Immunological Specificity of Carbohydrates Derived from Staphylococci. ∗

Abstract

During the course of studies concerning hypersensitiveness in patients with trachoma, it became necessary to determine the possibility of the existence of different immunological types among Staphylococci. Preliminary experiments undertaken with the agglutination reaction revealed that strains isolated from different sources and conditions are not differentiated satisfactorily by this technique, Consequently an effort was made to fractionate carbohydrates from this organism, and this was accomplished successfully.

The method finally adopted for the separation of carbohydrates from Staphylococci may be outlined as follows: (1) concentration of broth cultures (18–24 hours) by centrifugation in a Sharpies; (2) heating the thick suspension in N/16 HCl for 20 minutes in a boiling water bath; (3) after centrifuging, the supernatant is treated with 40% NaOH until flocculent precipitation occurs, which is at a slightly acid reaction; (4) to the supernatant N acetic acid and Na acetate, and 2 volumes of 95% alcohol are added; (5) after standing in refrigerator over night, the residue following centrifugation is taken up in H₂O, and the supernatant is reserved for further separation; (6) the water solution is treated with N acetic acid until precipitation occurs and to the supernatant after centrifugation are added 2 volumes of 95% alcohol and sufficient Na acetate to cause precipitation. The residue is re-extracted until all the soluble material is obtained; (7) up to 6 precipitations with acetic acid are sometimes required to cause complete removal of precipitable matter. The end product never precipitates in the usual protein precipitants; it is always ninhydrin negative, although from time to time biuret positive. The total nitrogen has varied up to 5%. The Molisch reaction is always strongly positive.