Aug 15, 2024
Acidified 0.5x Potato Dextrose Agar (APDA)
- 1Oklahoma State University
Protocol Citation: Gabriela S Paredes 2024. Acidified 0.5x Potato Dextrose Agar (APDA). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk82jdl5r/v1
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: August 14, 2024
Last Modified: August 15, 2024
Protocol Integer ID: 105297
Keywords: APDA, Plant Pathology, microbiology, cultivation of fungi
Funders Acknowledgements:
USDA
Grant ID: 2021-67013-33943
Disclaimer
This protocol is provided for research and educational purposes only. Ensure that all procedures are conducted by trained personnel following relevant safety and ethical guidelines. The authors and publishers are not responsible for any injuries, damages, or legal consequences resulting from the use of this protocol.
Abstract
Acidified 0.5x Potato Dextrose Agar (APDA) is a specialized culture medium used in microbiology for the selective cultivation of fungi, particularly in environments where lower pH is required to inhibit bacterial growth. This medium contains a reduced concentration of nutrients compared to standard PDA, making it useful for specific fungal isolations and studies.
APDA is not ideal for cultivating bacteria, fungi that prefer neutral pH environments, or nutrient-demanding fungi. It should also be avoided in situations where accurate quantification of fungal growth, enzyme activity studies, or broad-spectrum fungal isolation is required.
Guidelines
- Aseptic Technique: Ensure strict aseptic conditions throughout the preparation and handling of the medium to prevent contamination. This includes working under a laminar flow hood when pouring the medium into Petri dishes.
- Chemical Safety: Handle all chemicals, especially 85% lactic acid, with appropriate personal protective equipment (PPE) including gloves, lab coats, and eye protection. Be aware of the potential hazards associated with concentrated acids.
- Sterilization: Verify that the autoclave is functioning correctly and that all materials are properly sterilized to avoid cross-contamination. Allow the autoclave to cool completely before opening to avoid steam burns.
- Homogeneous Mixing: Ensure that all components are thoroughly dissolved and mixed before and after autoclaving, as well as after the addition of lactic acid, to achieve a uniform medium.
- Temperature Control: Monitor the temperature closely after autoclaving. Pour the medium into Petri dishes once it has cooled to between 55°C and 40°C to avoid issues with condensation and to ensure proper gelation.
- Disposal: Dispose of all waste materials, including unused medium and contaminated plates, according to institutional biohazard waste disposal protocols. Autoclave all biological waste before disposal to prevent the release of potentially harmful organisms.
Materials
- 10 g of potato dextrose broth
- 16-18 g of agar
- 1 ml of 85% lactic acid
- Distilled water (1 liter)
- Sterile Petri dishes
- Stir bar
Equipment
Adventurer™ Analytical Balances
NAME
Analytical balance
TYPE
Ohaus
BRAND
30100600
SKU
LINK
Equipment
8-Liter Autoclave
NAME
Portable Stainless Steel Pressure Steam Sterilizer
TYPE
China
BRAND
XFS-D-8L
SKU
LINK
Voltage: 220 V (AC)
Power: 1.2 kW
Working Medium: Steam
Design Pressure: 0.17 MPa
Working Temperature: 129 °C
Frequency: 50 Hz
Useful Life: 5 Years
Delivery Date: 3. Oct, 2019
SPECIFICATIONS
Equipment
Magnetic Stirrer RT touch series
NAME
Magnetic Stirrer
TYPE
Thermo Fisher
BRAND
88880013
SKU
Safety warnings
- Handling Lactic Acid: 85% lactic acid is highly concentrated and corrosive. Wear appropriate personal protective equipment (PPE) such as gloves, lab coats, and eye protection when handling to avoid skin contact or inhalation.
- Sterilization Safety: Ensure that the autoclave is properly secured before running the cycle. After autoclaving, handle the hot flask with heat-resistant gloves to avoid burns.
- Aseptic Technique: Maintain aseptic conditions during all steps, particularly after the autoclaving process, to prevent contamination.
Ethics statement
This protocol for preparing and using Acidified 0.5x Potato Dextrose Agar (APDA) is intended for use in research and educational settings. Researchers are responsible for ensuring that all work conducted with this medium adheres to institutional and national biosafety guidelines and ethical standards. This includes the proper handling, disposal, and sterilization of microbial cultures to prevent contamination and environmental release. Any work involving potentially pathogenic fungi should be conducted in a biosafety level-appropriate facility, with all necessary precautions taken to protect laboratory personnel and the environment. The use of this protocol must comply with all applicable laws and regulations governing the study and manipulation of microorganisms.
Before start
Ensure that all materials and equipment are available, clean, and sterilized. Prepare the lactic acid solution and ensure that the autoclave is set up and ready for use.
Prepare the Medium:
Prepare the Medium:
In a 1-liter Erlenmeyer flask with a stir bar, dissolve the following components in 1 liter of distilled water (dH2O):
- 10 g of potato dextrose broth.
- 18 g of agar.
Stir the mixture thoroughly until all components are fully dissolved.
Sterilize:
Sterilize:
Autoclave the solution at 121 °C for 20minutes using the liquid cycle to sterilize the medium.
20m
After autoclaving, remove the flask from the autoclave and place it on a magnetic stirrer.
Stir the medium for approximately 1 minute until the solution is homogeneous.
Acidification:
Acidification:
- Add 1 mL of 85% lactic acid to the medium.
- Stir the medium again for approximately 1 minute to ensure the lactic acid is evenly distributed.
- Place the flask in a room temperature water bath and stir continuously until the temperature of the medium cools to between 55 °C and 40 °C .
Pour into Petri Dishes:
Pour into Petri Dishes:
- Work aseptically to pour the cooled medium into sterile Petri dishes.
- Allow the medium to solidify at room temperature before storing or using.