154 UNIVERSITY OF WISCONSIN STUDIES 
quantity of a special methylene blue stain in a specially pre- 
pared centrifuge tube. The stain and milk are thoroughly 
mixed and allowed to stand for at least ten minutes. The 
sediment is thrown onto the cork by whirling the tube 
in a centrifuge. The sediment is carefully spread on a micro- 
scopical glass slide. This smear is allowed to air dry with- 
out coming in contact with water at any time. When dry 
it is examined under the microscope with a low power and 
later with the oil immersion objective. 
DetaiLs or MretHop 
Preparation of stain—Various stains have been tested 
out: safranin, methylene green, dahlia, thionine and methylene 
blue. On the whole methylene blue seems to be the stain 
best adapted to the test. Practically all of the work has been 
done with the German dye (Gruebler), but several American 
made dyes have been tested recently and have proved to be 
entirely satisfactory. 
At first quite concentrated solutions were used. Small 
quantities of these were added to larger volumes of milk. 
_ While this method produced satisfactory results, there was a 
tendency for these dyes to curdle the milk, and when this 
happened the test was worthless. 
I have found gradually that this danger can be overcome 
by using a more dilute stain and adding larger proportions 
of it to the milk. It is quite essential to the proper working 
of the test that the right amount of the dye be added to the 
milk. Fortunately this is indicated by the color of the 
milk. When methylene blue in any concentration is gradually 
added to milk the color becomes a deeper and deeper blue, un- 
til quite suddenly it becomes a ‘‘cadet blue’’. This deep blue 
color seems necessary to the proper working of the test. It 
may be arrived at in any way, but there is less danger of 
causing a precipitation if the dye is added to the milk in a 
weak solution. 
To make the solution needed, 1.5 grams of the methylene 
blue powder is added to a liter of distilled water. I have 
