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 OF METHOD 



Preparation of stain. Various stains have been tested 

 out : saf ranin, 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 



