SYNERGISTIC EFFECTS OF NONIONICS ON CATIONICS 579 A corollary to this definition could be that a combination of the two sur- factants would exhibit a specific germicidal activity in a significantly shorter period of time than that exhibited by the sum of the two individual sur- factants. Alternatively, a lower concentration of cationic germicidal agent would be required to produce the same antimicrobial activity in the presence of a nonionic surfactant as the higher concentration of cationic exhibits in the absence of the nonionic surface-active agent. Many popular test procedures are described in the literature for measuring germicidal activity. Some tests are of limited value in that the procedure is restricted to the use of a single test organism. Others are broader in scope. Germicidal activity is not a property of chemical composition, but is influ- enced by a number of physical properties, such as solubility, miscibility, de- gree of colloid dispersion, ionization, surface and interfacial tension, and some other undefined properties. It has been claimed (5) that the greatest single factor influencing the resistance of bacteria to germicidal agents is the chemi- cal composition of the medium in which they are grown. Therefore, differ- ences in data on germicidal agents, using different test procedures, are diffi- cult to interpret and reconcile. Thus, it has been reported (6) that quats which would maintain a definite germicidal activity against several types of pathogenic bacteria on one type of surface, porcelain tile for example, would be ineffective against the same microorganisms on another nonsimilar hard surface, such as plastic, linoleum, or rubber. If this is true, then one might question the reliability of the in vitro calf skin test, which is now so widely used when one wants to test human skin antiseptic properties of a new skin degermer. The variety of uses for which germicidal preparations are recommended is overwhelming. Therefore, no single bacteriological test method could be ex- pected to be adequate for evaluating all germicidal products in the various uses for which they are intended. The problem of testing, therefore, depends on (a) use of a test method that will provide meaningful results, and (b) cor- rect interpretation of the data obtained. If useful data are to be obtained by the test method selected, two criteria must be met. (A) Data must be inter- preted accurately in terms of practical value, and (B) data must be precise and reproducible. Although evaluation of data belongs in the laboratory, its effectiveness is the practical application. The data presented in this paper will not be restricted to those obtained from any one test procedure, but will be selected from all methods used to study nonionic cationic interactions. MECHANISM OF (•ERNIIC1DAL ACTIVITY Before it can be pointed out how the germicidal activity of a cationic sur- factant can be enhanced, it is important to know how cationic surfactants act to kill microorganisms. A good review of the subject was recently presented by James (7). The cationic germicidal agent consists of a small negatively

580 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS charged anion and a large positively charged surface-active cation. Germici- dal agents generally possess either one long alkyl chain, usually varying from 8-18 carbon atoms, or the four substituent alky] groups on the nitrogen atom may total 10 or more carbon atoms. Since the surface of bacteria and proteins possesses a negative charge due to the presence of one or more types of ionic groups on the surface, such as amino, carboxy], or phosphate, the cationic sur- factant is readily adsorbed onto the surface of a broad spectrum of micro- organisms. All bacterial cells consist of a cytoplasm enclosed by three layers, the proto- plast membrane, the rigid cell wall, and the outermost slime layer. The char- acteristic shape of the microorganism is governed by the cell wall. There are certain fundamental differences between the cells of gram-negative and gram- positive bactcria, both in composition and metabolic activity of the cell. The protoplast membrane of gram-positive organisms contains lipid as phospholipid, protein, and carbohydrate. The cell wall in gram-positive or- ganisms, it has been shown, does play a role in the biochemical activity of the cells, and also serves to protect the biochemically active protoplast. With gram-negative bacteria, the cell wall is a complex structure which may partici- pate in metabolic functions of the cell. The gram-negative organisms have a higher lipid content and a lower amino sugar content than gram-positive bac-' teria, and there are also differences in amino acid content between the two groups. However, there are strains of gram-positive bacteria which are resis- tant to bacteriostatic agents and which contain large quantities of cell wall [ipids. It has been suggested (• that the disintegration site in the protoplast mem- brane is on the phospholipids present in the membrane. Rearrangement in the permeability barrier of the cell results in damage to the cytoplasm. This is fol- lowed by leakage of intercellular metabolites and coenzymes. In other words, the cationic surfactant changes the charge distribution on the cell surface which causes disintegration. Initially, very little cell disintegration may occur on contact of the cationic surfact•tnt wlkh the micro'organism, but subsequent- ly, autolyric enzymes present in the 'e'ell speed up dissolution of the cell. The gram-positive organism geneSally loses the small molecules more rapidly than the gram-negative organism. Since most gram-ntgative cells generally have a higher 1ipid content in their cell walls than the gram-positive organisms, the cell wall polyproteins may be able to resist changes in charge distribution, and prevent the quat from reacting with the cell wall, thus giving a measure of protection to the protoplast membrane. The same mechanism is said to also apply to yeast. EXPERIMENTAL AND DISCUSSION One could therefore expect that the addition of a nonionic surface-active agent would speed up this process, or even decrease the concentration of