Carbohydrate Assays

REFERENCE: Wright and Rebers, Anal. Biochem. 49: 307-319, 1972.

OBJECTIVE: To determine the relative amounts of
LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars.

METHODS:

HEXOSE ASSAY: (sugars in the outer core and O-Ag)

1. Cool 200 µl of sample in an ice bath.
2. Add 900 µl reagent A (4 ml dH₂0 + 24 ml
   conc H₂SO₄). Shake carefully. Warm to room temperature (23ºC).
3. Boil in water bath for exactly 20 min.
4. Cool to room temperature under cold water tap.
5. Add 20 µl reagent B ( 0.3 g cysteine HCl in 10 mls
   dH₂O (fresh)). Shake and then let stand 30 min. in the dark at room temperature.
6. Assay the difference between 415 and 380 nm.
7. To zero the spec use all above ingredients except the bacterial sample. Standards are made of 1,2,3,4, and 5 µl of desired hexose sugar (eg. glucose) at 2 µg/ml.
8. To assay all three kinds of sugars do a wave length scan
   from 350 - 600 nm.

1. In step 3 above, boil for 5 min.
2. In step 6 above, let stand for 3 hours in the dark (this
   produces a green-yellow colour rather than pink)
3. Standards are the same as above using rhamnose.