SUMMARY: Screening tests indicated that Gram positive bacteria are inhibited by long chain fatty acids. No inhibition was demonstrated with Gram negative bacteria. The minimum inhibitory concentrations (MIC) for a series of the fatty acids are presented. Growth curves in the presence of linolenic acid showed increases in lag phase duration and calcium addition reversed this effect, thus indicating the arbitrary nature of the MIC values. Bactericidal studies showed lauric acid to be the most active saturated fatty acid but the activity was less than that of the C18 unsaturated fatty acids. Oleic acid was more effective than elaidic acid. Calcium ions, cholesterol and ergocalciferol reversed the activities of lauric and linoleic acids but magnesium ions effectively counteracted lauric acid only. A physicochemical explanation for the relative activities has been attempted.

SUM-Y.Long chain fatty acids stimulated oxygen uptake by Gram positive bacteria a t bactericidal and protoplast lytic concentrations and produced inhibition at higher levels. The order of activity between individual acids and effects of reversal agents on respiratory activity corresponded to those which produced bactericidal activity. Protoplasts were more susceptible to inhibition than whole cells. Gram negative bacteria were inhibited to a limited extent at high fatty acid concentrations, but spheroplasts were highly sensitive. Fatty acids inhibited amino acid uptake both aerobically and anaerobically a t sub-bactericidd levels. The effects were reversed by metal cations, and reflected the activity of dinitrophenol and sodium azide. The susceptibility of organisms to inhibition was of the same order as the sensitivity to other antibacterial effects. The probable mode of action of the fatty acids is discussed in terms of the interference with energy metabolism within the bacterial cell.

Fatty acids of chain length > C,, induced lysis of protoplasts at pH 7.4 when the concentration was nearly bactericidd. At p H 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysk was produced by the same acids below bactericidal concentrations. The lysis was immediate a t p H 8, but at pH 6 the effect was preceded by contraction of protoplasts. At pH 7.4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillwr megaterium relative to those of 2Mimcoccus lyeodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects then protoplasts. Reversal agents antagonized the lysis of protoplasts by fatty acids. A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed.ANTIBACTERIAL EIWECTS of long chain fatty acids have already been discussed in relation to bactericidal activity and physicochemical properties in solution (Galbraith, Miller, Paton & Thompson, 1971 ;Galbraith & Miller, 1973a).Several workers have produced evidence which suggests that antibacterial compounds with lipophilic and hydrophilic properties act on the cytoplasmic membrane (Hugo, 1967; Salton, 1968). The most convenient method of investigating the interaction between these antibacterial compounds and the bacterial membrane involves the use of protoplasts and spheroplasts. Gilby & Few (1957, 1960) have demonstrated the lysis of protoplasts by surface active agents and correlated the lytic activity with antibacterial activity on whole bacterial cells. In previous work with long chain fatty acids (Galbraith & Miller, 1973a) the reversible uptake of 14C labelled fatty acids by bacterial cells and protoplasts also indicated that the probable site of action of the fatty acids was the cytoplasmic membrane and so in the present work the lysis of protoplasts has been studied in order to investigate the relationship between antibacterial action and phyaicochemical activity on the membrane. The studies have included the effects of pH, aliphatic chain length, reversal agents and the sensitivity of the individual bacterial species which were used to determine bactericidal activity and uptake. An attempt was also made to investigate the physicochemical effects of fatty acids on whole cells in order to determine whether the overall response is similar to that found with protoplasts in the absence of the cell wall. Materials and MethodsCultures and media The organisms used were Bacillus megaterium NCIB 9521 and Micrococcus lysodeiktieus NCIB 9278. Bacterial suspensions and protoplasts of B. megaterium were prepared [6471

The bactericidal activity of long chain saturated fatty acids was antagonized by alkaline earth metals. The activity of linoleic acid waa less effectively antagonized but was more sensitive to reversal by ferric and stannous ions. With increasing pH value the bactericidal activity of lauric acid decreased but that of the longer chain saturated acids increased. Both Gram positive and Gram negative bacteria reversibly adsorbed fatty acids, Uptake increased with decreasing pH value and increasing chain length. Although adsorbed to a lesser extent, the intrinsic activity of linoleic acid was greater than lauric acid. The uptake appeared to be non-specSc and governed by the physicochemical properties of both the a i d s and the bacterial cell surfaces. Sensitivity to the fatty acids increased with decreasing pH value. Protoplasts of Bacillus mqaterium adsorbed fatty acids to a greater extent than whole cells. Resistance of the Gram negative Pseudomonas p k e o l i w l a was not due t o non-adsorption of the fatty acids. H. Galbraith and T. B. Miller ChemkdsFatty acids (l-14C labelled) were obtained from the Radiochemical Centre, Amersham, England. Lysozyme was a product of Sigma Ltd. Koch-Light Co. Ltd supplied the scintillation grade PPO (2,5 diphenyloxazole), POPOP [1,4-di(2-(5-phenyloxazolyl)) benzene] and naphthalene. The Maybridge Chemical Co. (Scarne Industrial Estate, Launceston, England) supplied the 2,4 dioxan which was double-distilled prior to use. All other chemicals were Analar grade or as previously specified (Galbraith et al., 1971). Preparation of organismsThe bacteria were grown, harvested and suspended in the buffered test systems as described by Galbraith et al. (1971), for bactericidal studies. Similarly, cations were added (as chlorides) and bactericidal activities estimated as before. Volumes (0.5 ml) of the bacterial suspension (c 1-2 x lo9 cells/ml) were added to 2.5 ml of the particular test system. Buffer solutionsPhosphate buffer at pH 6.0 and Tris-HC1 buffers at pH 7.0 to 8.0 were prepared at 0.1 M concentration by the method of Cruickshank (1968). The effect of cations was studied at pH 7.0 to 8.0 to avoid the precipitation found with phosphate buffers. Preparation of protoplustsProtoplasts of B. megaterium were prepared by a method similar to that of Baird-Parker & Woodroffe (1967). Cells (c. 1.2 x 109/ml) were suspended at pH 7.0 in 0.1 M Tris-buffered 0.5 M sucrose containing 30 pg/ml of lysozyme and 25 mM-NaC1 at 30". Microscopic examination showed that removal of the cell wall was complete at 25 min after which the protoplasts were sedimented by centrifuging (1000 g for 15 min) and gently resuspended to the original volume in the required buffer, containing 0-5 M sucrose and 2-5 m-NaC1. Where used, 0.5 ml of this suspension was added to 2.5 ml of the isotonic test system. Measurement of radioactivityRadioactivity was counted on a Beckman LS-150 liquid scintillation counter using 10 ml volumes of the scintillation solution containing naphthalene, 100 g; PPO, 5 g ; POPOP, 0.3 g/1 of 2,...

In 1913 Legendre and Pieron' reported that injection of cerebrospinal fluid (CSF) from a sleep-deprived dog into the cisterna magna of a normal animal induced sleep in the recipient for 2-6 hours following the injection. Recipients of fluid from normal dogs remained alert. The "hypnotoxic" factor was said to be nondialyzable and thermolabile. The Pieron phenomenon was reinvestigated in 1939 by Schnedorf and Ivy,2 who reported positive results in 9 out of 20 trials. The experimental conditions involved severe stress to both donor and recipient animals. Donor animals were deprived of sleep for 7-16 days and the technique of transferring relatively large quantities of CSF from donor to normal recipient without anesthesia undoubtedly involved severe trauma to the experimental animals. For these and other reasons, Schnedorf and Ivy questioned the relevance of the Pieron phenomenon to normal sleep, although they were convinced that the phenomenon was real.Monnier et al.3' 4 have recently reported that electrical stimulation of "sleep centers" in the thalamus of rabbits causes release of a dialyzable sleep-promoting factor in cerebral venous blood. The role of this substance in the initiation of normal sleep is unknown, as is its relation to the nondialyzable material described by Pieron. A humoral factor inducing sleep has also been postulated by Hayashi who reported that CSF obtained during the period of depression following convulsive seizures contains a factor which inhibits convulsions5 and promotes sleep6 when injected intraventricularly. Hayashi suggested that the active principle might be 4-amino-2-hydroxybutyrate or possibly 4-OH-butyrate.We have now conducted a new investigation of the Pieron phenomenon, taking advantage of new techniques which enable experiments to be carried out under more physiological conditions than were possible in the past. Crucial to our experiments is an abundant supply of CSF from goats which are provided with nylon guide tubes permanently implanted in occipital bone above the cisterna magna.7 8 CSF can be withdrawn repeatedly from each animal at the rate of 0.1 ml/minlute for many hours without the use of anesthesia and without causing the animals any apparent discomfort. For initial trials we prepared five cats with chronically implanted intraventricular cannulas of the Feldberg-Sherwood type.9 The ventricles of these animals were infused slowly (0.1 ml/min) with 1-3 ml of CSF from a normal goat or from the same goat which had been deprived of sleep for 72 hours. Cats which received fluid from sleep-deprived goats fell into a profound sleep or torpor which lasted 12-24 hours; the "sleep" appeared to be natural in the sense that the cats could be awakened by noise or handling, but reverted to sleep when left undisturbed. Some animals fell asleep during the infusion. Similar behavior was not observed when the same cats were infused with control CSF from the same (non-sleep-deprived) goats. These results supported Pieron's observations and indicated that the phenomenon was not specie...