TITLE: High-throughput 96-well assay to measure impact of polyphenolic compounds on fungal growth
AUTHORS: Megan Nickerson, Jana M U'Ren

[DESCRIPTION]
This protocol was developed to assess the growth of filamentous fungi in the presence of polyphenolic compounds--a class of secondary metabolites produced by plants with reported antimicrobial activity. Here, we use 96-well plates and a plate reader to screen each fungal isolate against six polyphenolic compounds at three concentrations (with four replicates per compound/concentration). Optical density is measured with a spectrophotometer to quantify fungal growth compared to a positive control containing only media. As numerous fungi do not readily produce spores in culture, here we harvest fungal hyphae from solid media with a cellophane overlay and blend tissue a liquid media prior to inoculation in 96 well plates

[STEPS]
SECTION: Media and Solution Preparation- Aspergillus nidulans Defined Media plus Carrageenan
1. Combine:
- 900mL nanopure water
-50mL 20X Sodium nitrate salts
-1mL Trace elements
SECTION: Media and Solution Preparation- Aspergillus nidulans Defined Media plus Carrageenan
2. Bring the pH to 6.5.
SECTION: Media and Solution Preparation- Aspergillus nidulans Defined Media plus Carrageenan
3. Add 2 grams of Carageenan Type II (0.2% final concentration)
SECTION: Media and Solution Preparation- Aspergillus nidulans Defined Media plus Carrageenan
4. Autoclave for 30 minutes on liquid cycle and allow to cool to room temperature. Store at 4C. 
SECTION: Media and Solution Preparation- Glucose solution
5. In a 15 mL conical tube, add 3.2g of glucose.
SECTION: Media and Solution Preparation- Glucose solution
6. Add enough nanopure water to bring volume to 10 mL and vortex to mix until completely dissolved. 
SECTION: Media and Solution Preparation- Glucose solution
7. In a biosafety cabinet, filter sterilize and make 1 mL aliquots. 
SECTION: Media and Solution Preparation- Polyphenol solutions (excluding Vanillin and Lignin)
8. Prepare 3 5mL tubes with 3 concentrations of each polyphenol dissolved in autoclaved nanopure water. Note: Concentrations (when diluted in wells): 2000ug/mL, 1000ug/mL, and 500ug/mL. Actual concentrations of stock: 40,000 ug/mL,20,000 ug/mL, and 10,000 ug/mL
SECTION: Media and Solution Preparation- Polyphenol solutions (excluding Vanillin and Lignin)
9. For inoculating two plates, dissolve 0.08g, 0.04g, and 0.02g of each polyphenol in 2mL of autoclaved nanopure water. Vortex thoroughly to mix until completely dissolved. 
SECTION: Media and Solution Preparation- Polyphenol solutions (excluding Vanillin and Lignin)
10. In a biosafety cabinet, using the 0.2um disc filters, filter sterilize 1mL of each solution into a sterile 1.5mL tube.
SECTION: Media Solution Preparation- Vanillin
11. Due to issues with solubility, the amount of vanillin used has to be scaled down. For inoculating two plates, dissolve 0.02g, 0.01g, and 0.005g of vanillin in 2mL of autoclaved nanopure water. Vortex thoroughly to mix until completely dissolved.
SECTION: Media Solution Preparation- Vanillin
12. In a biosafety cabinet, using the 0.2um disc filters, filter sterilize 1mL of each solution into a sterile 1.5mL tube.
SECTION: Media and Solution Preparation- Lignin
13. Due to issues with solubility, the amount of lignin used has to be scaled down. For inoculating two plates, dissolve 0.02g, 0.01g, and 0.005g in 20mL nanopure water in glass jars.
SECTION: Media and Solution Preparation- Lignin
14. Even at these concentrations, full solubility was not achieved so filter sterilization was not possible.  Autoclave on Liquid30 cycle.
SECTION: Media and Solution Preparation- Lignin
15. In a biosafety cabinet, aliquot into 2mL tubes. 
SECTION: Inoculate fungal cultures on cellophane (to be done ahead of time)
16. Inoculate ___ 60mm MEA plates +cellophane with ___ isolates and incubate at room temperature until growth is sufficient to proceed. List isolates in your notebook.
- Example: 24 plates with 4 isolates (6 plates per isolate)
- Allow to grow until sufficient mycelium for inoculation (e.g., 1 week)
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
17. Using a serological pipette add 20mL of A. nidulans defined carrageenan media to blender cup (20 mL is the minimum amount needed for blender cup to work).
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
18. Cut mycelium closely around the original inoculum to remove the agar square from the center of the cellophane. Carefully scrape the mycelium from the cellophane, without cutting into the cellophane, and gather into a ball using the broad scalpel. Pick up the mycelium ball using the fine forceps and place in blender cup. 
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
19. Blend four times at one-second pulses. Check fragment sizes to ensure sufficient blending.
SECTION: Read samples in spectrophotometer (at 600nm)
25. Place blank cuvette with just 1000uL of A. nidulans defined carrageenan media into reading position (with the cuvette side with the arrow closest to the screen) and press the button to blank the sample. 
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
20. OR
Once isolate has colonized entire plate, pipette 1-5mL of sterile water onto plate. Use sterilized rubber policeman to gently scrape the hyphae off the surface of the media. Using a wide-bore pipette tip, collect the liquid and place a sterile blender cup. 
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
21. Using serological pipette, add 0.2% carrageenan type II media until final volume is 20mL in blender cup.  (e.g., 20mL final volume - ___mL collected from plate = _____mL 0.2% carrageenan media to add.)
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
22. Blend four times at one-second pulses. 
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
23. Transfer 1000uL of blended culture (while pipette mixing) with a 5mL pipette to three spectrophotometer cuvette replicates. 
SECTION: Prepare liquid fungal inoculum for spectrophotometer  (all steps done in a biosafety cabinet)
24. Transfer 1000uL of A. nidulans defined carrageenan media from 5mL tube into cuvette (this will be the blank cuvette).
SECTION: Read samples in spectrophotometer (at 600nm)
26. Place sample in reading position and press the “arrow passing through the cuvette” button. Record absorbance.
SECTION: Read samples in spectrophotometer (at 600nm)
27. A final reading of  >0.44 is needed to reach the appropriate hyphal fragment density. If the absorbance of your sample is lower than this, harvest more hyphae from plates and repeat blending procedure in a new blender cup. 
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
29. Once dissolved, add 5uL of sterilized phenol solution to the respective wells.
SECTION: Dilute blended culture
28. When a reading is >0.66 you need to dilute the blended inoculum with A. nidulans defined carrageenan media to yield 4 ml of culture inoculum with an absorbance of 0.44. Use a 5 mL conical tube for the dilution. 
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
30. Add 5uL of autoclaved nanopure water to controls (media without phenols but with fungi).
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
31. Combine 1.8mL of blended culture at absorbance 0.66 and 12.5mL of A. nidulans defined carrageenan media. Mix well by inversion to evenly distribute the hyphal fragments in the media.
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
32. Add 0.89 mL volume glucose solution to a final concentration of 2% glucose. Immediately proceed to next step so that hyphae don’t settle in the media.
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
33. Inoculate 95uL of GLUCOSE CONTAINING solution for wells corresponding to the proper isolate +glucose. Also inoculate the corresponding positive control wells. (do not inoculate wells for the media only controls in Row A). 
- Do NOT push pipette plunger past the last stop because this will create bubbles, which will cause readings to be inaccurate. If bubbles form try to pop with a sterile pipette tip.
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
34. Place lid on the plate and parafilm for transport. Immediately measure the plate at 595, 660, and 750nm (cell density only) on plate reader. 
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
35. After reading, re-parafilm plate and place in plastic bag with wet paper towels to keep plates from drying out. Be careful to keep the plate upright at all times.
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
36. Store plates in an aluminum foil lined container to keep out the light.
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
37. Read plates every 3 days for a total of 24 days (tentative, depends on isolate growth). 
SECTION: Inoculate 96-well Plates (All steps to be performed in biosafety cabinet)
38. After 24 days, record visual observations and take picture of plates
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
39. Once dissolved, add 5uL of sterilized phenol solution to the respective wells.
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
40. Add 5uL of autoclaved nanopure water to controls (colored purple; media without phenols but with fungi).
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
41. Combine 2.0mL of blended culture at absorbance 0.66 and 14.0mL of A. nidulans defined carrageenan media. Mix well by inversion to evenly distribute the hyphal fragments in the media.
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
42. Immediately proceed to next step so that hyphae don’t settle in the media.
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
43. Inoculate 95uL solution for wells corresponding to the proper isolate +no glucose. Also inoculate the corresponding positive control wells. (do not inoculate wells for the media only controls in Row A). 
- Do NOT push pipette plunger past the last stop because this will create bubbles, which will cause readings to be inaccurate. If bubbles form try to pop with a sterile pipette tip.
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
44. Place lid on the plate and parafilm for transport. Immediately measure the plate at 595, 660, and 750nm (cell density only) on plate reader.
SECTION: Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet)
45. Follow steps above (for +glucose) for storage etc.