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Milk Phosphatase Kit




+32 1658 9045


0032 (0)16 41 44 07

+32 1650 9045


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0032 (0)16 41 44 07







Ready-to-use kit for detecting phosphatase activity in milk

Product No 182250
Size 2 x 500 ml

Intended use

Phosphatase test is performed to confirm that pasteurisation
has succeeded. Phosphatase is enzyme normally found in
raw-milk. Phosphatase enzyme is inactivated when
pasteurisation is carried out correctly at 72°C for 15 seconds. If
pasteurisation has not been carried out correctly or
pasteurised milk is contaminated with raw-milk, there is active
phosphatase enzyme in milk and it is indicated by
phosphatase test. Phosphatase test has to be performed
immediately after pasteurisation.

Principle of the reaction

The test is based on yellow color, which is developed when
there is active phosphatase enzyme in milk. If there is not
phosphatase enzyme activity in sample, color of the sample
does not change.

                                alkaline phosphatase
p-nitrophenyl- + H2O -----------------------------> p-nitrophenol
phosphate                     buffer pH 10,0            + phosphate

Sample type

Milk and milk products

Kit contents


170057 Diethanolamine-MgCl2-buffer:
1.0 mol diethanolamine /0.5 mmol MgCl2 -buffer, pH 10.0
2 x 500 ml

153006 p-NPP-Na2 –tabletti:
p-nitrophenylphosphate 230 μmol
50 tablets (103006)

Instructions for use


2…8°C protected from light, in which case the unopened
reagents are stable until the expiration date stated on the
label. Working solution is stable for a week when stored at
2…8°C and for 24 hours when kept at room temperature.


Water-bath, temperature 38-40°C
Test tubes
Pipettes, volume 0,5, 5 ja 10 ml
Measuring cylinder
Peleus ball for pipettes
Test tube rack

Working solution

Dissolve one p-NPP-Na2 –tablet into the 20 ml of

Preparing the negative blank

Inactivate phosphatase enzyme of the reference sample by
heating 10 ml of milk sample at 80°C (minimum) for
5 minutes. Cool the heated sample into room temperature.

Test procedure

Pipet 5 ml of working solution into two different test tubes.
Pipet 0,5 ml of negative blank into one test tube and 0,5 ml of
sample into the other tube. Do not pipet with your mouth since
saliva includes phosphatase enzyme. Close the test tubes with
parafilm and shake. Keep the test tubes in water-bath (38 –
40°C) for 10 minutes.

Sensitivity test (positive blank)

Background of sample and reagents is excluded with positive
blank. It is performed if necessary. Prepare positive blank as
negative blank but at the end add 0,2 % of raw-milk. Pipet 5 ml
of working solution and 0,5 ml of milk sample (phosphatase
enzyme inactivated) into which 0,2 % of raw-milk is added. The
color of the positive blank is only slightly yellow but when
compared to the negative blank the difference is clearly
distinguished visually.

Interpreting the result

Examine the color of the solution against white background. If
color of the solution turns yellow or yellowish in 10 minutes
(compare with the negative blank which must not turn yellow),
the reason might be that the temperature in pasteurisation was
not high enough or the milk is contaminated with
unpasteurised milk. The result is phosphatase positive in this
case. If the color of the solution is not changed in 10 minutes
wait another 20 minutes and compare the color again
(indicative of very low phosphatase enzyme concentrations).

Test performance

The test result is visually seen as positive when 0,2 % raw-milk
is added to pasteurised milk. (see Sensitivity test)

Interference/ Sources of errors

No known interferences or sources of error.


The tablet is harmful when swallowed. Avoid direct contact
with skin and eyes.
Buffer irritates eyes and skin. Danger of serious damage to
eyes. Harmful: danger of serious damage to health by
prolonged exposure if swallowed. In case of contact with eyes,
rinse immediately with plenty of water (for 15 minutes) and
seek medical advice. Wear suitable protective clothing, gloves
and eye/face protection. If swallowed, seek medical advice
immediately and show this container or label.

Do not use damaged reagent or tablets.
Buffer solution and working solution turn yellow if they are
stored exposed to light, or long periods at room temperature.
Do not use clearly yellow solution.



Copyright © 2002 GENTAUR Molecular Products
Last modified: 05/29/09