# Evaluating and Managing the Microbial Contamination of Eye Drops: A Two-Phase Hospital-Based Study
## Materials and Methods  
### Study Design  
This experimental study encompasses two phases. Phase 1 was conducted in situ and phase 2 in vitro. 1.1 Phase 1: The Assessment of the Microbial Contamination of In-Use Eye Drop Content and Dropper Tip and Inner Cap Surfaces  
The first phase involves conducting a comprehensive assessment of the level of microbial contamination of compounded eye drops used by patients and healthcare professionals at the Pharmacy Unit--UPSO2. The study was conducted over four months, from 17 January 2023 to 17 May 2023, following the outlined methodology illustrated in Figure 1.  
Step 1: Collection and storage of used eye drop bottles  
* This study was conducted collaboratively between the Ophthalmology Services and the Pharmacy Unit (PU)--UPSO2. * The storage temperature of collected eye drops was between +2 and +8 \({}^{\circ}\)C for a maximum of 48 h before analysis. * The information recorded for each eye drop included the collection date, active ingredient, batch number, source, packaging type, storage conditions during use, indication, and duration of treatment. Step 2: Sampling and culturing the eye drop content and dropper tip and inner cap surfaces to quantitatively and qualitatively determine the contamination level and identify the microbial contaminant. Validating the Assay Conditions  
The validation of the sampling and culture methods is a fundamental step to ensuring the reliability of the adapted method. All procedures were conducted within a Class A vertical laminar airflow hood within a Class B cleanroom. The selection of microbial contaminants  
The dropper tips and inner caps were intentionally contaminated with three microorganisms, as recommended by the _European Pharmacopoeia_, to determine the reliability and validity of the sampling and culturing methods [34]:  
Figure 1: Assessment process of used eye drops’ microbial contamination. 1. Gram-positive cocci: _Staphylococcus aureus_ (NCTC 10788);
2. Gram-negative bacilli: _Pseudomonas aeruginosa_ (NCTC 12924);
3. Fungi: _Candida albicans_ (NCPF 3179). The choice of _S. aureus_, _P. aeruginosa_, and _C. albicans_ as the test microorganisms was guided by a thorough review of the literature spanning three decades, which highlighted their frequent isolation as contaminants from eye dropper tips and inner caps across various studies [8]. Moreover, these three microorganisms are recommended for antimicrobial evaluation by the _European Pharmacopoeia_ (11 th edition) [34] and the ISO 22196:2011 standard [39]. Their inclusion ensures alignment with established guidelines for assessing microbial contamination on pharmaceutical packaging components that come into direct contact with the product during administration. 2. Establishing the number of microorganisms necessary for contamination Determining the number of microorganisms required for contamination is fundamental to ensuring that the number of microbial contaminants is countable after 24 h of contact with the surfaces. Precultures were prepared from Bioball(r) (Biomerieux, Marcy-I'Etoile, France), lyophilized hydrosoluble beads containing a determined number of microorganisms (30 \(\pm\) 3 colony-forming units (CFU) per microorganism). Bioball(r) was incubated in 9 mL of brain-heart infusion (BHI) broth (Biomerieux, France) for 24 h at 37 \({}^{\circ}\)C for _S. aureus_ and _P. aeruginosa_ and 48 h at 30 \({}^{\circ}\)C for _Candida albicans_ (Figure 2, step 1). From the enriched broths (average between 10\({}^{8}\) CFU and 10\({}^{9}\) CFU), a ten-fold dilution series was undertaken in sterile 0.9% NaCl (Biomerieux, France), and 100 \(\mu\)L of each dilution was then spread on blood agar (Columbia agar enriched with 5% horse blood, Biomerieux, France) at 0 h and after 24 h at room temperature. This culture medium was selected as it allows the growth of most microorganisms. The colonies were then counted after 24 h of incubation at 37 \({}^{\circ}\)C for _S. aureus_ and _P. aeruginosa_ and after 48 h at 30 \({}^{\circ}\)C for _C.