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Staining With Methylene Blue |
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Staining with Methylene Blue:
In this technique, only single dye e.g. methylene blue is used and still difference in colour between volutin (red) and cytoplasm (blue) is observed due to the metachromatic property of methylene blue. According to Bergeron and Singer (5), metachromatic granule has high affinity towards the cationic molecules of methylene blue.
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When dye molecules are tightly packed in granule, the orbital paths of electrons of dye chromophore groups are changed. This change finally causes change in the light absorbing property of the dye. Therefore, metachromatic granules appear red, eventhough dye used is blue. This method is at disadvantage when granules are smaller in size and cytoplasmic barring is absent. This will make it impossible to differentiate diphtheria organism from other bacilli.
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The procedure is as under. Prepare a smear and fix it by gentle heat. Flood the slide with Loeffler's alkaline methylene blue stain (SI8) and allow it to react for 5 minutes. Drain the stain and wash the slide with water. Air dry and examine under oil-immersion objective. Metachromatic granules appear red against pale blue cytoplasm.
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