Study area and period
The study was conducted at Yirgalem town, located 275 km south of Addis Ababa; the capital city of Ethiopia. The town’s geographical coordinates are 7o41’N latitude, 36o50'E longitude, and an average altitude of 1, 780 m above sea level. It lies in the climatic zone locally known as "Woyna Daga" (1,500-2,400 m above sea level) which is considered ideal for agriculture as well as human settlement. The town is generally characterized by a warm climate with a mean annual maximum temperature of 30°C and a mean annual minimum temperature of 14°C. The annual rainfall ranges from 1138-1690 mm. The maximum precipitation occurs during the three months from June through August, with minimum rainfall occurring in December and January. The study was conducted between November 2016 to August 2017.
A total of 160 food samples comprising of 40 Bayeaynet, 40 Injera firfir, 40 Vegetables, and 40 Spaghetti, were collected from different restaurants and food establishments in Yirgalem town, southern Ethiopia between November 2016 and August 2017. Food samples were collected using food serving utensils and placed into a sterile aluminum plate at a time between 5 PM and 6 PM after a brief description of the study purpose for the restaurant and food establishment owners. The samples collected were transported to the Yirgalem Hospital Medical College, Microbiology Laboratory in the date of collection using icebox and were kept in the refrigerator at 4oC until microbial analysis was conducted. The microbial analysis was conducted within one to three hours of collection.
Twenty-five grams of each food sample was cut into small pieces using food cutter and was added into 225 ml of 0.1% sterile peptone buffer water and homogenized for 2 minutes using a stomacher. One ml of the resultant homogenate was added into 9 ml of sterile 0.85% saline in a test tube and serially diluted up to 10-7.
Approximately 0.1 ml aliquot portions of the dilutions were spread onto duplicate sterile plates of plate count Agar, MacConkey Agar, Salmonella-Shigella Agar and Mannitol salt Agar, for total aerobic plate count, Coliform count, Enterobacteriaceae count, isolation of Salmonella and Shigella and isolation of Staphylococcus aureus, respectively. Also, Potato Dextrose Agar was used for the isolation of fungi. All cultures were incubated at 37oc for 24-48 hrs except that of Potato Dextrose Agar which was incubated for 3 to 5 days at 25oc. All the culture plates were checked for microbial growth when the appropriate time of the incubation was achieved. Thereafter, colonies between 30-300 were counted using colony counter (Gallenkamp, England) and the counts were expressed as colony-forming units per gram (CFU/g) of food. Cultures were purified by repeated plating and maintained on appropriate slants at 40C (14). Finally, the pure isolates were identified through gram staining and biochemically using (Catalase test, Oxidase test, coagulase test, Triple sugar iron agar, Lysine iron agar, Urea agar, Simmons Citrate agar, and SIM media) following standard microbiological methods (15, 25). Moreover, fungal isolates were identified based on their macroscopic and microscopic features. Reference was made standard identification keys and atlas.
Presumptive test: One gram of each food sample was transferred to sterile McCartney bottles containing Lactose broth and inverted Durham tube. Then, the tubes were incubated at 37oc for 24-48 hrs. Tubes showing gas production and/or color change of dye were streaked on Eosin Methylene Blue plates. The plates were incubated at 37oc and 44oc for 24 hrs for a confirmatory test. Colonies grown on the Eosin Methylene Blue plates were picked and inoculated into tubes containing lactose broth for a complete test and onto Nutrient Agar slants for further characterization. The inoculated slants and lactose broth tubes were incubated at 37oc for 24 hrs.
The data obtained were analyzed using SPSS software version 20.0. The significance of differences was considered at a 95% confidence interval (P < 0.05).
The study was approved by the Institutional Review Board of Yirgalem Hospital Medical College and an official permission letter was obtained from the Yirgalem town Hotel and tourism office. Written informed consent was also obtained from restaurant and food establishment owners after a brief explanation of the objectives and benefits of the study.
Data quality control
Aseptic techniques were employed in every step of the study. Samples were transported in an icebox and were processed without delay and in replicates. To ensure sterility, materials and media were autoclaved at 121oc for 15 to 20 minutes. The media were checked for sterility by incubating 5% of the batch at 37oc for 18-24 hrs.
Ready-to-eat foods: Foods that are available directly for consumption without further processing
Microbiological quality of food: a load of microbes existing in a certain food item
Microbiological safety of food: Safeness of foods ready for consumption from foodborne pathogens