Assays
Filter paper assay
Testing for utilization of pure unaltered cellulose
- control-noble (neg. control)
- filter paper
- PDA (pos. control)
CMC & Congo Red assay
Testing for cellulose-degrading enzymes
- control-salts (neg. control)
- CMC-salts
- CMC-PDB_0.25
- dextrose-salts
- PDA (pos. control)
MCC assay
Testing for cellulose carbon utilization (i.e., growth)
- control-noble (neg. control)
- MCC-salts
- MCC-PDB_0.25
- PDA (pos. control)
Media needed (all media per 1L, all with 20ml/L Penicillin&Streptomycin):
- control-noble: 15g noble agar, salts* 0NS
- control-salts: 15g normal agar, salts* 0S
- MCC-salts: 5g MCC, 15g noble agar, salts* MNS
- MCC-PDB_0.25: 5g MCC, 0.25g PDB, 15g regular agar M-green
- CMC-salts: 5g CMC, 15g normal agar, salts* CS
- dextrose-salts: 5g dextrose, 15g normal agar, salts*
- CMC-PDB_0.25: 5g CMC, 0.25g PDB, 15g regular agar C-green
- filter paper: piece of autoclaved Whatman filter paper 15g regular agar, salts* 0S
salts*
(prepare 10x solution ahead= 10X of the following per 1L, autoclave):
Salts Amount KH2PO4 0.2 g NH4Cl 0.25 g KCl – 0.5 g CaCl2 0.15 g NaCl 1 g MgCl 0.6 g (or 1.2g if hydrated) K2SO4 2.84 g include in media through filter after autoclaving: 1ml of 1000x trace minerals solution 10ml of 100x trace vitamins solution
Media based on Czapek-Dox
Czapek-Dox-CMC (cellulose degradation test)
| Ingredient | Amount | |
|---|---|---|
| penicillin & streptomycin | 20 ml of 100x stock/L | |
| KH2PO4 | 1g | |
| MgSO4*7H2O | 0.5g | |
| KCl | 0.5g | |
| FeSO4*7H2O | 0.01g | |
| cellulose | 30g | |
| NaNO3 | 2g | |
| noble agar | 20g | |
| water | 1L |
Czapek-Dox neg. control (no carbon source)
| Ingredient | Amount | |
|---|---|---|
| penicillin & streptomycin | 20 ml of 100x stock/L | |
| KH2PO4 | 1g | |
| MgSO4*7H2O | 0.5g | |
| KCl | 0.5g | |
| FeSO4*7H2O | 0.01g | |
| cellulose | 30g | |
| NaNO3 | 2g | |
| noble agar | 20g | |
| water | 1L |
Czapek-Dox-dextrose (positive control) Czapek-Dox neg. control (no carbon source)
| Ingredient | Amount | |
|---|---|---|
| penicillin & streptomycin | 20 ml of 100x stock/L | |
| KH2PO4 | 1g | |
| MgSO4*7H2O | 0.5g | |
| KCl | 0.5g | |
| FeSO4*7H2O | 0.01g | |
| dextrose | 30g | |
| NaNO3 | 2g | |
| noble agar | 20g | |
| water | 1L |
The Czapek-Dox doesn’t work for Raffaelea – doesn’t grow on positive control. Try different media – Czapek-Dox with proteins, or with little bit of yeast extract, or dextrose+salts+NaNO3.
Yeast Nitrogen Base media – Liquid
Tested media:
1) Prepare YNB&AA& antibacterials 10x stock: 6.7g YNB&AA and 20mL Streptomycin/Penicillin concentrate, in 100mL water. Use warm water, but do NOT autoclave! Store in dark and cold place.
2)YNB- CMC-liq (do not use MCC – not soluble, not usable in absorbance measurements)
- water 90mL
- CMC 3g
- after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter. pH: 5.7
YNB- dex-glu-liq
- water 90ml
- dextrose 1.5g
- glucose 1.5g
- after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter
YNB- blank-liq
- water 90mL
- after autoclaving: 10ml YNB&AA&antibacterials 10x stock through filter
Yeast Nitrogen Base media
1) prepare YNB&AA 20x stock: 26.8 g in 200mL warm water, do NOT autoclave! Store in dark and cold place.
2) autoclave 50 pieces of filter paper (the same size as last time) as you are autoclaving the media
3) media:
YNB-CMC
- water 950 mL
- agar 15g
- CMC 30g
- after autoclaving: 50ml YNB&AA 20x stock through filter
YNB-dex-glu
- water 950ml
- agar 15g
- dextrose 15g
- glucose 15g
- after autoclaving: 50ml YNB&AA 20x stock through filter
YNB-filter_paper
water 950 mL
- agar 15g
- one piece of filter paper,
- after autoclaving: 50ml YNB&AA 20x stock through filter
YNB-blank
- water 950mL
- agar 15g
- after autoclaving: 50ml YNB&AA 20x stock through filter
Congo Red assay
Prepare Congo Red solution (1g/L) and 1M solution of NaCl (58g/L). Overlay plate with Congo Red for 15 minutes, pour off, overlay with NaCl for 15 minutes, pour off, look for zone of clearing
