Identification of Gram-negative bacteria in histological sections using Sandiford's counterstain.

There exist many methods for the application of the Gram stain to histological sections, and with all of them there is generally no difficulty in recognising Gram-positive organisms. However, with most of the well-recognised methods Gram-negative bacteria are difficult to distinguish from the background. Twort's method distinguishes Gram-negative bacteria from the background but is relatively timeconsuming (Cook, 1974), and we therefore turned our attention to Sandiford's modification of the Gram stain (Cruikshank et al., 1975). Sandiford's counter stain was designed for use with bacteriological smears but appeared eminently suitable for applying to paraffin sections. In brief, the modification consists of staining bacteria which are not Gram-positive with pyronin and using malachite green as a counterstain. The resultant black and red organisms (Gram-positive and -negative respectively) both contrast well with the blue-green background and are easily recognisable. We have used this method for the last five years and have found it most helpful in the recognition of Gramnegative bacteria. A comparison of Twort's and Sandiford's modifications of the Gram stain shows that the results are virtually indistinguishable, but we prefer the Sandiford method because it is quicker. Recently, Sowter and McGee (1976) have described a Gram-methyl green-pyronin-light green method which gives results comparable to the GramSandiford technique but again the latter stain acts much more quickly. No doubt some histopathology laboratories already use the Sandiford modification but we are unable to find any description of it in the standard histology manuals and believe that it deserves wider recognition.