378 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS lation using 0.004% P.M.N. in fluorescein drops, sterility was achieved within a day. These workers did not use contact times of less than a day and the recovery media did not contain inactivators. Phenylethyl alcohol Lilley and Brewer (38) showed that 2-phenylethyl alcohol was partic- ularly active against gram-negative bacteria including Pseudomonads. Klein et al (27) tested phenylethyl alcohol against Ps.aeruginosa in the presence of atropine, eserine and fluorescein. In each case 0.6% sterilized within 1 hr while 0.5ø/0 required contact of up to 3 hr. Inactivators were not used during recovery. This preservative was rejected by Murphy et al (36) on the grounds that two strains of Ps.aeruginosa could be serially transferred in the presence of 0.5ø/0, and four strains of Staph.aureus grew in the presence of 0.6% in broth. Lawrence (29) tested the activity of 0.5% phenylethyl alcohol against 26 strains of Ps.aeruginosa and four species of Proteus using inocula of undiluted broth culture. Sterilization occurred after contact times of up to 6 days in the presence of a series of ophthalmic drugs. In the absence of drugs, sterilization occurred after longer contact periods and in some instances had not occurred by the end of the experimental period (6 days). Corneal ulcers were produced from intracorneal injections of a Ps.aeruginosa contaminated solution of 2ø/0 phenylethyl alcohol after 8 hours' exposure (longest experimental period) (11). These workers found that 0.75% solutions were irritating to the eye. Phenylethyl alcohol 0.6% failed to sterilize three strains of Ps.aeruginosa in aqueous suspension during exposures up to 1 hr, but sterilized one strain of Staph.aureus within 1 hr using cell concentrations of about 105/ml (37). Kohn et al (24) tested 0.5ø/0 phenylethyl alcohol against 13 strains of Ps.aeruginosa (2 x 106/ml) using Tweens as inactivating agents in the recovery media. Sterilization was not effected after 24 hours' contact, and in vivo experiments confirmed these in vitro results. Phenylethanol 0.9ø/0 sterilized inocula of 100 cells/ml of one strain of Ps.aeruginosa in aqueous suspension in 30 min at 18 ø. Inactivators were not used in the recovery medium (31, 32). Quaternary ammonium compounds There is much published evidence about the use of these compounds as ophthalmic preservatives. Klein et al (27) found that a concentration

THE PRESERVATION OF OPHTHALMIC PREPARATIONS 379 of 0.05% benzalkonium was required to sterilize broth suspensions (108/ml) of three strains of Ps.aeruginosa. Subcultures were taken after two days. These workers found that although 0.5% cetrimide sterilized, there was one instance when cetrimide was shown to have comparatively little effect against Ps.aeruginosa. Benzalkonium chloride was found not to be a uniformly effective bactericide against Ps.aeruginosa. Five out of 30 strains were not sterilized by contact for 24 hours with 0.01% of the agent, at pH 7.4 (36). Lawrence (39) in his review concluded that benzalkonium chloride, chlorbutanol and P.M.N. were the most suitable agents on the basis of published evidence. The same worker (29) tested benzalkonium chloride 0.02% and 0.01% against 26 strains of Ps.aeruginosa, and four species of Proteus using inocula of undiluted 24 hr broth cultures. Tests were made in (1) simple buffer, (2) in the presence of several ophthalmic drugs in buffer, and (3) in the presence of several drugs in aqueous solution. In every instance sterility was achieved in less than 30 min with 0.02% benzalkonium chloride. The majority of strains were sterilized by 0.01% in less than 30 min in all three systems but a few strains needed up to 6 hr. Chemical inactivators were used in the recovery broth. These results conflicted with those of Riegelman et al (11) who found that benzalkonium chloride was only slowly bactericidal. They suggested that the discrepancy between their results, and those of earlier workers, was because of inadequate inactivation of benzalkonium chloride in the recovery broth used by other workers. They found that 0.01% benzal- konium chloride failed to sterilize 108/ml Ps.aeruginosa cells in saline in one week when tested by in vivo methods whereas the use of nutrient broth subculture indicated sterility after five minutes contact. The use of a lecithin-Tween 80-thioglycolate medium resulted in concordant in vivo and in vitro tests. Riegelman et al (11) reported incidents when resistance to benzalkonium chloride by Ps.aeruginosa was demonstrated. In one case, active growth occurred and pigment was produced in the presence of un-neutralized 0.02% benzalkonium chloride from an inoculum of 1 ml of a 24 hr culture. Kohn et al (24) found that benzalkonium chloride 0.02% sterilized 13 strains of Ps.aeruginosa (2 x 10ø/ml) in aqueous suspension within 45 min, and that 0.01% required up to 9 hr. The effectiveness of the inactivators used in the in vitro recovery media was demonstrated by correlation with in vivo results. These workers found that of seven commonly used pre- servatives, only 0.02% benzalkonium chloride sterilized in less than 1 hr.