TITLE: Malassezia culture on modified Dixon media
AUTHORS: Hao Li, Genevieve Ong, Shirlyn Goh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes the procedures to culture Malassezia species in modified Dixon (mDixon) media. It includes the recipe for mDixon, how to start the culture from freezer stock, culture maintenance and how to make frozen stocks. </div></div>

[STEPS]
?. [Preparation of modified Dixon Broth]
Place a magnetic stir bar into an autoclaved beaker with Mili-Q water and turn on the magnetic stirrer.Set the temperature at 60-70ºC Temperature and stirring speed can be adjusted accordingly while preparing the media.
?. [Preparation of modified Dixon Broth]
Add the following ingredients while stirring.  AB1IngredientAmount needed for 1L2Peptone6 g3Desiccated Oxbile (Bovine Bile)20 g4Malt Extract36 g5Oleic Acid2 mL6Tween 4010 mL7Glycerol2 mLNote: Tween 40 takes a long time to dissolve completely, so it's recommended to prepare a working stock of 10% Tween 40 beforehand and add 100 mL of that. The same can be done for glycerol. Bacto Agar can be added to 1.5% (w/v) at the last step (after pH and volume adjustment) to make mDixon agar plates.
AB1IngredientAmount needed for 1L2Peptone6 g3Desiccated Oxbile (Bovine Bile)20 g4Malt Extract36 g5Oleic Acid2 mL6Tween 4010 mL7Glycerol2 mL
?. [Preparation of modified Dixon Broth]
Measure the pH of the media using a pH Meter and adjust accordingly using hydrochloride acid or sodium hydroxide to  .
?. [Starting Culture from Frozen Stock]
Retrieve the frozen stock from -80˚C and scrap some of the frozen stock using a sterilized pipet tip. If possible, this step should be performed in a Biosafety Cabinet to minimize contamination.
?. [Starting Culture from Frozen Stock]
Inoculate 12 mL of pre-warmed mDixon culture in a 125 mL flask with the frozen stock. Alternatively, the frozen stock can be streaked out onto a mDixon agar plate.
?. [Starting Culture from Frozen Stock]
Incubate culture at 32˚C. For planktonic culture, shake flask at 150 rpm. For fast growing Malassezia species (eg. M. furfur), planktonic culture will be confluent in 2 days after inoculation. For slower growing species (eg. M. globosa), planktonic culture will be confluent in 3-4 days after inoculation.
?. [Maintaining Planktonic Culture]
Warm up the media in a 37˚C water bath.
?. [Maintaining Planktonic Culture]
Retrieve the culture flask and aspirate most of the culture, leaving around 1 mL of culture.
?. [Maintaining Planktonic Culture]
Pipette in 12mL of the warmed media into the shaker flask and place the shaker flask back into the incubator shaker.
?. [Monitoring Viability]
Add 10 μL of trypan blue stain onto a glass microscopy slide and mix with 10 uL of the fungal culture.
?. [Monitoring Viability]
Use a light microscope with at least 40X lens to check fungal growth. Most of the fungal cells would be stained light blue, while the dead cells are stained dark blue. Note: cell viability and count can also be done at this point using a hemacytometer.
?. [Freezing Down Pellet]
Grow planktonic culture to late log/early stationary phase.
?. [Freezing Down Pellet]
Spin culture down at 8000 rpm for 5 mins and aspirate the supernatant.
?. [Freezing Down Pellet]
Resuspend culture pellet in the same volume of 25% glycerol in mDixon, aliquot in screw cap freezer tubes and freeze at -80˚C.