Per 1 L

• 100 mL 10X M9 salts
• 240 mL glycerol
• 300 µL 1M MgSO4
• water to 1L

Filter sterilize

Per 1 L:

• 100 mL 10X M salts
• 300 µL 1M MgSO4
• water to 1 L

Filter sterilize

Per Liter of medium (~75 plates):

• 975 mL Water
• 3 g NaCl
• 2.5 g Peptone (Fisher BP1420-500 $78.80)
• 17 g Bactoagar

Autoclave with stir bar inside

Cool to 55C in a 55C water bath

Add per L (see recipes in stock solutions):

• 1 mL cholesterol (5 mg/mL in 95% EtOH)
• 1 mL CaCl2 (1 M, STERILE)
• 1 mL MgSO4 (1 M, STERILE)
• 25 mL K-phosphate buffer (1M, pH 6.0, STERILE¹)

Swirl flask to mix

Dispense 10 mL into each 60mm dish.

Stack 10 high

Let stand for ~48 hours for condensation to evaporate

Pack in sterilized plastic boxes.

To make	NGM agar plates
at	mm plate diameter
pour	mL per plate
start with:	mL water
add:	g peptone, stir till dissolved
add	g NaCL, stir till dissolved
add:	g agar, leave stir bar in
autoclave for:	minutes
add:	mL 5mg/mL cholesterol
add:	mL sterile 1M CaCl2
add:	mL sterile 5mg/mL 1M MgSO4
add:	mL sterile K-phosphate buffer , pH 6

put bottles on stir plate near sterile hood until handle-able