THE PRESERVATION OF OPHTHALMIC PREPARATIONS 381 1 in 3000 and 1 in 6000 produced endothelial oedema which disappeared in 6 to 8 weeks. The use of a concentration of 1 in 7500 gave no visible evidence of damage. Bell (46) used a 1 in 10,000 concentration of a quaternary ammonium agent as a routine irrigant in eye surgery for three years, and observed no hypersensitivity or allergic reaction, nor was there any evidence of irritation. Lawrence (39) has quoted numerous other workers with similar experiences using quaternary ammonium compounds. Polymyxin Wiggins (47) found that 85 strains of Ps.aeruginosa were sensitive to polymyxin B sulphate. He found that the minimum inhibitory concen- tration at 37 ø in broth lay between 0.08 to 2.5 •g/ml. Antibiograms for 152 strains of Ps.aeruginosa showed that while there was marked resistance to several antibiotics, all strains were sensitive to polymyxin B (3). Riegelman et al (11) found that 1000 units/ml polymyxin B sulphate sterilized Ps.aeruginosa (108/ml) in aqueous suspension within 30 min. Lecithin was found to be an effective inactivator for polymyxin. In vivo tests confirmed the in vitro results. Polymyxin B sulphate was found to be only slowly bactericidal in aqueous solution against 13 strains of Ps.aeruginosa (2 x 106/ml). Sterility was achieved in 12 hr using 2000 units/ml, and in 18 hr using 1000 units/mi. The results of in vivo experiments were in agreement with results of recovery using lecithin broth (25). Agents not widely used as preservatives Anderson and Stock (37) showed that chlorhexidine acetate 0.01% in aqueous solution sterilized three strains of Ps.aeruginosa within 15 min, and one strain of Staph.aureus within 30 min using cell concentrations of about 105/ml. Egg yolk medium was used to inactivate the chlorhexidine on recovery. Much useful work on formulation of ophthalmic solutions with chlorhexidine has been done in Australia (48,49), and it has been included in the Australian Pharmaceutical Formulary. No instance of sensitivity had been detected after several years' hospital use of chlor- hexidine preserved drops (6). Crompton (6) has stated that" . . . Chlor- hexidine at present is the bacteriostatic of choice." He quoted evidence of the personal communications of two workers to support this belief but omitted to give sufficient detail to allow evaluation of experimental methods. Anderson et al (30) tested ch!orhexidine against single strains of Staph.

382 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS pyogenes, Proteus vulgaris and Ps.aeruginosa using inocula of about 103-104 cells/mi. Tests were made at 28 ø in the presence of numerous ophthalmic drugs in 75 formulations. Counts were made of recovered cells, and the preservative was regarded as satisfactory if the count remained static or was reduced. In most instances sterility was achieved within the shortest test period of one day. Increased counts occurred with both serum and methylcellulose formulations in the presence of 0.005% chlorhexidine. Growth occurred in the presence of lignocaine in one out of two formu- lations. It was suggested that growth in the presence of pilocarpine was due to incompatibility with the 0.005% chlorhexidine. Chlorhexidine is not in fact used in the Australian Pharmaceutical Formulary for eye drops containing pilocarpine (50). The experimental procedures of these workers are open to question. They did not use conventional inactivators in their recovery medium but attempted to inactivate the agents tested, including chlorhexidine, using a washing procedure, the efficiency of which apparently was not demonstrated. They recovered chemically treated Pr.vulgaris cells in medium containing 0.1% phenol which might well reduce recovery as well as swarming. One other unusual aspect of their experimental pro- cedure was an 18 hr recovery incubation period. Without evidence to the contrary, this would seem to be a short time for the recovery of chemically treated organisms. Hugo and Foster (32) found that inocula of Ps.aeruginosa (100/ml) were sterilized in aqueous suspension at 18 ø by 0.005% chlorhexidine, and at 30 ø by 0.002%. Kohn et al (25) tested 51 chemicals not previously employed widely in ophthalmic solutions as preservatives. Thirteen strains of Ps.aeruginosa (2 x 106/ml) were used, and in vitro and in vivo methods employed. Six out of 37 quaternary ammonium compounds were found to possess equal or superior activity to benzalkonium chloride which they used as a standard. All of eight amphoteric surfactants were found to be in- adequate while three iodophors were found to possess sterilizing times of less than 1 hr. Chlorhexidine and Colistin were each found to sterilize in less than 1 hr. Richards (35) found that chlorhexidine was less active in final eye drop preparations at acid pH than it was in simple solution. Wiseman (51) attempted to increase the resistance of several gram- positive and gram-negative species to chlorhexidine by training in its presence. He failed to increase the resistance of gram-positive organisms but found that gram-negative species showed a substantial increase in resistance. In particular the minimum inhibitory concentration against