Feb 28, 2024

Public workspaceGeneral Media Recipes: WA, PDA, LB, CMA, V8 V.3

This protocol is a draft, published without a DOI.
  • 1USDA ARS;
  • 2USDA
Nimalka Weerasuriya
Open access
Protocol Citation: Rebecca Bennett, Nimalka Weerasuriya 2024. General Media Recipes: WA, PDA, LB, CMA, V8 . protocols.io https://protocols.io/view/general-media-recipes-wa-pda-lb-cma-v8-c9ubz6snVersion created by Nimalka Weerasuriya
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95843
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
Abstract
These are current general media recipes for (acidified) water agar, (acidified) potato dextrose agar, LB broth, cornmeal agar, and clarified V8 juice agar.
Guidelines
These are media recipes.
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 Temperature50 °C .
  • Prepare hood by spraying surfaces with ethanol and allowing hood to equalize for Duration00:15:00 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.
Select media types from below:
Step case

Water Agar (1.5%)
6 steps

WA and aWA (1.5%)
Preparation
Preparation
Water Agar preparation:
ABC
500 mL250 mL
Bottle size (mL)1000500
Agar (g)7.53.75
RO Water (mL)500250
500 mL ~ 1.5 sleeves of 90 mm plates or 2 sleeves of 60 mm plates.
Add stir bar and stir contents well.
Place a loose orange cap. Label cap with autoclave tape (1.5% WA) and date.
Mix
Autoclave liquid setting for 20 minutes to sterilize.

Acidified WA (aWA)
Acidified WA (aWA)
To make acidified WA (aWA):
Adjust to Ph3.9 after autoclaving with Concentration10 % (v/v) lactic acid
  • Amount~0.5 mL for 500 mL of media
After Pouring Media
After Pouring Media
  • Mark plates with media color codes.
Bennett Lab 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.

When media has solidified, place in bags labeled with media name, your initials, and date.
Protocol references
Singleton, L. L., Mihail, J. D., & Rush, C. M. (Charles M. (1992). Methods for research on soilborne phytopathogenic fungi. APS Press.

Clarified V8 Juice Broth
- Ayers, W. A., & Lumsden, R. D. (1975). Oospores of three Pythium species. Phytopathology, 65, 1094–1100.

P5ARP/P10ARP
- Ferguson, A. J., & Jeffers, S. N. (1999). Detecting Multiple Species of Phytophthora in Container Mixes from Ornamental Crop Nurseries. Plant Disease, 83(12), 1129–1136. https://doi.org/10.1094/PDIS.1999.83.12.1129
- Jeffers, S. N., & Martin, S. B. (1986). Comparison of Two Media Selective for Phytophthora and Pythium Species. Plant Disease, 70(11), 1038–1043. https://doi.org/10.1094/PD-70-1038
- https://wiki.bugwood.org/PARP_or_PARP

CMA
- Tuite, J. (1969). Plant pathological methods. Fungi and bacteria. In Plant Pathological Methods: Fungi and Bacteria. Burgess Publishing Co. .

PDA
- Nelson, P. E., Toussoun, T. A., & Marasas, W. F. O. (1983). Fusarium species: an illustrated manual for identification.

WA/aWA
- Singleton, L. L., Mihail, J. D., & Rush, C. M. (Charles M. (1992). Methods for research on soilborne phytopathogenic fungi. APS Press.

Some modifications made by Bennett Lab.