The Clinical and Laboratory Standards Institute susceptibility method prohibits the use of pharmacy preparations, but obtaining pure powders is difficult. The activity of natamycin against isolates of Aspergillus and Fusarium species isolated from keratitis was assessed by using both powder and pharmacy eye drop preparations. Eye drop preparations may be a viable option for testing natamycin activity.Keratitis is a leading cause of monocular blindness worldwide (11,12,14,15), and recent reports suggest that there has been a steady increase in the percentage of infectious keratitis caused by fungus (1,12,17). Fusarium and Aspergillus species are the most common fungal isolates associated with this infection (1, 17). Classes of antifungals used for treatment of fungal keratitis include the polyenes, triazoles, and echinocandins (13). Natamycin (NAT), a tetraene polyene, has long been considered the mainstay of treatment for filamentous fungal keratitis and is the only available antifungal medication that has been approved for this indication by the U.S. Food and Drug Administration. Although previous reports regarding the efficacy of NAT in fungal keratitis are available (4-6), there are few studies that describe the in vitro activity of NAT against the common ocular isolates (2,7,8,9).The Clinical Laboratory and Standards Institute guidelines stress the use of pharmaceutical-grade powder solely for susceptibility testing. This is due to unknown purity and potency values and additives that may impact results (3). Pharmaceutical-grade powder is very difficult to obtain, especially for drugs that are not frequently used, such as NAT. We studied the in vitro activity of NAT against Fusarium and Aspergillus species isolated from cases of corneal ulcers seen at the Aravind Eye Hospital, Madurai, India, and compared the MICs obtained by using both pharmaceutical-grade NAT powder (NAT-P) and commercially available NAT eye drops (NAT-D).A total of 100 fungal isolates recovered from clinical cases of corneal ulcer were evaluated in the present study. The fungal isolates included 41 Fusarium, 32 Aspergillus flavus, 18 Aspergillus fumigatus, 5 Aspergillus terreus, and 4 Aspergillus niger isolates. The reference strain of A. flavus ATCC 204304 was included and was tested with both formulations in each series of assays. Antifungal susceptibility testing was performed exactly as outlined in CLSI M38-A. Pharmaceutical-grade NAT was obtained from Alcon Laboratories (Ft. Worth, TX). A 5% NAT suspension of topical eye drops was purchased from Sun Pharmaceutical, Ltd. (Mumbai, India). Amphotericin B was used to assess quality control against A. flavus ATCC 204304 strains. Inocula were prepared without the use of Tween 20. NAT-P was weighed and dissolved in dimethyl sulfoxide. Stock solutions were stored frozen at Ϫ70°C until needed. Drug dilution tubes were prepared from a 3,200-g/ml stock and diluted 1:2.5 with water to achieve a top concentration of 1,280 g/ml. For NAT-D, 1 ml of 5% NAT suspension was mixed with 9 ml of 100% dimet...