TITLE: Refractive index adjusted imaging medium: Sorbitol (RI ~ 1.4) - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare imaging medium for Saccharomyces cerevisiae with adjusted refractive index. This medium is optimized for fluorescence imaging by the reduction of auto-fluorescence through an abundance of Adenine [1] and the repression of of the Met-promoted pp7- CP expression [2].

[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)

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

Methionine 200x 50 ml:
Concentration: 17.12 g/l
mix 856 mg into 50 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 30ml refractive index adjusted imaging medium with Sorbitol
1. Compound medium for autoclave
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.1. Fill a 50 ml flask with 3.9 mLddH2O
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.4. Add 3 mL SC-XX 10x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.5. Add 1.5 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).


SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.6. Add 18 mLSorbitol.

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
3. When the medium cooled down to around 80 °C add 1.5 mL sterile Glucose 20%.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
4. 
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.1. Fill a 50 ml flask with 9.9 mLddH2O
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.2. Add 0.3 mLAdenine 100x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.3. Add 0.3 mLMethionine 200x solution.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.4. Add 57.6 mg SC-XX powder.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.6. Add 18 mLSorbitol.

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
1.5. Add 201.6 mg Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB).
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
3. When the medium cooled down to around 80 °C add 1.5 mL sterile Glucose 20%.
SECTION: Preparation of 30ml refractive index adjusted imaging medium with Sorbitol
4.