| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| Dimethylformamide permeabilizes the cells. Carboxypeptidase Y in the cells cleaves the ester linkage in N-acetyl-DL-phenylalanine β-naphthyl ester. Free β-naphthol then reacts with the diazonium salt of Fast Garnet GBC to produce an insoluble red dye. |
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1. Grow colonies or patches of colonies on YPD plates (see Hint #2).
2. Carefully pour the Overlay Mix over the surface of the plate to completely cover the cells.
3. After the agar hardens in 5 to 10 minutes, carefully flood the surface with fresh Garnet GBC solution.
4. Color will develop in 5 to 15 minutes.
5. Wild-type strains will turn red whereas carboxypeptidase Y-negative strains will appear yellow or pink. If color development takes longer than 30 minutes the Garnet solution is not fresh.
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| Garnet GBC Solution |
| Prepared in 0.1 M Tris
Prepare just before use
5 mg/ml Fast Garnet GBC Sulfate Salt
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| 0.1M Tris-HCl, pH 7.4 |
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| Overlay Mix |
| 4 ml of 0.6% Molten Agar
2.5 ml Naphthyl-Ester
Keep solution at 50°C until use.
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| 0.6% Molten Agar |
| Heat until liquid, keep at 55°C
0.6% Agar
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| Naphthyl-Ester |
| Prepared in Dimethylfomamide (CAUTION! see Hint #1)
1 mg/ml N-acetyl-DL-Phenylalanine β-Naphthyl Ester
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| 10 g/l Yeast Extract |
| Autoclave 20 min, cool to room temperature
20 g/l Peptone
20 g/l Glucose (Dextrose)
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Glucose
Fast Garnet GBC Sulfate Salt
Dimethylfomamide
N-Acetyl-DL-Phenylalanine beta-Naphthyl Ester
Yeast Extract
Dextrose
Peptone
Tris-HCl
Agar
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1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Three days for colonies to grow and one day for colony patches is usually sufficient.
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