Gram staining is a method to stain or colour bacteria in order to make them better visible or to differentiate bacteria when analyzing them under a microscope. In a first step of the staining procedure the bacteria are coloured with a dark-blue/purple dye.

This staining is stable for so called gram-positive bacteria and they keep this staining also during a following washing step. This is in opposite to so called gram-negative bacteria which lose most of the dye during washing step. These bacteria can be stained with another suitable red dye in a subsequent second staining step. Thus, they are also clearly visible under a microscope, where now gram-positive bacteria will appear dark-blue/purple while gram-negative bacteria will appear red.

The reason for the different behavior during staining is a different structure of the outer cell sphere. Simplified, the outer cell sphere of the gram-positive bacteria has an inner membrane which is covered by a layer of molecules which can build stable binding with the dark-blue/purple gram dye. This layer of dye binding molecules is thinner and also covered by a smooth second membrane at gram-negative bacteria. Therefore, the first gram dye can be washed off and the second dye can be absorbed.
This method has been developed in the late 19th century by the Danish Hans-Christian Gram. Today there are also other staining procedures available which are using other molecular properties of the bacteria for dye adsorption and distinction.

In the field of wastewater treatment Microthrix and Nocardia, which may cause floating sludge, are bacteria of the gram-positive ones. Gram-negative bacteria in this field are for example Sphaerotilus spp. and Haliscomenobacter hydrossis, which are well known to cause bulking sludge. The enterobacteria coli are also gram-negative.


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