We'll call this DIY Murashige and Skoog inspired medium “PTC20 agar, v0.1”. It consists of 7 g/L agar, 0.2 g/L Miracle-Gro (SKU# 1001123), 20 g/L sucrose, and enough vitamin B-complex to ensure at least 0.5 mg/L each of pyridoxine HCl and niacin and 0.1 mg/L of thiamine. The main idea is to see what can be done using only ingredients we can buy at the grocery store or the dietary supplements section of the pharmacy.
1 L volumetric flask, measuring cup, or beaker (in order of preference)
500 mL graduated cylinder
1 L culture media bottle with cap
autoclave or pressure cooker (e.g. Instant Pot®)
For pouring plates:
sterile plates or plant culturing containers
At BosLab, we can use sterile petri dishes (cheap, plentiful) or Falcon tubes (expensive and scarce).
At home, I plan to use pressure-cooked 1 cup mason jars with glass lids.
non-contact thermometer, such as an infrared thermometer
sterile work area such as a biosafety cabinet (best), laminar-flow workstation, the BosLab PCR hood, or work on a sanitized surface within 50 cm of a bunsen burner flame
This is deliberately less than 500 mL to allow room for adding the 25 mL of vitamin B-complex stock solution right before using the media.
Pour the beaker contents into the culture media bottle.
Add 3.5 g agar to the media bottle.
Cap the bottle and shake to mix the agar into the water, but note that it will NOT fully dissolve until we autoclave/pressure-cook it.
Loosen the cap of the bottle.
Label the bottle with “PTC20 agar v0.1 for plant tissue culture” and write your initials and the date.
Check that the culture media bottle cap is loose before autoclaving or pressure-cooking. This is important to prevent it from exploding.
Autoclave or pressure-cook 121°C and 15 psig for 1 hour.
Allow to cool to room temperature.
Tighten the media bottle cap.
Store at room temperature or refrigerate at 4°C until needed.
If refrigerating, it may be helpful to store the media bottle next to the vitamin B-complex stock solution as they will be used together when culturing.
Microwave in 30-second intervals until the media is fully melted.
Boil the media in the microwave for a total of 1 minute to ensure it is completely liquified.
Using an infrared (IR) thermometer, wait until the media cools to 50°C.
If you don't have an IR thermometer, wait until the bottle is hot but OK to touch for 3 seconds without pain, but this is obviously subjective and not very repeatable. If you take this approach, be sure to note this in your lab.notebook.
IR thermometers may not be as accurate as an instant-reading meat thermometer dipped into the media, but if you choose this route, obviously you have to