Plate pouring protocol for NGM and HGMA.

General notes

• 6 cm plates take 13.5 mL of solution
• 10 cm plates take 32.5 mL of solution

Preparing NGM and HGM media mixes

The dry ingredients can be pre-measured into the plastic bottles labeled "For NGM Prep" (Shelf 2). Label the bottle with tape stating the type of media and final volume (e.g. NGM-1L or NGM-2L), the date of preparation, and your initials.

NGM recipe

HGM recipe

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Ambiguous wording for the A variants. The source says: for NGMA plates, "add 7 g of Agarose and 10 g of Agar per 1 L"; for HGMA plates, "add 8 g of Agarose and 12 g of Agar per 1 L". In both cases the two figures sum to the base recipe's agar amount (17 g and 20 g respectively), which suggests these replace the agar row rather than being added on top of it. Confirm before pouring — reading it as "add" would double the gelling agent.

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Preparing media for pouring

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Renumbered during migration. The source numbered this section 1, 1, 4, 5, 6 — two steps numbered 1, then a jump past 2 and 3.

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1. Combine all reagents in a flask that can hold 2× the volume of your media.

   • Put a plastic funnel in the mouth of the flask.
   • Dump the contents of the bottle into the flask.
   • Pour sterile water into the bottle and swirl to get any powder reagents off the sides.
   • Pour the water from the bottle into the flask, rinsing any powder stuck on the funnel into the flask.
   • Repeat the previous two steps until you've poured in all the sterile water.

2. Cover the mouth of the flask with foil and tape in place with autoclave tape. Use label tape to label the flask with the media type, date of pour, and your initials. You can simply move the tape label from the plastic bottle to the flask, adding the date of pour.

3. Add a stir bar.

4. Autoclave the following with a 60 minute liquid cycle:

   • 1 L H₂O in a 1 L bottle.
   • Liquid dispenser hoses, capped and wrapped with aluminum foil.
   • The NGM or HGM media.

5. Turn on the water baths in lab room 230 and set to 55°C while the media is still in the autoclave. Once sterilization is complete, bring the flask to the lab and allow it to cool to 55°C in the water bath for at least an hour, maximum four hours. Then add the following in the exact order listed, gently swirling to mix after each addition:

   Reagent	1 L	2 L
   1 M KH₂PO₄ (K phosphate buffer)	25 mL	50 mL
   Cholesterol (5 mg/mL in ethanol)	1 mL	2 mL
   1 M CaCl₂	1 mL	2 mL
   1 M MgSO₄	1 mL	2 mL

   If these solutions are already made, make sure they are autoclaved — except cholesterol — or filter-sterilized before adding to the media.

Operating the PourBoy