Muramic Acid Assay

• 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

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.