Detailed Differentiation of Bacteria by Means of A Mixture of Acid and Basic Dyes at Different pH-Values

As previously reported by the author (1927), a mixture of methylene blue and eosin Y can be used for the differential staining of bacteria. It gives a fairly deep staining of bacteria at about pH 3 and above. Below pH 3 the eosin Y stains bacteria only a very pale pink; at such high H-ion concentration, the eosin is present as undissociated color acid, and for this reason not enough eosin is in solution to stain bacteria. To improve the staining at such reactions, the eosin was replaced by a stronger acid dye, namely acid fuchsin. The mixture of methylene blue medicinal Merck and acid fuchsin can be successfully used at a pH-value as low as 0.8. The method of staining by this new mixture is entirely the same as with the old mixture. It is sensitive enough to detect the difference in the isoelectric points: (1) of the single bacteria from the same pure culture, (2) of different strains of the colon and typhoid organisms. Some strains of the colon organism were found by this method with an isoelectric point at a pH-value as low as that of the Staphylococcus. Others, on the contrary, have their isoelectric point as high in the pH-scale as that of the typhoid organism. The new mixture can also be used for the study of the chemical composition of the different parts of bacterial body. Applying it at a definite pH-value, the author was able to stain differentially polar bodies of the typhoid group and of the diphtheria organism. This new mixture can be recommended in staining of B. diphtheriae as a substitute for Neisser's stain. It is interesting to note that polar bodies of the colon group consist of more alkaline protein than the body of the bacteria itself, i. e., they are stained by acid fuchsin. The polar bodies of the B. diphtheriae on the contrary are composed of more acid protein than the bacterial body; i. e., they are stained by methylene blue. The impossibility of detecting the above mentioned variations in the isoelectric points of bacteria using the Gram method is explained by the absence of pH variations in the latter technic. The differentiation of bacteria by the Gram stain depends chiefly on the varying stability of the compound formed (Gram-positive or Gram-negative bacteria plus gentian violet and iodine) in the presence of organic solvents, such as alcohol, acetone, etc.