This is a suggested sample preparation method for milk (liquid). 


NOTE: The volume of supernatant used in the assay can be adjusted to suit the sensitivity of the assay being used.


1. To a 1.5 mL polypropylene microfuge tube, add the following with mixing after each addition:

Pipette into microtube


sodium phosphate buffer (0.4 M)

0.20 mL

Distilled water

0.70 mL

milk sample

0.50 mL

concentrated Carrez II solution

0.05 mL

concentrated Carrez I solution

0.05 mL

Mix by vortex and then centrifuge at 13,000 rpm for 10 min. 

Carefully pipette the clear supernatant for use in the assay.


1. Sodium phosphate buffer (0.4 M, pH 7.6, 4 mM magnesium sulphate): 500 mL
Add 24 g of Na2HPO4 (Sigma cat. no. 71640), 4.3 g of NaH2PO4.
H2O (e.g. Sigma cat. no. 71506) and 0.5 g of MgSO4.7H2O (e.g. Sigma cat. no.
M5921) to 450 mL of distilled water. Adjust the pH to 7.6 with
1 M NaOH (40 g/L) and make the volume up to 500 mL with distilled
water. Store at 4°C.

2. Concentrated Carrez I solution: 200 mL
Dissolve 30 g of potassium hexacyanoferrate (II) {K4[Fe(CN)6].3H2O}
(e.g. Sigma cat. no. P9387) in 200 mL of distilled water. Store at room

3. Concentrated Carrez II solution: 200 mL
Dissolve 60 g of zinc sulphate {ZnSO4.7H2O} (e.g. Sigma cat. no. Z4750)
in 200 mL of distilled water. Store at room temperature.