# Evaluating and Managing the Microbial Contamination of Eye Drops: A Two-Phase Hospital-Based Study
## Materials and Methods
### Study Design
albicans_ to estimate the microorganism concentration in each dilution tube. Figure 2: Process for the validation of microbial contamination of eye drop tip and cap. The first agar plate with a countable number of colonies was used to calculate the number of microorganisms in the upstream tubes. The 4 th dilution (\(10^{-4}\)) was chosen as the dilution of interest because it enables counting and detecting microorganisms (no more than 330 CFU/plate) even after 24 h.  
* Microbial contamination of the dropper tip and inner cap of sterile eye drops The protocol for the contamination of the dropper tips and inner caps from the dilution of interest is as follows (Figure 3):
* Volumes of 0.2 mL of the selected dilution, \(10^{-4}\), were deposited on the inner surface of eye drop caps. * The eye drop bottles were positioned upside down to bring the eye drop tips into contact with the suspension by immersion. * The eye drop bottles were then repositioned upright to evenly distribute the suspension over the entire surface of the tips and caps. * Finally, the eye drop bottles were placed horizontally and rolled on the bench. * The eye drop bottles were incubated upright for 24 h at room temperature. In parallel, the last 3 dilution tubes, including the dilution of interest, were treated as follows:  
* Inoculated (0.1 mL) at H0 onto blood agar plates. These were incubated for 24 h at 37 \({}^{\circ}\)C for _S. aureus_ and _P. aeruginosa_ and for 48 h for _C. albicans_, and they were then analyzed to enumerate the microorganisms (MO) in the dilution of interest. * Incubated for 24 h at room temperature and then inoculated (0.1 mL) onto blood agar plates. These were in turn incubated for 24 h at 37 \({}^{\circ}\)C for _S. aureus_ and _P. aeruginosa_ and for 48 h for _C. albicans_, and they were then analyzed to enumerate the MOs in the dilution of interest after 24 to 48 h in 0.9% NaCl. The agar plate corresponding to the dilution of interest was the control. * Sampling and culturing the eye drop tips and inner caps The Bacteriology Department of the Pharmacy Unit--UPSO2--established the microorganism recovery protocol. * The excess solution in the caps was removed after 24 h of contact with the indicated microorganism. * The eye drop tips and inner caps were swabed separately using eSwab() (Deltalab, Spain). * The agar plates were inoculated by direct inoculation from the swab, which was spread over the entire surface; then, the swab was turned by 90\({}^{\circ}\) and spread a second time by turning the Petri dish 90\({}^{\circ}\). * The blood agar plates were incubated for three days at 37 \({}^{\circ}\)C for _S. aureus_ and _P. aeruginosa_ and at 30 \({}^{\circ}\)C for _C. albicans_. The method was considered validated if the number of MOs collected was equivalent between the 3 vials (in terms of log) for each microorganism in the same run. Collection and Culture of In-Use Specimens  
* Identification of Microorganisms by MALDI TOF-MS  
Figure 3: The protocol for the contamination of the eye drop caps and tips. The validated method for the recovery of the 3 test microorganisms was applied to all collected eye drops, maintaining them under controlled storage conditions. The agar plates were inspected after 72 h of incubation to detect microbial growth. Microorganisms isolated on agar plates were identified using the MALDI-TOF microflex(r) spectrometer (Bruker, France) in collaboration with the Hygiene Department. This technique utilized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the obtained spectra were compared with reference spectra in the database. Evaluation of Residual Content Contamination  
When the presence of microorganisms (MOs) was detected on the dropper tip and/or the inner cap, the residual contents of the eye drop bottle were sampled under sterile conditions using a syringe for the sterility test. The sterility test is a microbiological test required by the _European Pharmacopoeia_ to verify that sterile products do not contain viable microorganisms [34].