This page outlines the methods that may be needed for anyone working on the mycoprotein (waste to food) project. It outlines things like plate and media preparation, autoclave use, simple microbial transfer techniques. Page will be updates once work has on the project has started.
Safety Training Requirements[edit | edit source]
- Dr. Pearce Safety Lab Tour
- Worker Health & Safety Awareness
- WHMIS 2015 - Workplace Hazardous Materials Information System
- Laboratory Safety & Hazardous Waste Management
- Biosafety training
Safety Issues With Method[edit | edit source]
Personal protective equipment[edit | edit source]
PPE is to keep YOU safe so that you can keep doing what you want to do.
- Safety glasses – always required
- Lab coat - always required
- Gloves - may be required
SDS and other[edit | edit source]
Knowing what chemicals are in the lab and how they interact with each other is critical when accidents happen.
- Appropriate SDS sheets should be viewed online.
- Note the hazards listed on the door to the lab. If you introduce any new equipment or materials you must clear them with the responsible person listed on the lab door. If the responsible person is out of date, contact the departmental administrators to get it updated.
Using Autoclave[edit | edit source]
Location: Biology and Geographical Sciences Building Room 3071
Important: MAKE SURE THE LIQUID ONLY FILLS UP TO HALF OF THE GLASSWARE CONTAINER!!!
Operation:
After preparing media in glass, autoclave-safe flask / jar, cover with aluminum foil. Label the glassware with what is inside, and attach a small piece of autoclave tape on the foil "lid". Jars may be lidded, and if so, do not close the lid tightly or it may explode in the autoclave. Place the media-filled glassware in a metal tray found on the counter near the autoclave, fill the tray with water (up to about an inch deep), and carefully place in the autoclave. Close the autoclave and choose the cycle. Depending on the volume in each flask / jar, a different cycle may be needed. Typically, for volumes under 500 mL, use Liquid30 cycle which takes about an hour. Once complete, open the autoclave and leave it open for a few minutes. TAKE A STEP BACK TO AVOID HOT STEAM FROM BURNING YOUR FACE! Using heat-safe gloves (also found on the counter near the autoclave), carefully take out the tray. Remove your media and dump the water in the sink. After leaving the autoclave open for a few minutes, do not forget to close it again. Be sure to record information on the sign in/out sheet found on the counter and record any notes/abnormalities or just put a check mark if everything is okay. The autoclave tape on the media should now have black stripes, indicating it has been autoclaved.
FOR DRY MATERIALS (unlikely to need this but just in case):
Very similar to liquids except a few key differences:
- Do not add water to the tray
- Use the cycle called gravity (might be gravity20, grav, or something similar... just not a liquid cycle)
Please note that autoclaves may vary. Always ensure that you have the appropriate training and/or are under the supervision of someone that knows how to use it. Misuse can be dangerous.
Preparation of malt extract agar plates for growing Fusarium venenatum[edit | edit source]
To prepare malt agar plates for growing F. venenatum stocks, measure out appropriate mass of malt extract and agar, and add to DI H2O. For 200 mL, measure 6 g malt extract and 3 g agar. Add to 100 mL DI H2O to a flask, stir until dissolved, and top up to 200 mL with more DI H2O. Cover with aluminum foil, add autoclave tape, and autoclave. Once autoclaving is complete, let the liquid cool to about 50°C before pouring plates (it will solidify by the time it gets near room temperature).
In a sterile workspace, arrange petri dishes so that lids can be lifted easily with one hand and molten agar can be poured with the other hand. Pour enough liquid until the surface is mostly covered and cover with lid. Swirl to cover entire surface, and let the plates sit until agar has solidified. Depending on how good the sterile environment is, it may be beneficial to leave the lid slightly open to avoid condensation build-up. Once solidified, seal with parafilm (optional but will help protect against contamination, although not perfect) and store plates upside down. Label each plate with type of media and date made (label plates on bottom, not on lid). Store plates at 4°C or in a cool place.
Preparation of liquid culture media for Fusarium venenatum[edit | edit source]
To prepare liquid media for growing F. venenatum, malt extract and alternative media will be used. The preparation of media is the same, except for the components being dissolved (e.g., malt extract media will contain malt extract, whereas the alternative media would have glucose, magnesium sulfate, etc.). Start by measuring out components and dissolving in 200 mL DI H2O in a labelled glass jar. Cover with aluminum foil and loosely attach the lid before autoclaving media. Once autoclaved, let it cool and close the lid completely. Store until needed.
Transferring F. venenatum growth from plate to plate[edit | edit source]
In sterile conditions, open the petri dish containing F. venenatum mycelium and cut out a small piece of agar. Take it out and place it upside-down on a new malt agar plate. Incubate the newly inoculated plate at room temperature. It should grow in about 4-5 days.
Transferring F. venenatum to small-volume liquid media[edit | edit source]
In sterile conditions, open the petri dish containing F. venenatum mycelium and cut out a piece of agar containing growth. Take it out, break it into a few small pieces, and put it in a jar containing 200 mL of sterile media. Close the jar lid and incubate at room temperature. It should take a few days to grow, and it is best to give it a swirl every few hours (or keep it on a shaking incubator at a low speed if available).
Transferring from small-volume to large-volume[edit | edit source]
Simply pour the jar of 200 mL media with F. venenatum growth into the larger volume, close the lid, and incubate at room temperature. Larger volumes will likely grow slower. Ensure the media is swirled every few hours if not on a shaking incubator.
Depending on the end-goal volume, may need to scale up gradually. Some organisms simply do not grow in too large volumes as it dilutes it too much, so you may need to transfer 200 mL to 1 L, and then 1 L to 5 L, etc.
Experimental Overview[edit | edit source]
The goal is to see if Fusarium venenatum, a fungi used in the production of mycoprotein, can be grown on various waste media. The outline of the experiments will consist of 2 major parts: maintaining the F. venenatum stock on malt agar petri dishes and growing F. venenatum on various media.
Maintaining the F. venenatum stock[edit | edit source]
Materials required: prepared malt extract media agar in petri dish and a petri dish containing F. venenatum growth.
Method: see the "Transferring F. venenatum growth from plate to plate" section above. One plate should be viable for a few weeks if stored in clean conditions, but always check for contamination before use. This can also be used to check for contamination of sample used for liquid growth, as anytime a petri dish containing F. venenatum is opened for transfer to liquid media, a sample can also be plated. This will help maintain the stock, but also if anything unexpected grows on the plate, it is possible that liquid media inoculated at that time was contaminated.
Growing F. venenatum on various media[edit | edit source]
Materials required: sterile, prepared media and petri dish with F. venenatum.
Method: See "Transferring F. venenatum to small-volume liquid media" section above. This part also consists of two parts: growing in small-volume (in a glass jar) and then transferring to a larger volume (in bioreactor). This is because starting with a large volume may be difficult for growth as some organisms cannot grow in very dilute conditions.
Start with malt extract media, then try an alternative media. If both work, then experimentation can begin with all sorts of media; pressure-cooked cardboard, plastic waste, food waste are all good starting points to consider.
Here is some information on the composition of the malt extract and alternative media:
| Component | Concentration | Mass for 100 mL | |
|---|---|---|---|
| Malt Extract (BD 2118630) | 30 g/L | 3 g | |
| Agar | 15 g/L | 1.5 g |
Dissolve in distilled water and autoclave. For liquid media, do not add agar.
| Component | Concentration | Mass for 100 mL | |
|---|---|---|---|
| Glucose | 25 g/L | 2.5 g | |
| MgSO4 | 0.15 g/L | 0.015 g | |
| KH2PO4 | 1.6 g/L | 0.16 g |
May also need to add (NH4)H2PO4 at a concentration of 4.5 g/L if available (nitrogen source). Dissolve in distilled water and autoclave.
Following F. venenatum growth[edit | edit source]
After growing F. venenatum in a relatively large amount, it needs to be processed to actually produce mycoprotein. This still needs to be looked into, but it will likely involve filtering out the liquid media to separate the biomass, drying the biomass and heating it to decrease RNA content, and then mixed with a few other ingredients (e.g., vegetable oil, egg).
References[edit | edit source]
