First samples must be  treated as per the sample preparation example instructions in the assay protocol (page 9-10) as follows:

1. Mill yeast, mushroom or plant sample to pass a 0.5 mm screen using a Retsch centrifugal mill (or equivalent).

2. Add milled sample (approx. 100 mg, weighed accurately) to a 20 x 125 mm Fisher Brand culture tube (or equivalent). Tap the tube to ensure all of the sample falls to the bottom of the tube. 


Option A: For samples that require sugar removal - ethanol washing:


3A. Add 8 mL of aqueous ethanol (80% v/v) to each tube and stir the tubes vigorously on a vortex mixer. Incubate the tubes at

~ 80°C for 15 min. Ensure that the solution does not boil out of the tube. Add another 8 mL of aqueous ethanol and stir the

tubes vigorously on a vortex mixer. Cool and centrifuge at 1,500 g for 10 min. Carefully decant and discard the supernatant

solution.

4A. Resuspend the pellet in 8 mL of aqueous ethanol and stir vigorously. Add another 8 mL of aqueous ethanol and stir.

Centrifuge the tubes at 1,500 g for 10 min. Carefully decant the supernatant. Invert the tubes on absorbent paper to ensure

complete removal of all free liquid.


For samples not containing reducing sugar (like commercial yeast β-glucan preparations that are already washed), the alcohol washing steps may not be required.

Therefore after step 2 above proceed as follows:


Option B: For samples that do not require sugar removal:


3B. Wet the sample with 0.2 mL of aqueous ethanol


For either sample option, then proceed to solubilisation of polysaccharide as follows:


Solubilisation of polysaccharide


1. Add a magnetic stirrer bar (5 x 15 mm) to each tube and place the tubes in a test-tube rack in an ice water bath over a magnetic stirrer (Figure I). Add 2 mL of ice-cold, 60% (v/v) sulphuric acid to each tube with vigorous stirring on a magnetic stirrer (to ensure complete dispersion/dissolution). Continue stirring for 1 h.

2. Add 12 mL of water to each tube and continue stirring for a few min.

3. Loosely cap the tubes and place them in a boiling water bath at ~ 100°C. Allow the remaining alcohol to boil off over 5 min,

then tighten the cap and continue the incubation for 2 h. 

4. Cool the tubes to room temperature and carefully loosen the caps.

5. Quantitatively transfer the contents of each tube to a 100 mL beaker and adjust the volume to approx. 60 mL with

distilled water. Adjust the pH to approx. 7.6 with 2 M NaOH (using a pH meter). Quantitatively transfer the solution to a

 100 mL volumetric flask using a water wash bottle and adjust to volume with distilled water. Mix the contents well by inversion.

6. Filter an aliquot of the suspension through Whatman GF/A glass fibre filter paper or centrifuge at 1,500 g for 10 min.



Subsequently the sample should be applied to the enzymatic determination procedure on page 5 of the assay protocol to determine mannose released. Typically, no further dilution is required and a sample volume of 0.2 mL is satisfactory

The mannan content can be calculated as follows:

 

Where:
100 = volume correction factor (total volume after solubilisation with acid adjusted to 100mL)
100 / W = conversion back to 100 mg of sample (i.e. as % w/w).
W = weight of sample analysed
162/180 = a factor to convert from free D-mannose, as determined, to anhydro-mannose, as occurs in mannan.