Box 12 The Gram stain

The Gram stain involves the sequential use of two stains (see below). The critical stage is step 3; some cells will resist the alcohol treatment and retain the crystal violet, while others become decolorised. The counterstain (safranin or neutral red) is weaker than the crystal violet, and will only be apparent in those cells that have been decolorised.

The Gram stain is a differential stain, which only takes a few minutes to carry out, and which enables us to place a bacterial specimen into one of two groups, Grampositive or Gram-negative. The reason for this differential reaction to the stain was not understood for many years, but is now seen to be a reflection of differences in cell wall structure, discussed in more detail in Chapter 3.

Specialised forms of microscopy have been developed to allow the viewer to discern detail in living, unstained specimens; these include phase contrast and dark-field microscopy. We can also gain an estimate of the number of microorganisms in a sample by directly counting them under the microscope. This is discussed along with other enumeration methods in Chapter 5.

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