Muramic Acid Assay

(Purpose to assay muramic acid, a component of peptidoglycan)

Stock Solutions:

• 1.5% (w/v) p-hydroxydiphenyl in 96% ETOH (PHD)
• 1.0 M NaOH
• concentrated H₂SO₄
• 0.5 M H₂SO₄
• 4% (w/v) CuSO₄-5H₂O in H₂O

Procedure:

1. Bring sample to 0.5 ml with 1.0 M NaOH.
2. Incubate at 38^oC for 30 min.
3. Add 0.5 ml of 0.5 M H₂SO₄ + 5 ml conc. H₂SO₄, stopper tube well and place in boiling H₂O bath for 5 min.
4. Cool and add 0.05 ml CuSO₄ + 0.1 ml PHD.
5. Stopper tube and incubate at 30^oC for 30 min.
6. Read absorbance at 560 nm.

Standard curve:

0 – 20 µg muramic acid.