The sequence of steps to be followed is as under. Place a clean slide over one centimeter square, drawn on a paper and place 0.01 ml of milk sample in the centre of the square. Spread the sample with needle to cover the square. Dry the smear with gentle heat and immerse the slide in xylene or chloroform to remove fat. Fix the smear with 95% alcohol for 3 minutes. Stain the smear with Breed's methylene blue (S20)
for 2 minutes. Wash the smear with 90% alcohol till smear appears faintly blue. Dry in air and examine under oil immersion objective. Bacteria are seen dark blue against light blue background.
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