NGM Agar Medium with Amphotericin B and Nystatin

Note 1: for our cephalob cultures from Deep Canyon, we use a pH that is consistent with our desert soil (7.8-8.0)

Note 2: These plates can be stored (4 C) and used for about 1.5 months. Often we make a “half batch” of plates rather than the full 500 ml as indicated below.

Note 3: 1M CaCl2, 1M MgSO4, and 1M KPO4 buffer are prepared by filter sterilizing before use (can be stored in a sterile tube once filtered)

For 500 (250) ml of medium in a 1 liter flask

Plain agar        8.75 g (4.375)

NaCl                1.5 g (0.75)

Peptone           1.25 g (0.625)

Plain agar (50% Bacto Agar 214010, 50% SuperPure Agar US Biotech Sources A01PD-500)

Peptone (Tryptone) US Biotech Sources P004-1001

Add 488 ml (244) of distilled or Reverse Osmosis water

Cover flask with aluminum foil

Autoclave on liquid cycle; Cool flask in water bath at 55 C for 15 minutes or more. When equilibrated at 55 C, add the following aseptically:

0.5 ml (0.25) Cholesterol soln (5mg/ml in 100% ethanol) – store stock solution at 4C (make new every 4 months) Cholesterol Sigma C-3045

0.5 ml (0.25) filter sterilized 1M CaCl2 (Fisher C70-500)

0.5 ml (0.25) filter sterilized 1M MgSO4 (Fisher M63-500)

12.5 ml (6.25) filter sterilized 1M KPO4 buffer (pH 8.0 buffer)

NOTE: (94 ml 1 M dibasic; 6 ml 1M monobasic) yields media that without agar has a pH of 7.7)

2 ml (1.0) Amphotericin B (250 ug/ml) = 2x amphotericin B

2.5 ml (1.25) Nystatin (10,000 units/ml) = 1x or 25U/ml Nystatin

Fungicides (store frozen):

Amphotericin B, Fisher BP2645-20 (250 ug/ml), Nystatin, Sigma N1638-100 ml (10,000 units/ml)

Mix by swirling and pour plates. Incubate plates at 37C overnight. Then repackage (store in original bags, but with agar side “upside down”) in refrigerator.