THE PRESERVATION OF OPHTHALMIC PREPARATIONS 375 with Pr.vulgaris in glycerine eye lotion. More useful information might have been obtained if tests had been made at intervals less than daily. Hugo and Foster (9) have shown that the rate of growth of one strain of Ps.aeruginosa was little affected by the presence of the concentration of esters used in Solution for Eye Drops B.P.C. (0.034%), and that they could be used as a sole carbon source at the same concentration. These workers, [Foster (31), Hugo and Foster (32)], found that a mixture of two parts methyl and one part propyl phydroxybenzoate sterilized aqueous suspensions containing 10 and 100/ml Ps.aeruginosa cells of one strain in 30 minutes at 18 ø using concentrations of 0.125% and 0.2% respectively. They did not use an inactivator in the recovery medium. Chlorocresol pChloro-m-cresol was the first official ophthalmic antibacterial preser- vative used in Britain. Its present replacement by Solution for Eye Drops in the B.P.C. has been discussed (4). Davis (33) found that 0.05% chlorocresol was as effective against $taph.aureus as was 0.5% phenol. In each case, sterility was achieved within one day. Details of reaction menstrum and recovery conditions were not given. Klein et al (27) used a broth dilution procedure and found that 0.1% chlorocresol sterilized broth suspensions (108/ml) of three strains of Ps.aeruginosa. They also tested 0.1% and 0.03% chlorocresol in the presence of three eye drop preparations. In each case, sterility was achieved within 1 hr using 0.1%. 0.03% chlorocresol sterilized atropine drops within 4 hr, while fluorescein and eserine were sterilized between 6 and 24 hr. They found that 0.1% chlorocresol caused smarting. 0.05% chlorocresol sterilized inocula of 10/ml Ps.aeruginosa cells of one strain in aqueous suspensions at 18 ø within 30 min, but 0.07% was required to sterilize 100 cells/ml within the same time (32). Crompton and Anderson (34) have reported that injections of 0.05% ½hlorocresol in normal saline into the anterior chamber of rabbits' eyes caused the cornea to become opaque. This disadvantage would seem not to apply in ophthalmic solutions preserved and used with intact eyes. Richards (35) reported that chlorocresol was more active in terms of reduction of viable count in final eye drop preparations at acid pH, than it was in simple solution.

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%