TITLE: YPS+CRAD Media preparation
AUTHORS: Gabriela Paredes

[DESCRIPTION]
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.

[GUIDELINES]
Aseptic Technique: Strict aseptic techniques must be followed throughout the preparation process to avoid contamination. Ensure that all materials and equipment are sterile.
Component Quality: Use high-purity reagents and freshly prepared solutions to maintain the integrity and effectiveness of the medium.
Homogeneous Mixing: Continuous slow stirring during the addition of CRAD components is essential to ensure that the antibiotics and other chemicals are evenly distributed throughout the medium.
Timing: Prepare and use the medium as soon as possible after preparation to ensure the viability of the antibiotics.

[STEPS]
SECTION: Dissolve YPS Components:
1. (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):

1 g of yeast extract.

1 g  of peptone or tryptone.

1 g  of glucose or sucrose.

0.1 g of chloramphenicol.

18 g of agar.

Stir and heat the mixture until all components are fully dissolved.

SECTION: Sterilize:
2. 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.

SECTION: Add CRAD:
3. Add the following components to the cooled medium:

Rifampicin: Incorporate the solution prepared in DMSO (0.025 g dissolved in 500 µLof DMSO).

Ampicillin: Add the required amount as per protocol.

Danitol: Add of the pre-prepared stock solution.



SECTION: Pour into Petri Dishes:
4. Work aseptically and pour the medium into sterile Petri dishes.
Allow the medium to solidify at room temperature.