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Materials

Standard lab procedures are explained in Appendix A, B, C and D. Novel protocols are mentioned and explained at the specific topic pages, since they cannot be explained without the mention of intermediate results and can be considered results. A detailed version of these protocols is also given here.

Strains

A. niger N593
This strain is cspA and pyrA deficient. Media for N593 are supplemented with 5 mM uridine. Unlike citric acid producers, this strain produces mainly oxalic acid.

A. niger strains expressing GFP
Three strains have been applied that express Green Fluorescent Protein (GFP) in various constructs. GFP is widely used as a localisation tool. These strains have been constructed using strain AB4.1 (Vinck, A., M. Terlou, et al. (2005). "Hyphal differentiation in the exploring mycelium of Aspergillus niger." Molecular Microbiology 58(3): 693-699). The GFP constructs are made using the sGFP gene. This mutated GFP variant has an excitation peak at 488 nm and an emission peak at 510 nm. Information is given in table 1.

Table 1) Different Aspergillus niger strains expressing GFP

A. nidulans WG505
This is a pyr-deficient strain, so 5 mM uridine was added to the medium. Use of this strain is mentioned by Nyyssola (Nyyssola, A., R. Heshof, et al. (2012). "Methods for identifying lipoxygenase producing microorganisms on agar plates." AMB Express 2(1): 17). No further data available.

Media

The standard protocol for media for the SSB group is provided as Appendix D. Media and supplements used here are:

Uridine solution
Per 100 ml; 12.2 g uridine. Filter-sterilised using a 0.2 µm filter.

Vishniac solution
Per litre; 10 g EDTA; 4.4 g ZnSO4_7H2O; 1.0 g MnCl2_4H2O; 0.32 g CoCl2_6H2O; 0.32 g Cu¬SO4_5H2O; 0.22 g (NH4)6Mo7O24_¬4H2O; 1.47 g CaCl2_2H2O; 1.0 g FeSO4_7H2O. pH adjusted to 4. Filter-sterilised using a 0.2 µm filter.

Vitamins solution
Per 100 ml; 10 mg thiamine; 100 mg ribovlavine-5P; 10 mg p-aminobezoic acid; 100 mg nicotinamide; 50 mg pyridoxine-HCl; 10 mg panthothenic acid; 2 mg biotine. Filter-sterilised using a 0.2 µm filter.

Minimal medium salts (MMS)
For preparation of medium, 10x MMS stock solution is used. This contains per 1000 ml; 60 g NaNO3; 15 g KH2PO4; 5 g KCl; 5 g MgSO4_7 H2O and is adjusted to pH 5.45.

Minimal medium (MM)
Stock 10x MMS solution is diluted 10x, autoclaved for 20 minutes at 121°C and supplemented with 50 mM carbon source, appropriate supplements (e.g. 5 mM uridine) and 1 ml/l Vishniac trace element solution.

Complete medium (CM)
Stock 10x MMS solution is diluted 10x, supplemented per litre with 2 g meat peptone; 1 g yeast extract; 1 g Casamino acids; 0.3 g yeast ribonucleic acids and then autoclaved for 20 minutes at 121°C. After autoclaving it is supplemented with 50 mM carbon source, appropriate supplements (e.g. 5 mM uridine), 1 ml Vishniac solution and 2 ml vitamins solution.

Cultures

Microscopes

Appendices

A. Preparation spores suspension/inoculation cultures and making spore plates
B. Preparation and storage of Aspergillus glycerol stocks
C. Media for Aspergilli
D. HPLC, sample preparation
E. DAPI staining of A. niger
F. Calcofluor staining of A. niger
G. Experimental evolution protocol “Mycelium to single cell”
H. RNA extraction protocol

A. Preparation spores suspension/inoculation cultures and making spore plates

Materials:
Sterile Pipettes and Pipetting tips
Sterile saline-Tween solution (0.9 % NaCl + 0.005 % (v/v) Tween-80
Sterile saline solution (0.9 % NaCl)

Method for plates:
1. Inoculate on Thursday six 15 cm CM plates (protocol 30) with a total number of 4x105 spores (this is ~22 spores per mm2 on a 15 cm plate; for 9 cm plates, add total 1.4x105 spores). Make sure to pour the CM-agar while it is relatively cold, as this will enhance the spore scraping procedure. Do not add too many spores: with >50 spores per mm2 the plates will wrinkle and spore concentration will decline since it is more difficult to harvest the spores!
2. Place the plates for 4 days at 30 °C (until Monday).
3. Put the spore plates overnight in the cold room @ 4 °C for maturation (Monday overnight).
4. Pipette 10 ml saline-Tween solution on the spore-mat.
5. Scrape off the spores using a Drigalski spatula.
6. Transfer the spore suspension to a sterile 50 ml Greiner tube with a sterile 10 ml pipet.
7. Mix 30 seconds on Vortex and filter over funnel with glass wool. Centrifuge 10 minutes, 5000 rpm at room temperature.
8. Pour off supernatant and resuspend in 40 ml saline solution, mix 30 seconds and repeat the wash once more.
9. Prepare a 50-fold dilution (20 µl spore suspension + 980 µl saline) and determine the concentration of spores by counting the diluted sample in a heamocytometer (see remarks).
10. Calculate the concentration of spores.

Neubauer improved heamocytometer:

depth: 0.1 mm
Minimal area (A): 1/400 mm2

Pipette spore dilution between coverslip and slide enough liquid until the space is flooded (~20 ul). NB If the coverslip is chipped or in any way damaged, it must be replaced- it is no longer functional.
Count the spores (magnification 400 X) in 16 fields.
Calculate the spore concentration in the spores suspension:
spores/ml = count x d x 2.5 x 105 (d = dilution)

B. Preparation and storage of Aspergillus glycerol stocks

1. Harvest spores from a plate.
2. Transfer the spore solution in ST into a sterile glass tube with aluminium cap
3. Spin down the spores at low speed (3000 RPM in table top centrifuge in rec lab)
4. Resuspend the spores in 1 ml sterile glycerol medium (1 part glycerol 87% : 2 parts 1% pepton).
5. Divide over two Eppendorff tubes and label well
6. Store immediately in the -70⁰C freezer and update the collection database
7. Sterilize all tubes used and fungal material left

Taking a strain from glycerol stock:
1. To take a strain from the stock use a sterile platinum needle.
2. Take an inoculum from the stock and transfer to a plate
3. Distribute the spores with a sterile Drigalski spatula
4. Return the stock to the freezer as quickly as possible

C. Media for Aspergilli

Minimal medium salts(MMS) for 1000 ml:
6.0 g NaNO3
1.5 g KH2PO4
0.5 g KCl
0.5 g MgSO4. 7 H2O


when 10xMMS is adjusted to pH=5.45, a 1x MM solution will have pH ~6.0

Minimal Medium for 1000 ml:

100 ml Minimal medium salts 10x

pH adjusted to 6.0
For solid medium 15 g agar is added
autoclave

Before use:
50 mM carbon source and appropriate supplements, 1 ml Vishniac trace-elements solution (lab fridge)
Complete Medium for 1000 ml:
100 ml Minimal medium salts 10x +
2 g meat peptone (pepton 100)
1 g yeast extract
1 g pepton 140 or Casamino acids (vitamins free)
0.3 g yeast ribonucleic acids (cold room shelf)
2 ml vitamins solution (fridge)

pH adjusted to 6.0
For solid medium 15 g agar is added
autoclave

Before use:

50 mM carbon source and appropriate supplements (added after sterilization) 1 ml Vishniac trace elements solution

Vishniac solution (Vishniac and Santer, 1957): 1 litre
10 g EDTA
4.4 g ZnSO4.7H2O
1.0 g MnCl2.4H2O
0.32 g CoCl2.6H2O
0.32 g CuSO4.5H2O
0.22 g (NH4)6Mo7O24.4H2O
1.47 g CaCl2.2H2O
1.0 g FeSO4.7H2O

pH adjusted to 4.0 and stored at 4°C.

Vitamins solution: 100 ml, store in protected from light
10 mg thiamine
100 mg ribovlavine-5P
10 mg p-aminobezoic acid
100 mg nicotinamide
50 mg pyridoxine-HCl
10 mg panthothenic acid
2 mg biotine

D. HPLC, sample preparation

Materials:
Samples and if necessary defrost them on time
HPLC vials and caps
micro centrifuge vials
Demi water
HPLC standards

Media

For Organic acids:
Crotonate Internal Standard (MICFYS): 30 mM crotonate in 0,3 n H2SO4
per liter: 2,58 g crotonate + 8,4 µl (0,96%) H2SO4

External Standards organic acids: (example) 100mM stock concentration
#1; oxalic acid, a-ketoglutaric acid, sorbitol, fumaric acid
#2; cis-aconitic acid, citric acid, xylose, , itaconic acid
#2; oxaloacetic acid, glucose, malic acid, succinic acid
#4; pyruvic acid, formic acid

External Standards sugar: (example) 100mM stock concentration
#1; sorbitol, Xylose (for Carbopac-MA1)
#2; sorbitol, arabinose, galactose, glucose, xylose, mannose (for Carbopac-PA20 max 100 µM)

Protocol
1. Take your samples, if necessary defrost them, centrifuge for 15 min max. speed
2. Label your HPLC vials or 96 well plate (ICS 5000)
3. Make a H2O HPLC vial
4. Prepare the external standards in the following concentrations in a micro centrifuge

table 2)

5a.
For the HPLC vials take 800 µl standard dilution, add that to the HPLC vial + 200 µl Crotonate I.S.
Do this also with the samples.

Take sample/controls + Crotonate I.S. in a 4:1 ratio,
The minimal total volume in the HPLC vial should be 500 µl

5b.
For 96 well plate take 200 µl standard dilution, add that to the 96 well plate + 50 µl Crotonate I.S.
Do this also with the samples. When done cover the plate with a sheet

Take sample/controls + Crotonate I.S. in a 4:1 ratio,
The minimal total volume in the 96 well plate should be 250 µl

6. For HPLC-vials, Cap the vials
7. Mix the HPLC vials well, not necessary for a 96 well plate

The samples are now ready for measurement on HPLC to measure organic acids.

E. DAPI staining of A. niger

Made using A. niger strain N400

Materials:
• Gloves
• 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen D1036)
• pH meter
• Fluorescence microscope

Protocol
1. Use gloves! DAPI is carcinogenic. Prepare a 10x Hoechst stock solution (1,0 g/ml) in demi water and store protected from light at 4°C.
2. Sample a minimum of 200 µl of mycelium in an eppendorf.
3. Correct the pH to 7 (between pH 4 and 11 fluoresence remains unchanged)
4. Cultured cells are examined immediately after incubation with DAPI at
0.1g/mL in phosphate buffered saline at 30 °C for 15-30 minutes.
5. Use the fluorescence microscope with block A. Consider using Vectashield mounting medium if bleaching of the stain occurs too quickly.

Calcofluor staining

Calcofluor white (fluorescent brightener 28, Sigma; 100 μl) (0•3 g l−1) was added to 1 ml diluted sample in a 1•5 ml Eppendorf vial covered with tin foil. Samples were mixed and incubated at room temperature for 5 min. A few drops of the solution were placed on a glass slide and covered with a coverslip for analysis. The dye fluoresces when bound to chitin and glucans, and thus stains cell walls and septa. Images were obtained using fluorescence microscopy on a Leika microscope using a mercury burner and a filter block with 330–380 nm excitation wavelength and dichroic mirror with emission >420 nm.