**2. Materials and methods**

## **2.1 Sample collection and bacterial identification**

A prospective cross-sectional study was conducted at Aminu Kano Teaching Hospital, which is a tertiary hospital situated in Kano state, Nigeria, and Murtala Muhammad Specialist Hospital, a secondary healthcare institution and as well a referral centre for a number of primary healthcare centres, also situated in Kano state. Socio-demographic data collection and relevant clinical evaluations of 88 patients with bacterial ocular infections were done using structured questionnaire. The bacterial ocular infections were clinically defined after patients were examined by an ophthalmologist.

*Prevalence of Bacterial Ocular Infections among Patients Attending Eye Clinic of Aminu… DOI: http://dx.doi.org/10.5772/intechopen.108243*

The specimens were collected from eyelids and conjunctiva using sterile cotton swab moistened with sterile saline. The swab was rolled over the eyelid margin from medial to the lateral side and back again. Pus from lacrimal sac (dacryocystitis) and blepharitis was collected using dry sterile cotton-tipped swab either by applying pressure over the lacrimal sac to allow purulent material to reflux punctum or by irrigating the lacrimal drainage system [12]. The swabs were then transported immediately to the laboratory.

Bacterial identifications from eye discharge specimens were performed using standard procedures. Direct gram staining was done for all specimens. The specimens were inoculated on a proper culture media; MacConkey agar, Mannitol Salt Agar, Blood agar and Chocolate agar (Oxoid Ltd. Basingstoke, Hampshire, UK). The inoculated plates was incubated for 24 hours at 37°C. The aerobic atmospheric condition was maintained for the MacConkey agar and mannitol salt agar, while the chocolate agar and blood agar were incubated at 5–10% CO2 atmosphere.

All the plates were initially examined for growth after 24 hours. After getting pure colonies, further identification was conducted using standard microbiological techniques, which include Gram stain, colony morphology and biochemical tests, namely lactose fermentation, mannitol fermentation catalase, coagulase, oxidase and indole tests.

## **2.2 Susceptibility testing**

Once pure culture was obtained, a loopful of bacteria was taken from colony and transferred to a tube containing 5 ml of physiological saline and mixed gently. The suspension was incubated at 37°C until the turbidity of the suspension becomes adjusted to 0.5 McFarland standards. The suspension was uniformly rapped on to Mueller-Hinton agar for non-fastidious organisms and Mueller-Hinton agar with defibrinated sterile sheep blood (10% V/V) for fastidious organisms. Antimicrobial susceptibility test was carried out on each identified bacterium using the Kirby-Bauer disk diffusion method on Muller Hinton agar (Oxoid Ltd. Basingstoke, Hampshire, UK) based on clinical and laboratory standard institute (CLSI) 2014 guideline. Identifications and antibiotic sensitivity tests were performed according to the manufacturer's instructions. The antimicrobials used include the following: ceftazidime (30 μg), cefuroxime (30 μg), erythromycin (15 μg), cefixime (5 μg), gentamicin (10 μg), ciprofloxacin (5 μg), amoxicillin-clavulanic acid (30 μg), nitrofurantoin (300 μg), ceftriaxone (30 μg), oflaxocin (5 μg) and cloxacillin (10 μg) (Oxoid, England). Reference strains of *E. coli* ATCC 25922 and *Staphylococcus aureus* ATCC 25923 were used as quality control measure for identification criteria and antimicrobial susceptibility tests.

## **2.3 Quality control**

Prior to actual data collection comprehensiveness, reliability and validity of questionnaires were pre-tested on 10 patients at AKTH and MMSH. All specimens were collected following standard operating procedure for ophthalmic specimen collection. The sterility of culture media was ensured by incubating 5% of each batch of the prepared media at 37°C for 24 hours. Performances of all prepared media were also checked by inoculating standard strains such as *E. coli* (ATCC 25922), *S. aureus* (ATCC 25923) and *P. aeruginosa* (ATCC 27853) obtained from AKTH (Ramesh *et al.,* 2010). The qualities of biochemical testing procedures were checked by these reference strains.
