376 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Chlorbutol Klein et al (27) used a broth dilution test, and found that three strains of Ps.aeruginosa (108/ml) were sterilized by 0.2% chlorbutol. They found that three eye drop preparations inoculated with 108/ml Ps.aeruginosa were sterilized after 1 hour's contact with 0.5% chlorbutol. Recovery media did not contain inactivators. These workers found that heat signi- ficantly reduced the antibacterial activity of this compound. Chlorbutol is slowly soluble in water, and heat is often used. This phenomenon may account for the contamination of 0.25% chlorbutol preserved eye drops reported by Crompton (6). All of 35 strains of Ps.aeruginosa failed to grow on subculture after 24 hours' contact with 0.5% chlorbutol (36). These workers measured the percentage hydrolysis of buffered 0.5}/0 chlorbutanol solutions after auto- claving at 121 ø. There was about 5% hydrolysis after 15 min at 121 ø at pH less than 5. At about pH 6, and higher, the percentage rose consider- ably. They found that autoclaving at acid pH (up to pH 6) did not inactivate the antibacterial effect of chlorbutanol. Murphy et al (36) reported an absence of endothelial damage or other ill effect upon the tissues within the eye, despite repeated use of 0.50//0 chlorbutol lotions during anterior chamber washes. This preservative had been in continuous hospital use for twenty-nine years. Lawrence (29) found that 0.5% chlorbutol sterilized heavy inocula of 26 strains of Ps.aeruginosa and four species of Proteus in simple buffer after contact times of up to two days. The bactericidal action was more rapid in the presence of non-buffered solutions of several drugs, and sterility of four strains of Ps.aeruginosa took up to 6 hr. Recovery media did not contain inactivators. Riegelman et al (11) found that 0.5% chlorbutol apparently sterilized broth suspensions of Ps.aeruginosa in times which varied with the in- activating recovery broth. Negative in vitro tests were obtained between 8 and 24 hr using optimum recovery broth, and these were correlated with in vivo tests. Apparent sterilization occurred after 45 rain when recovery was made in nutrient broth alone. These workers found that concentrations in excess of 0.5% were irritating to the eye. Anderson and Stock (37) found that chlorbutol 0.5% in water sterilized three strains of Ps.aeruginosa (about 105/ml) within 15 min, but was ineffective against one strain of Staph.aureus even after 1 hr. These workers recovered in nutrient broth without any inactivator. Kohn et al (24) used 13 strains of Ps.aeruginosa and found that 0.5%

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-