Making Agar Media for Yeast Culturing

First, a note on plastics (if you choose to buy plastic tubes instead of the glass vials listed above):

Polyethylene (PE) and polystyrene melt in an autoclave. Polypropylene (PP) is autoclavable. Be advised that PP tubes may have PE caps. MAKE SURE (i.e., ASK).... If the caps are not autoclavable, you will need to use the solid black rubber stoppers. My recent experiences with the PP tubes (first using the PE caps that I thought were PP, second using rubber stoppers that didn't quite fit right because they were the closest I could find to the right fit) was, to say the least, highly unsatisfactory, proving, IMHO, that borosilicate glass vials/tubes with autoclavable screw-on caps is the only way to go.

In addition to what's already been listed, ou will need a box or other platform with which you can make a slanted surface to allow the slants to cool/solidify at an angle. This is what I came up with:

The Slant Box (front view)

The Slant Box - Front View

The Slant Box (side view)

The Slant Box - Side View

There are two cigar boxes in the cardboard box, and a rigid cardboard tube (same size and shape as a paper towel roll, but thicker and stronger). Anything that elevates the front to about a 30 deg. angle will work, as long as it can be secured so it doesn't slip.

Mixing agar:

  1. First, make starter wort using a good yeast starter wort pack (e.g., the ones available from HomeBrew Den, which make about 900mL per pack). Add one hop pellet in a stainless steel tea strainer (available from any Thai market, probably any Asian market, British market, etc.). Boil for 10 minutes or until hot break forms (foam on surface clears as colloidal proteins coagulate and form solid chunks), whichever is longer.

  2. Gently pour (you do NOT want HSA here) into an Erlenmeyer flask (or flasks—whatever fits in your fridge) and cap securely with solid rubber stoppers, plastic wrap, aluminum foil, or whatever combination of these gives you a good, secure seal that will prevent microbiological nasties from being blown into your wort. Cool wort in fridge or ice bath to form the cold break.

  3. Filter 250mL of the wort into a 600mL beaker using a sanitized coffee filter (I use the re-usable type found in the coffee section of just about any grocery store). The idea here is to get the cold break and hot break material out.

  4. Filter remaining wort into 250mL Erlenmeyer flasks, cap with solid black rubber stoppers (number 6) and set aside to autoclave.

  5. Bring wort back to a boil (simmer in a boiler with water in the boiler). Remove from heat and add 3 level tsp (1 TBSP) of the agar-agar.

  6. Swirl or stir (do NOT shake) to dissolve agar into wort

  7. Use a sanitized 10mL syringe and fill each tube to about 1/4 full (keep the agar's flask in a boiler w/ simmering water while you're working, or the agar may solidify before you finish...mixing it too thick will also bring about premature solidification)

    For plates, fill each plate with 10mL of prepared agar.

  8. Keep the slant tubes' lids slightly loosened (just enough to vent, about 1/4 turn max).

  9. Load tubes and plates into 600mL beakers (keeps them out of the water) and the beakers into the autoclave (pressure cooker) and autoclave at 250 deg. F (15 PSI) for 30 minutes. Allow to cool, as usual, until no hissing when weight is tipped.

  10. Remove tubes and place on a rack or other platform such that they are laying down at approximately 30–40 degrees to form the slant.

  11. Remove plates and place on a level platform.

  12. Cool at that angle in the fridge—should solidify in a few hours or overnight. Close lids on slants completely AFTER cooling, not before, particularly if stoppers are used (they may be pulled into the tube as the tube's contents cools).

  13. Stretch Parafilm around plates (around lid/plate edge) and slants (around point where tubes and caps/stoppers meet) to seal. For slants, plastic wrap and/or aluminum foil is ok, too.

  14. Put in a ziploc bag and into the fridge until needed.

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