TITLE: Cultivating Melanized Fungi from Biological Soil Crust and Rock Surfaces
AUTHORS: Tania Kurbessoian, Jason E. Stajich, Renata Haro

[DESCRIPTION]
As the interest to understand melanized fungi becomes more of a focus due to pathological diseases, there needs to be a clearer method to isolate and identify the fungi from their surroundings. Culture independent analysis has helped bloom scientist understandings of the vast quantities of microorganisms around us, but culturing and identifying has always been a struggle. We must look into utilizing different microbiological techniques to help better understand, isolate, and to apply Koch’s postulates to prove they are the cause of said disease. Growing and isolating fungi has always been an issue especially if the fungi in question is a slow growing fungi, where fast growing fungi or other microorganisms can grow and surpass the field of view and compete with the slow growing fungi. A series of dilutions, antibiotics and oligotrophic media can all counteract the issues to provide you with a clear window to help isolate your fungi in question. 
There is still a need to verify that living strains of organisms are actually present in the environment. One approach is to use “culture dependent” method to obtain strains of organisms present in the biocrust. This is achieved by plating biocrust soils onto microbiological media in order to further isolate life strains. Serial dilution is used to isolate the fungi from the soil. This method involves making a soil slurry by diluting the soil sample with increasing higher proportion of water or media in order to get a low starting concentration of starting spores or material growing on the Petri dishes. 

[STEPS]
SECTION: Prepare Media
2. Prepare Nutrient Poor Media Glucose Asparagine Agar (GAA) - Krainsky's Media/1 L
0.500 g 
0.500 g
10.00 g 
15.00 g 

Autoclave

Prepare filtered antibiotics for 1 L agar solution. Either Gentamycin, Tetracycline, Kanamycin,  Streptomycin or Chloramphenicol will work. 

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3.  

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4. 
0.1 g of sample  (either biocrust, soil or rock surface can be used) placed in microcentrifuge tube. 

  

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4.1.  Add 1000 µL into the same tube.

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4.2. Label this 10^0.
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5. Vortex (on highest setting) sample for 10 minutes until solution becomes a slurry. 
 

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6. Preparing the Serial Dilution
Prepare 3 other microcentrifuge tubes with 900 µL .




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6.1. Label them 10^-1, 10^-2, 10^-3.
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6.2. Take100 µL  from 10^0 and add it to 10^-1. Vortex 10^-1. 


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6.3. Then take 100 µL and add it to 10^-2. Vortex 10^-2.

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6.4.  Then take 100 µL and add it to 10^-3. Vortex 10^-3. 

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7. Set up 12 empty Petri Plates and label them with appropriate labels. I.e. Date, Media (list antibiotic used), Crust type, Dilution, Iteration (Plate 1, Plate 2 or Plate 3).
 

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7.1. Pipette out 100 µL into first three plates that were properly labeled for 10^0. 

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7.2. Pipette out 100 µL into next three plates labeled for 10^-1.

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7.3. Pipette out 100 µL into next three plates labeled for 10^-2.

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7.4. Pipette out 100 µL into next three plates labeled for 10^-3.

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8. Make sure to have media cooled (to 50 °C*) and ready to go at this point.
 

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8.1. Add the media slowly to avoid bubbles. Fill until agar closes all gaps on plate. Fill up to three plates at a time.
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8.2. 

When three plates have been filled start to slowly mix the plate and slurry solution. Do a very careful "Figure 8" motion. Make sure to mix slowly and evenly without touching the lid of the plate. 
 

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8.3. Continue repeating 5.1 and 5.2 until all plates have been filled and are cooling. 
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9. Let plates air dry for 24 hours, then parafilm to allow for long-term storage. 
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10. New fungi should start popping up on undiluted and next dilution plates in the next couple of days. Most dilute plates will also have sparser filamentous fungi growing. 

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10.1. Melanized fungi will take about 1-2 weeks to show up on plates.
Make sure to check on the underside of plates for black dots. Those are the fungi you are looking for!

 
 
SECTION: Prepare Media
1. Prepare Nutrient Rich Media Malt Extract Yeast Extract Agar (MEYE)1 L
20 g 
1 g
20 g 
20 g