Develop color

4. Add 4.5 ml of the 6:1 sulfuric acid/water solution to each tube and cool on ice.

5. Add 1 ml of the standard rhamnose solutions, the water-only control, and each of the diluted unknown PG-PS suspensions to the appropriate tubes (in duplicate, including the tubes for addition of cysteine and the controls without cysteine).

6. Heat all tubes in boiling water bath for 3 to 10 min, then cool in tap water bath.

7. Add 0.1 ml of 3% cysteine solution to appropriate tubes. Do not add cysteine to the tubes designated as controls without cyteine.

A green-yellow color, characteristic of methylpentoses, develops in tubes to which cysteine is added and is stable for at least 24 hr. Other colors may be noted in the control tubes.

Measure rhamnose concentrations

8. Measure the absorbances of each reaction tube at 396 and 430 nm.

Methylpentoses generate uniquely colored products that can be distinguished from other sugars by measuring the optical densities at these wavelengths.

9. Subtract the absorbances of the controls without cysteine from the absorbances of the corresponding reactions with cysteine to generate corrected optical densities at 396 and 430 nm.

10. Calculate the differences between the corrected absorbances at 396 nm and the corrected optical densities at 430 nm (A 396-430).

11. Plot the concentrations of the standard rhamnose solutions versus A 396-430.

A graph with approximately linearly increasing A 396-430, as a function of rhamnose concentration, should result.

12. From the A 396-430 of the unknown PG-PS suspensions, estimate the approximate rhamnose concentrations from the graph generated from the standard solutions.

Streptococcal Cell Wall Arthritis

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