The presence of lactic acid is the most important cause of a positive lizarol test. At the higher acid level (lower pH) the
protein suspension in milk will be less stable with the result that protein flocculation (coagulation) will occur when acid milk is
mixed with alcohol.
Mastitis
Mastitis milk differs from normal milk with regards to, amongst others, the protein and mineral composition. These differences
cause the casein (protein) to flocculate more readily in mastitis milk. As the pH of mastitis milk is normally higher than that of
normal milk the alizarin colour will turn violet if such milk is tested.
Sweet curdling
Certain contaminating bacteria are capable of producing rennin-like enzymes, which increase the viscosity of the milk and
even flocculate casein in the absence of acid. This phenomenon is known as sweet curdling. Such milk can be identified
by inoculating aseptically a small quantity there-off into sterilised milk and incubating the inoculated milk at room
temperature for a day or more on which the viscosity of the milk will increase at the normal pH of fresh milk.
Large numbers of these bacteria e,g. 800,000 /ml milk was found to create a positive reaction in previously sterilised milk.
Mineral imbalance
Too high or too low a concentration of minerals such as calcium, phosphate and citrate will increase the ease with which
casein will flocculate in the presence of alcohol.
This aspect is complex and not well defined. It is well known that the addition of very small amounts of Calcium (0.2 ml M/4
Ca-acetate / 25 ml milk = 0.6g / 1 l milk) and Magnesium (0.1 mlM/4 MgCl 2 / / 25 mlmilk = 0.95 g / 1l milk ) will normally result
in a positive test while the addition of phosphate and citrate salts may alleviate the situation. In more extreme cases excess
phosphate and citrate may however be the cause of a positive test.
Small variation e.g. 0.01% in the normal Calcium content of milk may influence the test.
It is claimed that in some instances supplementing a cow’s ration with mono-calcium phosphate e.g. 20 – 40 g /day /
cow may improve the situation.
If a positive reaction is due to mineral imbalance a very slight souring of the milk, causing a portion of the colloidal fraction of
the minerals to become soluble, may result in a negative test.
Heating of alizarol positive milk to 60°C for 30 minutes will cause partial precipitation of Ca 3 (PO 4 ) and as result the test may
turn negative.
Stage of laction;
The chemical composition of early and late lactation milk differs from that of normal milk in the sense that it is characterized
e,g. by higher levels of albumin, globulin and chloride.
Due to these differences the protein suspension in such milk is usually very unstable against alcohol.
Milk is normally very unstable to alcohol in early lactation, thereafter gradually becoming more stable and remaining at a fairly
constant level of stability which is specific for each cow. Towards the end of lactation milks from some cows become more
stable but a decrease in stability is more common.
Other factors that may cause alizarol positive reactions include:
Irritation of the udder tissue
Infected uterus
Abortion
PRINCIPLES OF THE TEST
The stability of the protein dispersion in milk is maintained by hydration (combining with water) and by the negative electrical
charges on the protein particles. If either of these two factors undergoes a change, the proteins will flocculate. Alcohol is a
dehydrator and therefore destabilizes the protein. If the protein is already slightly unstable due to souring of the milk,
dehydration with alcohol will lead to the precipitation of the protein in the form of flakes.