value, but soon enough it becomes valuable to look into why certain steps are performed and what function they fulfill.

Understanding why different methods share things in common even if that is not immediately clear, helps to figure out what is better or worse for your particular situation and how to integrate these things into your own style.

What’s your favorite part of mushroom growing?

Haha the pinning (baby mushrooms) and fruiting of course, but also trying new things. Even thinking about them / coming up with them, as I am a sucker for novelty.

Who do you think should grow mushrooms and why?

Anyone who is interested in one or more of the following: self-sufficiency, meat substitutes, creating/growing in general, experimenting, recycling, nature/biology. And mushrooms probably.

What are you learning about mycology or planning to learn in the future?

One thing I have been working on and will keep doing is to experiment with substrates (materials to grow on as food for the mushrooms) and to learn how this connects with what different species are used to growing on in nature.

Furthermore I am learning about reproduction and how this applies to crossing or hybridizing fungi in order to make my own variety or hybrid. This also involves breeding which I like to learn about. However, there is a difference in making such a new combination, and stabilizing or optimizing it for cultivation. It seems relatively doable to make something new (although for now I am struggling a bit), and surprisingly much work to optimize afterwards.

Also some more about taxonomy (classifications of species and other such categories), however I am beginning to learn just how much confusion there is even among experts, and it also makes me doubt the importance of fixating too much anyway on whether 2 fungi are the same species or not. It is a matter of perspective and focus of what is different versus what is shared in common.

There is a lot to be done in new applications, including bio-materials, bio-art and new foods and I have barely touched yet on this but I can already see the avenues.

What is the biggest challenge for new growers and what can newcomers do to overcome those challenges?

I can discuss some practical matters like learning how to do sterile procedure if you have never done this before, and how it could be frustrating especially if you hardly have the right tools invested in, or expect too much too fast…

But in my opinion the real challenge is one of attitude. It can be very hard if you are focused on results and look at everything as a means to complete a goal just beyond the horizon. While it is good to have direction, learning to grow is much less bewildering if you try to enjoy the process and accept that depending on your goals you have to invest a certain amount of time and dedication and most of all: faith and patience.

Occasionally fungi appreciate neglect, but overall it will work best if you like to learn to grow and are not just interested in getting the mushrooms as soon as possible.

How would you recommend someone who is starting from scratch get started?

Some are apprehensive about investing and like to get a kit of some oysters on coffee grounds, or do a little more yourself like injecting liquid culture of fungi from a syringe into bags of substrate bought pre-made. Which might help to inspire but tends to be relatively costly and I don’t think it is representative of doing your own growing and more symbolic.

Beyond that a real pressure cooker (for canning, reaching at least 15 PSI) tends to be essential although there are ways to go even without one. I have never done this myself but brown rice is said to be one of the least naturally contaminated grains and can be sterilized fine in lesser pans.

Doing agar in petri dishes or makeshift dishes is also part of solid basics for growing, for this it is also possible to be frugal before getting professional gear (if you do at all).

Not doing your own agar, you could buy liquid cultures of gourmet mushrooms and use this to inoculate brown rice as your spawn (“seeding material”), which you could expand to substrate from just hardwood fuel pellets you quick pasteurize with poured boiling water in filterbags.

It is not something I’ve done in particular but it’s what I come up with stringing together some really low threshold methods.

What, if any, economic opportunities are there for mushroom growers?

I am not one to ask. Still finding my own way exploring different business models but preferably niche ones, and the project at the academy is a pretty unique one. I also don’t plan to become a large scale producer who just competes with other producers, that is not my strong suit and it is not interesting to me.

As far as I know mushroom growing is on a rise though, and there are holes in the market – the white button industry is quite big but they are grown differently than woodlovers.

A market also needs to be created a bit, people don’t seem to be quite so used to oysters and shiitake yet at least not in all regions.

Other than mushroom growing, what other diybio or amateur science projects are you interested in?


I’m interested in plant tissue culture which I experiment with but I have only had limited success so far, I find it quite challenging and it can be more work than mushroom lab work. The goal I have for it is to be able to acquire small tissue samples of rare cactus genetics and culture them to get new plantlets. For example one professional Ariocarpus grower/breeder said he was interested in it and would send me tissue samples for free if I would keep him posted. I’ve ordered some seeds but have not yet gotten tissue yet, but I also haven’t pursued or reminded him.

I’ve also investigated bio-materials a bit and it is something i want to continue once the show is on the road at the academy. I made a prototype wallet out of SCOBY leather, so made from bacterial cellulose out of kombucha. I still want to isolate the bacteria responsible for it out of kombucha but all of this got on the back burner.

How can folks stay connected with you and everything you are working on?

Right now anyone can just e-mail me at [email protected] on Instagram @mycomatters.

Categories
Blog Microbiology Mycology

Creating a DIY Mycology Lab Notebook

I’m currently growing five different species of mushrooms with multiple experiments running and new ones under development. Ensuring that I track all of this work is a must; for sanity and science.

In addition to the project management benefits of writing down the details of my work, keeping a good lab notebook is a necessity for an amateur scientist.

Here’s a brief overview of requirements for keeping a lab notebook, what I track in my own lab notebook, options for keeping a lab notebook, and some thoughts about how I plan to improve tracking my lab work in the future.

Adam Savage Writing Meme
Adam Savage Meme

General Requirements for a Lab Notebook

My Personal Requirements

Given that this is my first mycology notebook, I wanted to capture some information for myself that may or may not also meet the normal requirements of a commercial laboratory notebook (or maybe they do!).

My background in data and computer science has also trained me to see the potential value in tracking more data than I may seem immediately necessary. Here are some of the data points I wanted to be sure and capture off the top-of-my-head:

  • Date and time of procedures
  • Temperature and humidity
  • Detailed procedures with tools, methods & materials used
  • General observations
  • Side-effects of experiments that involved the procedures and materials I was new to or unfamiliar with
  • Related to the above: mistakes made and adaptations or workarounds as each step advanced
  • General inventory counts

What information did I hope to learn by capturing all of this meta data about my experiments?

By tracking all of the above, I could presumably finish a complete grow cycle of mushrooms and determine the following:

  • Total time from initial culture to the end of a grow
  • Total yields
  • Biological efficiency
  • Total costs and costs per unit of final product
  • Initial capital / startups costs per species
  • Most and least efficient techniques
  • Gross costs
  • Net profits
  • Return On Investment (ROI)

You can see how helpful it is to measure the feasibility of starting a fungi-related business if you track and interpret the data as you go along. I’ve seen a lot of new entrepreneurs across as businesses try to wing it and it usually causes them a lot of lost time and money, greatly reducing their runway and general chance of being financially successful in the long run.

Although learning the science of synthetic biology and genetic engineering is my first priority for EverymanBio, I do need to be mindful of how I spend my limited resources and eventually need to generate some kind of revenue to keep this dream alive. Having the detailed data tracking my moves gives me valuable insight into the return on my time and money.

Keeping a good lab notebook is just as much about ensuring the science you employ can be reproduced by ones peers. The same data used to ensure reproducibility also allows me to more easily and more efficiently keep iterating on what works and drop what isn’t.

Options for Keeping a Lab Notebook

My first inclination when logging anything, especially data I want to analyze over time, is to go electronic. However, several of the chemistry and biology textbooks I own recommend that students start with something written.

In my experience, writing the journal is faster and more convenient. It has a different psychological component to it as well that contributes towards my overall desire to keep pressing ahead with the work.

With a hand-written lab notebook, I don’t have to worry about bringing in my dirty laptop into my clean space and all that entails decontaminating it and myself as I go back and forth.

I also don’t need to worry about any technical issues. No internet connection is needed. I don’t need to expend mental energy on learning a new tool or customizing it to suit my needs. I can easily move my notebook between work spaces and not worry about connectivity or power constraints.

Excerpt From Hand-Written Mycology Lab Notebook
My Mycology Lab Notebook

But it isn’t perfect. There are real trade-offs that make it difficult for me to imagine using a hand-written notebook for everything over the long-run. I cannot, for instance, share my lab work easily on EverymanBio.com.

Trade-offs between Written Lab Notebook & Electronic Lab Notebook

The main trade-off is that if you want to do all of the calculations and analyses that I mentioned above, you would need to transcribe all of your handwritten data to something like an Excel spreadsheet, SQL or some other database.

And that’s a pretty big trade-off. Imagine writing notes about a grow-cycle that takes four months start to finish. At the end of the grow cycle is where mushroom growers are doing daily harvests, sometimes multiple times a day. It’s just not feasible with even a moderately sized grow to go back through all of the lab notes and manually calculate something like ROI when its just so much easier to use Excel to quickly perform calculations or plot graphs.

Looking Ahead

I’m still searching for the right balance of data tracking that accommodates my love of hand-written lab notes vs the loss of electronic analysis. There are many cloud-based lab notebook offerings, but they come with the overhead that I’m not entirely sure I’m ready to take on. For example, I’ve considered using Markdown in Git, Jupyter or eLabFTW, which can be displayed natively on GitHub. But there are massive trade-offs that result in added time and energy on matters which don’t add much value towards my core objectives. Have you ever tried to write out a large data table in Markdown? Not the funnest of exercises.

I may end up with a hybridized approach where I use a cloud-based solution for quantitative data and keep the written portion for qualitative data. Which solution I use for which is still up in the air.

Molecular florist and fellow diybio Sebastian S. Cocioba puts his lab notes in a Google Doc for all to see: https://tinyurl.com/yxzzfq2m

I really like this approach from an ease-of-use standpoint. It’s a plain document that he keeps appending to every day; however, I’m not sure how this approach would solve for the kind of quantitative analysis that I’d like to do. As of today, his notebook is over 500 pages long. Sebastian has several diy projects that I’d love to follow and reproduce in my lab, but it’s incredibly difficult to do that with such a large document because it requires that I scroll back through the entire history to find where the details pertaining to a specific project live.

Perhaps one day he’ll see this post and fill me in with his thoughts on this matter. I’m sure he’s tried many solutions over the years. If I were a bettin’ man, I’d say he landed on this format because of its simplicity and that is worth more than the problems the more complex solutions aim to solve.

If you have any opinions or suggestions on the matter, please reach out and let me know. I’d love to hear your ideas. For now, I continue to write things down and track them in my written notebook while simultaneously experimenting with different quantitative tracking options. And if I happen to land on the perfect solution, I’ll be sure to let you know.

Categories
Blog

My First diyBio Lab and Upcoming Improvements

Some years ago, when I first started growing mushrooms using a basic still-air box for sterile work, I began to think about how I might expand my working space to accommodate a wider array of lab work.

So after Cat and I moved into a new home last year with a large semi-finished attic space, I pounced on the opportunity to build a clean-room where I could put a custom-built laminar flow hood and have space for mycology work.

I started by building the flow hood, which I will show pics and provide more info on later. But once it was built, I began to think about what I needed and how I could build a mini-cleanroom that would work in the attic at minimal cost.

My chief needs and concerns at the time included:

  • The attic has poor ventilation with several skylights that stay open year-round to help dissipate heat.
  • The attic space is full of contamination; mainly various forms of mold and rot.
  • The attic space is unconditioned with no filtration, heat or A/C.

My original thought process was such that if I could create a barrier between the attic air and the clean sterile air put out by my flow hood, I would be able to do proper sterile work without losing time and materials to contamination.

I also read Paul Stamets’ Growing Gourmet and Medicinal Mushrooms which discusses the idea of creating progressively clean spaces and techniques for mitigation the transfer of contamination into a cleanroom.

Using the limited information and ideas I had at the time, I ended up building a little tent-like cleanroom using pvc pipe and 3.5mil plastic sheeting.

Here are some photos of the build and where I’ve been working for the last six months or so.

diyBio Lab v1 Hallway
Front-facing View of My First diyBio Lab
diyBio Lab v1 Backside
Rear-facing View of My First diyBio Lab

You can kind of see that what I built was essentially a room inside of a room. It’s nothing more than a PVC frame with plastic sheeting taped all over it.

Notice the skylights above the tent. I originally thought that my cleanroom needed to have a roof so that any airborne contaminates that might come in from the skylights would be deflected.

I also made it so that there is an initial room where one could potentially wipe themselves down before entering into the main room of the lab. This comes from Stamets, whose own lab has upwards of five separate rooms that progressively lead into the cleanest area.

The plastic strips were designed to help keep dirty air from entering the tent. Once you enter into this room and do an initial clean and wipe-down of yourself, the idea was that you would then unzip the ZipWall and enter into the cleanest portion of the lab.

First Entrance to diyBio Lab
Entrance to the First Room of the Lab
Zip Entrance to Main Area of Clean Room
View of the ZipWall Leading to the Main Room

All of this is completely unnecessary, by the way. And I’ll get to that in a moment, but building this really helped me gain confidence and had I not built this, I might have put off getting back into mycology work altogether.

Anyway, once you enter through the first room and past the ZipWall, here’s what you see.

Inside View of diyBio Lab
Main Working Space Inside of the Lab
Inside View of diyBio Lab Looking Back
Inside the Lab Looking at the Entrance

By and large, this lab has served me well. It definitely smells sterile compared to the rest of the attic. My rate of contamination has been approaching 0%. This lab has also provided everything else I wanted: shelving space, workbench space, room for my flow hood and isolation from the dirty attic air.

But, I’ve learned so much since building this first lab. First and foremost, I’ve learned that proper sterile technique is far more effective at mitigating contamination.

I’ve seen photos of amateur mycologists doing sterile work on a dirty counter in their bathrooms in a run down trailer house. And they were completely successful because they knew good sterile technique and were able to work using a basic still-air box (SAB) to mitigate contams from the surrounding environment.

I’m not at all proposing that cleanrooms aren’t necessary or that SABs are the answer to anyone wanting to do sterile work, but it certainly makes a good argument that having a tent isn’t necessary to be successful.

One thing I completely disregarded was that no amount of tent would be able to prevent the biggest vector of contamination: me.

We carry on our bodies, in our breath, on our phones, etc an enormous amount of organisms and my cleanroom was never going to mitigate that vector on its own. The one advantage of the tent is that it does reduce the amount of attic air it has to filter, improving the longevity of my HEPA filter.

Psychological Benefits of My First Lab

I don’t want to discount the first version of the lab completely. This lab gave me some piece-of-mind, misguided as it may have been. It allowed me to move forward with one less concern weighing on me as I proceeded with more and more mycology research and experimentation.

It also provided me a dedicated work-space that was also easy to clean. And it really does have its own energy. It puts me in a focused flow state where I can more easily visualize what’s happening at a micro-level and what actions and movements I need to make in order to accomplish a task successfully. I don’t think I would have had this sort of experience had I just put the flow hood up on a table in the open air of my attic.

For new diy science folks, I suggest never discounting the psychological barriers to be considered when developing a strategy for learning. It is more important to manage your mind, well-being, expectations, etc and incorporate them into your methodologies so that you can overcome the mental hurdles associated with learning something new, often difficult and riddled with failure, and maintain the motivation to keep moving forward.

So while this tent really wasn’t and isn’t necessary to do mycology work, it served a variety of needs that I had and for that, I’m grateful. I wouldn’t be able to be here talking about what I want and need in the next iteration of my lab had I not had this experience.

Now Under Construction: EMB Lab v2

There is an adjacent room in my attic space that was finished some thirty years ago. I’ve decided that it is time to update this space and take what I’ve learned and where I plan to go and use that to expand the lab. I have a lot of new needs for a lab space and it doesn’t hurt to make some much needed improvements to this hundred year-old house in which we live.

I’m excited to announce the construction of the new lab space has commenced!

Plans for the New EverymanBio Lab

Here’s what I’m planning to do to prepare the space for the new lab build-out:

  • Refinish the beautiful douglas fir wood floors and seal them (this will make cleaning the floors much easier as well as brighten things up)
  • Install a Portable A/C to keep the space comfortable in the summer (this also allows me to close some sky-lights which help with cleanliness as well)
  • Add a fresh coat of paint
  • Install new bright and energy-efficient lighting
What Kind of Work the Lab will Support

The lab will be partitioned to support the various kinds of research and experiments I plan to do with a primary wet-lab workbench as the central hub. The room will also accommodate the following:

  • Space for my flow hood that is open to the rest of the lab
  • PCR thermocycler for DNA replication, sequencing and more
  • Electrophoresis gel-box and lighting
  • Microscopy section for creating, viewing and photographing slides
  • Mushroom grow tent with humidity and fresh air controls
  • Small culture incubator
  • Fridge for culture library and stock cultures
  • Greenhouse for botany and microgreen experiments
  • Maker table for diy electronics and instrumentation prototyping
  • Plenty of storage for disposables
  • Racks for forceps, pipettors, reagents and more
  • Dark-room box for photosensitive projects and photography
  • Space for a laptop and large monitor
  • Mounted cameras for live-streams and videos

I feel incredibly blessed and grateful to have this opportunity and my hope is that eventually, the lab will be buzzing with ongoing meaningful work that inspires and educates a new cohort of amateur learners.

Perhaps this lab space will evolve into a community lab that serves the South Los Angeles and greater area. This is only the beginning of many great things to come!

For now, its back to work on the lab, starting with those floors! Be sure to follow the EverymanBio Instagram for ongoing updates and photos tracking the progress of the build.

Working on the Floor of New Lab
Refinishing the New Lab Floors
Categories
Blog Experiments Mycology

Bioluminescent Mushroom Grow (Update #1)

It’s been a few weeks since I started my experiment in growing a bioluminescent mushroom and I thought it’d be a good time to give an update on my progress.

Not everything has gone according to plan, but that is to be expected for a DIYer working in a largely uncontrolled environment. In spite of the challenges, I was able to successfully capture a glowing petri dish and my friends, it was well worth the effort.

Update on the Goals of the Experiment

Let me review the goals and briefly update you on where I’m at with each one:

Replicate results from published research on the optimal growing conditions for maximizing luminosity of Panellus stipticus

I can say definitively that I was unsuccessful in replicating the two published research papers in their entirety.

Here’s a summary of what I’ve observed:

  • The growth of my cultures have taken 4x longer than published
  • I’ve been unable to see any light emitted from the liquid cultures
  • The MEA petri cultures grew quicker and do emit light

* Prepare and inoculate PG (potato glucose) liquid culture
* Test my new custom lids with self-healing injection ports
* Test which jar labels survive the pressure-cooker sterilization
* Calibrate my cheap pH meter found on Amazon

All of these goals were successful! I made PG liquid culture, although as mentioned above, the expected growth rates did not match the published research.

My new custom lids with injection ports and 2-ply breathable micropore tape appear to be working with one caveat: when I shake the jars to mix and breakup the growth, sometimes a little bit of culture seeps through the tape. This is also problematic when I draw a sample from the culture with a syringe. The needles I use aren’t very long and after a good shake, much of the culture floats to the top requiring that I tilt the jar on the side while I draw in a sample. If I’m not careful, I’ll tilt the culture jar so far that it can leak from the breathing port covered in the micropore tape.

As for the labels, both the tape and marker labels survived the pressure cooker. While I prefer the label that is written directly on the jar with a marker because it’s less work to add and remove (it wipes off easily with a little alcohol), it’s prone to smudging with all of the handling that happens during a grow cycle. The label written and affixed to tape takes longer to make but is generally more durable over time.

As for pH, I did calibrate my cheap meter with the reference solutions and use it to create cultures at varying pH levels around the published ideal of 3-4. But, I’ve spoken to many folks who strongly recommend using a better meter as these cheap devices are prone to unreliable readings. Unexpectedly, the jars where I did not do any pH adjustments appear to be showing the fastest colonization. These were also the cultures jars that showed a baseline reading of 7.3 – 7.6 pH.

What Might Account for the Differences in Results

I’m obviously not working under highly controlled laboratory conditions, but I have a hunch about what might be causing some of the variations in my results vs the published research.

  • My colonization temps have been about 10 degrees cooler.
  • I used a cheap, probably faulty, pH meter.
  • I adjusted the pH only before sterilization and not after.
  • pH was just adjusted using lime juice concentrate as opposed to HCL (hydrochloric acid) and NaOH (sodium hydroxide). Maybe the citrus concentrate had other compounds that interfered with growth.
Quick Note on Tracking the Experiment

I originally intended to track all of the experiment data in a structured and organized way, first starting with a spreadsheet and then working my way into something I could more easily update and share on the site.

Unfortunately, I still haven’t found a great solution. I don’t know yet how to properly quantify growth in a liquid culture given the manner in which it grows. I also don’t have meter to measure luminosity. So even if I had a good way to track and share the experiment data, it wouldn’t be so useful without these two important variables. Nevertheless, resolving these challenges are on my roadmap to sort out.

Now, onto the fun stuff – photo updates!

I’ve decided to remove these members from the experiment. 1-A is definitely contaminated and the rest are either showing little to no growth or questionable growth altogether.

Contaminated or Slow Growth Jars
Contaminated or Slow Growing Members Removed from the Experiment

This experiment required that I create a liquid culture for the first time. Based on everything I’ve read, healthy fungi liquid cultures should be crystal clear with the exception of clearly differentiated fungal growth suspended in the solution.

If we compare these two jars, it’s clear to me that 1-A is definitely not healthy and is contaminated.

Healthy Liquid Culture (2-A) vs. Contaminated Culture (1-A)

Let’s take a look at the remaining healthy liquid cultures.

Note how all jars show nice clear liquid substrate and coagulated clumps of the Panellus stipticus culture hanging out.

Healthy Liquid Culture Experiment Members
Healthy Liquid Culture Experiment Members
Close Up of Liquid Culture Growth
Close-up of Healthy Growth in Liquid Culture

OK – now, recall from that first part of this experiment that I added some members: some grain jars inoculated from healthy cultures and a couple of plates. Let’s take a quick peak at how those are coming along.

Panellus Stipticus Grain Jar
Visible Signs of Colonization
Close-up of Mycelium Growth on Grain

As you can see, we have some decent growth in these jars. The grains I’m using here are what I have available in my area: winter rye berries.

Note how the jar says 5mL on 6/1 and then +10mL on 6/8. That is because I added extra culture after a week of not seeing any growth. My advice is don’t do this! I was impatient and not only did I lose the data from the 6/1 inoculation, I introduced another opportunity for contamination.

Luckily, this jar is looking good! This species exhibits a light cottony wispy-like growth on the grain.

The Challenge of Capturing Bioluminescent Light

There’s a lot of information on the net and many cool pics of bioluminescent mushrooms, but much of it is conflicting. Some things say the LC culture glows, some say it doesn’t. Same for cultures on solid mediums. Some say the glow can be viewed with the naked eye.

I’ve heard some people say that the glow is so bright, it can illuminate a hallway enough to act as a nightlight.

In my experience, the glow is quite dim. It was not until I created a dark room in my closet and stood in it at night in complete darkness that my eyes adjusted enough to see a subtle glow from one of the petri dishes.

The night I discovered that the petri dish was glowing, I was ecstatic. I called my wife, Cat, into the closet and we just stared in awe of one of natures gifts. It was incredible.

Once I knew that the dish was glowing, I became semi-obsessed with taking a decent photo. First, I tried taking the photo with a paid iPhone app that provides fine-grained manual controls not available in the native app. This is the best I could achieve using an iPhone 11.

iPhone Attempt at Capturing Bioluminescence
iPhone Attempt at Capturing Bioluminescence

If you look closely, you can see a little bit of light but the overall image quality is terrible. The high ISO generates a lot of noise.

So I switched over to an old DSLR with a 50mm lens and started working on which manual settings would give me a better pic. Here’s what the first round of attempts looked like:

DSLR Pics of Bioluminescense Round 1
First Round of DSLR Pics

When I saw these, boy was I excited! For the first time, you could clearly see the green glow of the petri dish with no image processing.

But I didn’t stop there. I knew that it could get better. Specifically, the light was still so faint and blurry.

A little more trial and error, spending upwards of two hours in a dark closet, anxiously awaiting the result of a new round of manual settings, when finally, a wonderful preview comes across the display:

Photo of Bioluminescent Mushroom (Lights On)

Photo of Bioluminescent Mushroom on a Petri Dish
Photo of Bioluminescent Mushroom (with post processing)
Successful Capture of Bioluminescent Fungi Culture on MEA (No Image Processing)

The photo was taken with a Canon EOS Rebel T3i with a 50MM lens, manual settings of ISO 3200 f/1.8 with a 30-second exposure time.

What’s Next?

I’m absolutely thrilled to have successfully cultured and captured a glowing mushroom on camera. I’m moving forward with fruiting this culture and capturing more amazing shots while also expanding my learning of science, mycology and experiment replication.

If you have any thoughts about how to address some of the challenges I mentioned above or you want to jshare any feedback, drop me a comment below or on the EverymanBio instagram page.

See you soon!

Categories
Blog Experiments Mycology

An Experiment in Growing a Bioluminescent Mushroom

As a new member of the Los Angeles Mycological Society I was excited when I saw an email hit my inbox with an invitation to attend a presentation on the topic of “glow-in-the-dark”, or bioluminescent, mushrooms by Assistant Professor Brian A. Perry at Cal State East Bay.

Brain’s Presentation, dubbed Illuminating Fungi – The Science of Fungal Bioluminescense, walked us through the fascinating world of glowing mushrooms around the world and the current phylogenetic research he and his team are working on to better understand how luminescent fungi evolved this incredible ability independent of one-another, as well as any clues that might reconcile any taxonomic uncertainties.

He also gave an introduction to luciferase and luciferine, the enzymes and compounds that give rise to the bioluminescent reaction. I’ll be diving much deeper into these as I ramp up my DNA research in my home lab.

At the end of the presentation, I had the opportunity to ask Brian which species of luminescent mushroom would he recommend for home cultivation. His recommendation was to have a go at Mycena Chlorophos and ensure there is adequate caffeic acid available for the fungi to facilitate the enzymatic process of luminescence.

Up until that point, I had never heard of caffeic acid before. After doing a little research, I discovered that it’s a naturally occurring organic compound that naturally exists in all plants. Interestingly enough, it’s also used quite a bit in a cosmetics. If you search eBay for caffeic acid, you’ll see sellers marketing it as such.

Unfortunately, getting my hands on a culture of any Mycena fungi has proven to be difficult. I did; however, find an alternative that I’m moving forward with: Panellus stipticus aka The Bitter Oyster.

Determined to grow a glowing mushroom and capture some cool photos, I have decided to use this experiment as an opportunity to learn a variety of new mycological skills. Let’s dive into it.

Goals of the Experiment

This experiment presents an opportunity to learn and try many new tools, techniques and mediums which have been on my to-do list for some time. Although my main goal is to successfully cultivate and capture a bioluminescent mushroom, I’d like to take advantage of this project to learn some new skills that will needed for some of my future plans.

Here are some of the goals I put together for this experiment:

  • Replicate results from published research on the optimal growing conditions for maximizing luminosity of Panellus stipticus
  • Prepare and inoculate PG (potato glucose) liquid culture
  • Test my new custom lids with self-healing injection ports
  • Test which jar labels survive the pressure-cooker sterilization
  • Calibrate my cheap pH meter found on Amazon
  • Compare the luminosity of two strains across varying pH in liquid culture and in fruiting bodies
Panellus stipticus LC inoculation
Getting ready to inoculate the prepared LC jars

The Research

I am using two published research papers for this experiment:

1. Prasher, i.b. (2012). Prasher IB, Chandel VC and Ahluwalia AS. Influence of culture conditions on mycelial growth and luminescence of Panellus stipticus (bull.) P. Karst. Journal of research in Biology (2012) 3: 152 – 159. Journal of research in Biology. 3. 152-159.

2. Se, Medvedeva & Ks, Artemenko & Aa, Krivosheenko & Ag, Rusinova & Ek, Rodicheva & Ap, Puzyr & Bondar, Vladimir. (2014). Growth and light emission of luminous basidiomycetes cultivated on solid media and in submerged culture. Mycosphere. 5. 565−577. 10.5943/mycosphere/5/4/9.

I found these two publications by searching Google Scholar for Panellus stipticus and finding the most recent publications which were focused on assessing the affects of growing conditions on luminosity. Said differently: I wanted a paper that would tell me how to grow my cultures to maximize the brightness of the fungi.

The main findings I’m using to determine how I organize this experiment are as follows:

The greatest light emission was obtained when Panellus stipticus mycelia were grown on PGA and SabA (Fig. 2).

Panellus stipticus mycelium growing on all of the media exhibited long-term luminescence (more than 40 days) (Fig. 2, 4). Although the luminescence differed in the time of occurrence and intensity, its maximum was reached at certain times depending on the composition of the culture medium: after 4–5 days of cultivation on FA, after 9– 11 days of cultivation on PSA, after 11–14 days on PGA, after 10–11 days of cultivation on MEA, after 13–15 days on SabA and YMA. As noted above, Panellus stipticus mycelia did not reveal any considerable differences in growth of colonies on media with different compositions.

Se, Medvedeva & Ks, Artemenko & Aa, Krivosheenko & Ag, Rusinova & Ek, Rodicheva & Ap, Puzyr & Bondar, Vladimir. (2014)

Temperature and pH affect growth and bioluminescence to a great extent. Glucose-peptone medium has been found to be the best for optimum mycelial growth as well as luminescence. The fungus exhibits luminescence at 20-24°C. The maximum mycelia dry weight (mg/25ml of the basal media) and luminescence observed at pH 4.0. The fungus exhibits luminescence after eight days of incubation at 24°C and pH 4.0, whereas it intensified to maximum after 13 days of incubation (pH 4.0 and temperature 24°C).

Prasher, i.b. (2012). Prasher IB, Chandel VC and Ahluwalia AS. (2012)

I was originally only going to run this experiment with liquid cultures but after re-reading the papers, it looks like I’m going to need to grow out these species on solid agar mediums in order to maximize brightness. The solid mediums showed a significantly higher level of luminosity compared to their liquid culture counterparts.

Materials and Methods

For the experiment, I’m growing two strains of Panellus stipticus.

  1. Panellus Stipticus ME-P8 from MushroomEmporium.com 4/28 (Shipped from Maine)
  2. Panellus Stipticus from fungi_supply on eBay 4/24 (Shipped from Canada)

Here are the general materials and methods used to prepare the liquid cultures:

  • Boil 2 large peeled Russett potatoes on medium for an hour
  • Prepare 2L, 1L batch-wise, liquid culture (LC) using 200g/L strained potato broth and 20g/L Karo Dark Corn Syrup
  • Prepare 250mL Potato Glucose LC for 8 jars
  • Pressure-cooker sterilize culture jars for 20 mins at 15 PSI
  • Inoculate each jar with 1mL LC culture syringe (2 strains * 4 jars)
  • Store jars at room temp (my closet) and allow to incubate in the dark
Preparing Potato for PG Liquid Culture
Liquid Culture Prep
Experiment Setup

Updated 5/2/2020

After re-reading the aforementioned studies, it occurred to me that liquid cultures yielded the lease amount of luminescence over solid growth mediums. In order to improve my chances of capturing the luminescence with the naked eye, I have added two grain and two MEA agar cultures into the mix.

The plates and grain jars were innoculated from a selection meeting the following criteria:
* Jars and plates would represent both A & B strains
* LCs would have pH closests to 3-4 which the research showed optimized luminosity
* I would take the LCs which shows the most aggressive growth so far

Accordingly, here is a photo of the new additions to the experiment with the source liquid cultures. Note the growth that is visible at the bottom of the jars.

The grain jars contain winter rye berries PC for 90 mins @ 15PSI. The jars were inoculated with 5mL and the plates with 1mL of LC via syringe.

Additional Cultures of Experiment
Solid and Grain Media Cultures Added to the Experiment on 6/2/2020.

Results

I am working on finding a better way to track and share the experiment results. For now, here’s an initial overlay of the experiment variants.

Come back here to find updated results and *hopefully* some really cool glowing mushroom photos as the experiment progresses.

Updates
Be sure to check out the following for updates to this experiment, including cool photos and more:

Bioluminescent Mushroom Grow (Update #1)