TITLE: General Media Recipes: WA, PDA, LB, CMA, V8 
AUTHORS: rebecca.bennett, Nimalka M Weerasuriya

[DESCRIPTION]
These are current general media recipes for (acidified) water agar, (acidified) potato dextrose agar, LB broth, cornmeal agar, P5ARP/P10ARP, and clarified V8 juice agar.

[BEFORE_START]
Prepare the following:
Turn on water bath if making more than 3-4 bottles of poured media, or if making P5ARP/P10ARP. Set to 50 °C. 
Prepare hood by spraying surfaces with ethanol and allowing hood to equalize for 15 min minutes.
Open the necessary quantity of Petri plate bags and stack in hood (500 mL agar =~ 1.5 sleeves of 90 mm plates, 2 sleeves of 60 mm plates).
Standardize pH meter, if applicable (aWA and aPDA).
Thaw any antibiotics, if applicable.

[GUIDELINES]
These are media recipes.

[STEPS]
SECTION: Making Media
1. Select media types from below:
SECTION: Preparation
4. Autoclave liquid setting for 20 minutes to sterilize.


SECTION: Preparation
3. Add stir bar and stir contents well. 
Place a loose orange cap. Label cap with autoclave tape (1.5% WA) and date. 
SECTION: Preparation
2. Water Agar preparation:
 
500 mL ~ 1.5 sleeves of 90 mm plates or 2 sleeves of 60 mm plates.
SECTION: Preparation
2. To make 1/4 strength PDA:
 


SECTION: Preparation
3. Add stir bar and stir contents well. 
Place a loose orange cap. Label cap with autoclave tape (1/4 PDA or aPDA), and date. 
SECTION: Preparation
4. Autoclave liquid setting for 20 minutes to sterilize.


SECTION: Acidified WA (aWA)
5. To make acidified WA (aWA):
Adjust to pH 3.9 after autoclaving with 10 % (v/v) lactic acid 
~0.5 mL for 500 mL of media

SECTION: Acidified PDA (aPDA)
5. To make acidified PDA (aPDA):
Adjust to pH 3.9 after autoclaving with 10 % (v/v) lactic acid 
~0.5 mL for 500 mL of media

SECTION: Making Media
2. To make LB Agar:
 

SECTION: Making Media
3. Add stir bar and stir contents well. 
Place a loose orange cap. Label cap with autoclave tape (LB), and date. 
SECTION: Making Media
4. Autoclave liquid setting for 20 minutes to sterilize.


SECTION: Preparation
2. To prepare Cornmeal Agar (CMA) for Pythium:

 


SECTION: Preparation
3. Add stir bar and stir contents well. 
Place a loose orange cap. Label cap with autoclave tape (CMA), and date. 
SECTION: Preparation
4. Autoclave liquid setting for 20 minutes to sterilize.


SECTION: After Pouring Media
6. When media has solidified, place in bags labeled with media name, your initials, and date.
SECTION: Making Media
3. For V8 Juice Clarified Broth:
 

 

SECTION: Making Media
3.2. Optional:
Add cholesterol from a 1.5 % (v/v) solution:
2 mL Cholesterol
2 mL 95% Ethanol
SECTION: Making Media
4. Add stir bar and stir contents well. 
Place a loose orange cap. Label cap with autoclave tape (CMA), and date. 
SECTION: Making Media
5. Autoclave liquid setting for 20 minutes to sterilize.

SECTION: Making Media
3.1. To V8 juice, add calcium carbonate and clarify by centrifugation at 13200 rpm, 30 min. Add agar. Bring to 1 L RO water.

SECTION: Making Media
2. Set water bath to 50 °C to 55 °C.

SECTION: Making Media
7. Prep pH station by standardizing probe. See protocol at pH bench.
Adjust to pH 7.2 ± 0.2

 

SECTION: Making Media
6. Place in water bath and cool to 55 °C. 
SECTION: After Pouring Media
7. When media has solidified, place in bags labeled with media name, your initials, and date.
SECTION: After Pouring Media
7. When media has solidified, place in bags labeled with media name, your initials, and date.
SECTION: After Pouring Media
5. Mark plates with media color codes. 
  
Turn off water bath.
Turn off autoclave, if no longer needed.
Spray inside of hood with ethanol.
Close filling port in pH meter, if necessary.
Tightly cap empty media bottles so agar doesn’t dry out.

SECTION: After Pouring Media
6. When media has solidified, place in bags labeled with media name, your initials, and date.
SECTION: After Pouring Media
5. Mark plates with media color codes. 
  
Turn off water bath.
Turn off autoclave, if no longer needed.
Spray inside of hood with ethanol.
Close filling port in pH meter, if necessary.
Tightly cap empty media bottles so agar doesn’t dry out.

SECTION: After Pouring Media
6. Mark plates with media color codes. 
  
Turn off water bath.
Turn off autoclave, if no longer needed.
Spray inside of hood with ethanol.
Close filling port in pH meter, if necessary.
Tightly cap empty media bottles so agar doesn’t dry out.

SECTION: After Pouring Media
6. Mark plates with media color codes. 
  
Turn off water bath.
Turn off autoclave, if no longer needed.
Spray inside of hood with ethanol.
Close filling port in pH meter, if necessary.
Tightly cap empty media bottles so agar doesn’t dry out.

SECTION: After Media Preparation
8. Mark plates with media color codes. 
  
Turn off water bath.
Turn off autoclave, if no longer needed.
Spray inside of hood with 80% ethanol.
Close filling port in pH meter, if necessary.
Tightly cap empty media bottles so agar doesn’t dry out.