YPS+CRAD Media preparation
Gabriela Gabriela Paredes
Disclaimer
This protocol is provided for educational and research purposes only. The procedures described herein should be conducted by trained personnel in a properly equipped laboratory, adhering to all relevant safety and ethical guidelines. The authors and publishers of this protocol are not responsible for any injuries, damages, or legal consequences that may result from the improper use of this protocol. The use of chemicals, reagents, and equipment should be done with caution, and users must ensure they are compliant with all local, national, and institutional regulations.
Users are responsible for ensuring that their work complies with applicable biosafety and bioethics standards, particularly when handling genetically modified organisms (GMOs), pathogens, or other hazardous materials. This protocol does not constitute medical, legal, or professional advice, and should not be used as a substitute for consultation with qualified professionals in those fields.
Abstract
YPS (Yeast Extract, Peptone, Sucrose) is a culture medium specifically designed for the general isolation of fungi. This medium is composed of yeast extract, peptone (or tryptone), and a carbohydrate source such as glucose or sucrose, providing a nutrient-rich environment that supports the growth of various fungi. The addition of CRAD (Chloramphenicol, Rifampicin, Ampicillin, Danitol) to the medium after sterilization creates a selective environment, inhibiting the growth of bacteria and other unwanted microorganisms, while allowing fungi to proliferate under controlled conditions.
CRAD in the context of YPS medium includes the following antibiotics and chemicals:
- Chloramphenicol : A broad-spectrum antibiotic that prevents the growth of Gram-positive and Gram-negative bacteria.
- Rifampicin : An antibiotic effective against Gram-positive bacteria, dissolved in DMSO to ensure complete incorporation into the medium.
- Ampicillin : A broad-spectrum beta-lactam antibiotic that inhibits bacterial cell wall synthesis.
- Danitol : An acaricide and insecticide used to control specific organisms, such as insects or mites.
Before start
Before starting the preparation of YPS+CRAD medium, the following steps must be completed:
Prepare Rifampicin Solution in DMSO :
- Dissolve 0.025 g of rifampicin in 500 μl of DMSO. Ensure the solution is completely homogeneous.
Prepare Danitol Solution :
- Prepare a stock solution by diluting 500 μl of Danitol 2.4 EC in 100 ml of acetone. From this stock solution, take 100 μl to add to the medium.
Ensure that all necessary materials and equipment are available and sterile before beginning the procedure.
Steps
Dissolve YPS Components:
(For 1 liter of medium)
In a 2-liter Erlenmeyer flask with a stir bar, dissolve the following components in 1 liter of distilled water (dH2O):
1g
of yeast extract.1g
of peptone or trypton1g.
of glucose or sucr0.1gse.
of chloramphe18gicol.
of agar.
Stir and heat the mixture until all components are fully dissolved.
Sterilize:
Autoclave the solution at 121°C for 20 minutes to sterilize the medium.* Allow the medium to cool to approximately 50°C in a room temperature water bath, with slow stirring to facilitate the dissolution and mixing of ingredients.
Add CRAD:
Add the following components to the cooled medium:
Rifampicin: Incorporate the solution prepared in DMSO0.025g( dissolved i500µL of DMSO).
Ampicillin: Add the required amount as per protocol.
Danitol: Add of the pre-prepared stock solution.
Pour into Petri Dishes:
Work aseptically and pour the medium into sterile Petri dishes.* Allow the medium to solidify at room temperature.
