Notes on the Gram Stain. II

The earlier investigations of the senior author have been continued, and the writers come to the following conclusions:1. The pH of the environment in which organisms are grown may affect the Gram reaction.2. Organisms should be classed as Gram positive or Gram negative according to the staining reaction when grown in the optimum pH range for the species. Since the reaction may change with growth the media should be strongly buffered.3. An alkali should be incorporated in the Gram staining technic in order to nullify the effect of an acid environment. We frequently wish to stain organisms from an environment not subject to control.4. If alkali is added to the staining technic and the best American dyes are used the reaction of the medium upon which organisms are grown should not affect the Gram reaction.5. There is no relation between acid and alkali production and the classification of bacteria regarding the Gram reaction. Acid and alkali producers are found in both groups.6. The selection of a Gram staining technic should be based upon reliability of the methods under unfavorable conditions of acidity as well as on results obtained with borderline organisms.There is included a discussion of the requirements of a practical Gram stain.