TITLE: Pre-Imaging Solid Growth Medium - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare solid culture medium for Saccharomyces cerevisiae. 
This solid medium is used for cultures that are going to be investigated by fluorescence imaging. Of that reason the medium is prepared with an abundance of Adenine to reduce auto-fluorescence [1].

[BEFORE_START]
Have the following solutions premixed:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Sodium Hydroxide 1M solution 50 ml:
Concentration: 39.997 g/l
mix 1.99985 g NaOH into 50 ml ddH2O

Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 ml ddH2O

Optional:
SC-xx 10x 100ml solution:
Concentration: 19.2 g/l
mix 1.92 g into 100 ml ddH2O

YNB 20x 100ml solution:
Concentration: 134.4 g/l
mix 13.44 g into 100 ml ddH2O

[STEPS]
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1. Compound medium for autoclave
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.1. Fill a 500 ml flask with 444 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.2. Add 3.36 g Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB)
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.3. Add 0.86 g SC-XX powder.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.4. Add 10 g Agar 
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.5. Add 5 mLAdenine 100x solution
 


SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.6. Add 1 mL NaOH 1 molar solution.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3. Cooling and platting

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.1. Add a sterile stirring bar and thermometer. Let the medium stir to homogenize its temperature, while cooling.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.2. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.3. When the medium cooled down to 60 °C , plate 25 mL per  10 cmPetri dish (~20 dishes).
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.4. Let the medium solidify and cool down to Room temperature.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
4. Seal the prepared dishes in a plastic bag to prevent condensation and store them in 4 °C fridge.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.1. Fill a 500 ml flask with 369 mLH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.2. Add 25 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.3. Add 50 mL SC-XX 10x solution.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.4. Add 10 g Agar 
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.5. Add 5 mLAdenine 100x solution
 


SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
1.6. Add 1 mL NaOH 1 molar solution.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3. Cooling and platting

SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.1. Add a sterile stirring bar and thermometer. Let the medium stir to homogenize its temperature, while cooling.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.2. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.3. When the medium cooled down to 60 °C , plate 25 mL per  10 cmPetri dish (~20 dishes).
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
3.4. Let the medium solidify and cool down to Room temperature.
SECTION: Preparation of ~20 agar plates of yeast pre-imaging growth medium
4. Seal the prepared dishes in a plastic bag to prevent condensation and store them in 4 °C fridge.