Understanding Sterile Workflow

Part 1 : Unit 6 : Understanding Sterile Workflows

The first few steps of mushroom cultivation require very clean conditions, as close to sterile as possible. This is to ensure that spores and mycelium are allowed to grow in a competition free environment, with no other rogue fungi, bacteria or other contaminants. While a completely sterile environment is hard to achieve,  you can reach almost sterile conditions with the help of a Still Air Box, and a good understanding of sterile workflows.

In this module we will discuss the procedures for selecting a good workspace and keeping it clean and ready for cultivation work. We will also discuss how to prepare and work your Still Air Box to achieve close-to-sterile conditions. Rinse up, scrub down and grab a spray bottle, we’re almost ready!

Sterile workflow procedures
Sterile Workflow Procedures

Table of Contents - Understanding Sterile Workflow

Understanding Sterile Workflow in Cultivation

Video Tutorial : Preparing and Cleaning your Work Environment and SAB

The video markers above 👆🏼correspond to the the various Steps in this Unit. Click on them to skip to a Step.

Step 1 : Select your Mushroom Cultivation Workspace

Your first task is to choose your workspace – the area where you will be working, cutting, pouring, injecting and mixing stuff. You want to find a small room or corner which is relatively clean, and has little airflow around it. A small desk which can be wiped down would work great as well. If nothing comes to mind, the bathroom is always a viable option – a small room which can be easily washed down and cleaned up. Before you start working in your workspace, close the windows and turn off the fan or AC in order to minimize the amount of airflow and avoid introducing contaminants from outside.

Step 2 : Prepare Yourself for Clean Work

Statistically speaking, your body is the most common source of contamination. Coodies in your saliva, germs in your breath, dead skin falling off your arms and dirt under your fingernails – you are literally a cultivation disaster waiting to happen. For this reason you should strive to shower or clean up before any processes which require cleanliness or sterility. Wash your hands and forearms well, trim fingernails, collect your hair (if you have any remaining) and wear a face mask and nitrile / latex gloves when you work. All these measures will help minimize the chances of early contamination of your Petri Dishes and Liquid Cultures.

Step 3 : Clean and Disinfect your Work Surfaces

Now that you and your environment are clean, we can start focusing on your work surfaces and specific work areas. Before using your Still Air Box, fill one spray bottle with 70% alcohol, and another with 3% or 5% hydrogen peroxide. Start by wiping down all surfaces with 70% alcohol and let the alcohol dry out. Once dry, you can preform another wipe down with 3% or 5% hydrogen peroxide. Do this with any surface you or your tools will come into contact with during your work.
Disinfecting table and happy because of it

Step 4 : Clean and Disinfect your SAB and Work Tools

Next, you need to clean and disinfect your Still Air Box and the tools you will be using inside the SAB. Both the inside and outside surfaces of the SAB should be be sprayed down with 70% alcohol and left to dry out, followed by another spray down and dry out using hydrogen peroxide. Once the SAB is dry, it’s ready for use. Continue by disinfecting any tools, jars or containers which you are going to use. Spray all of these with alcohol, give them a gentle wipe down, place them in the SAB allow them to dry. Slowly add each of the items and tools which you’ll need into the SAB after disinfecting or sterilizing them. Lastly, disinfect the alcohol spray bottle itself, and place it in the SAB, together with a few alcohol wipes, or some paper towels sprayed with alcohol.

Step 5 : Maintain a Sterile Workflow Mindset

Now that everything is clean and ready, you’re ready to start clean work inside your Still Air Box. Place your hands in the SAB, and spray your gloves with 70% alcohol. Now spray the air in the SAB with Alcohol in order to bring down any contaminants in the air, wait for a few seconds, and using minimal movements, complete the task at hand – whether it’s scraping spores into agar or pouring out petri dishes. During this time, you need to be very aware of every movement you make. Did you take your hands out of the SAB to scratch your nose? Contamination! You need to re-sanitize your hands. Did you wipe some sweat off your forehead? Contamination! You need to re-sanitize your hands. Every movement that you make needs to be calculated from the perspective of avoiding contamination. The more you pre-plan and predict the actions you’ll need to take, the smoother and more sterile your work will be. Every so often during your work in the SAB, it’s not a bad idea to re-mist the air in the SAB enclosure with water or alcohol to help bring any airborne particles down. Re-sanitizing your hands inside the SAB as you touch various items or between operations is also a good habit to pick up. Remember that a SAB works by having the air inside it fairly clean and still, and thus less contact with contaminants will be made. As such, avoid sudden movements, or any actions inside the SAB that may draw air in or out of the SAB. Admittedly, this strict mindset makes the first few times of using a SAB a bit stressful, however you soon get the hang of it and it becomes second nature.  Today, I find SAB work to be very meditative and relaxing. I like putting on my headphones and listening to music while doing all my clean work. To see some Still Air Box action, head on to the “Germinating Mushroom Spores in Agar” section.
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Next Unit: You’ve finished all your preparations for the grow! You can now head on to the mushroom cultivation section and experience the lifecycle of a speck in the kingdom fungi. 🍄❤️

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