THE PRESERVATION OF OPHTHALMIC PREPARATIONS 377 chlorbutol sterilized suspensions of about 2 x 106 cells/ml in 12 hr. These workers used inactivators in their recovery media and obtained good agreement between in vivo and in vitro experiments. Organic mercurials Phenylmercuric nitrate, phenylmercuric acetate and thiomersalate all sterilized three strains of Ps.aeruginosa (108/ml) at a dilution of 1 in 50,000 in broth (27). It was found that 0.005% thiomersalate sterilized this organism in 2 hr in fluorescein drops but a contact of 6 hr was necessary to sterilize atropine and eserine drops. These sterilizing times must be viewed critically because the efficiency of the recovery media was apparently not demonstrated. Lawrence (29) found that orgamc mercurials were less active in the presence of common ophthalmic drugs than in their absence. 0.01% P.M.N. and 0.01% thimerosol sterilized four strains of Ps.aeruginosa in several ophthalmic solutions in contact times varying up to 48 hr. In the absence of any drug, 0.01% P.M.N. sterilized in times varying up to 3 hr. Recovery was in Brewer's fluid thioglycolate medium. Riegelman et al (11) showed that 0.01% P.M.N. produced apparent sterility after 1 hour's contact with Ps.aeruginosa (108/ml) when sub- cultured into thioglycolate broth. However, •orneal ulcers were produced by these apparently sterile suspensions. The use of lecithin-polysorbate 80-thioglycolate medium eliminated the discrepancy between in vivo and in vitro results, and the sterilizing time was in excess of a week. 0.01% P.M.N. failed to sterilize one strain of Staph.aureus and three strains of Ps.aeruginosa in contact times up to 1 hr using about 105/ml cells (37). Ridley (16) stated that 0.004% P.M.N. had been used successfully in hospital practice and had been shown to be effective against several pathogenic species but omitted to give experimental procedures. In vitro tests using inactivating recovery media together with in vivo tests showed that P.M.N. 0.01% and thimerosal 0.02% sterilized Ps.aeruginosa (2 x 106/ml) in aqueous suspensions in 6 hr. A two-fold dilution of these mercurials had little effect upon the sterilizing time (24). Anderson et al (30) tested the activity of P.M.N. (0.001-0.004%) against Staph.pyogenes, Proteus vulgaris and Ps.pyocyanea (103-10•/ml) using 18 eye drop formulations involving 12 drugs. Sterility was achieved in less than one day in all cases excluding fluorescein. P.M.N. 0.002% did not sterilize fluorescein drops after several days contact. In the one formu-

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