Study area
The study area encompassed urban environs of Arsi Negele, Hawassa and Yirgalem (Fig. 1) located at about 231, 270 and 315 km south of Addis Ababa, respectively. Water samples were collected from smallholder dairy farmers and milk retail shops. Hawassa is capital city of South Nations and Nationalities People Regional State (SNNPRS) with an estimated human population of 316,842. In the study area, raw milk is collected from smallholder farmers of the milkshed by milk retailers and again sold to consumers without further processing. In some cases, the raw milk is processed to produce traditional dairy products such as ergo (curd milk), ayib (soft cheese) and butter. Recently, few milk pasteurization plants are also emerging in the area.
Study design
A cross-sectional microbiological quality assessment of water used in hygienic measures related to milk handling and processing in milk retail shops and dairy cattle keeping households was carried out. Milk collecting and retail shops in Hawassa were asked for their cooperation and accordingly a total of 26 of them were identified. Thereafter, 53 smallholder dairy farmers supplying milk to the collectors/retailers were identified and traced back to collect water samples. Overall, a total of 79 water samples to be used for cleansing and washing of milking and milk storage containers were collected (26 from milk retail shops, 53 from the houses of smallholder dairy farmers).
Water sample collection and transportation
The water samples were collected from milk collecting houses and selected individual households with dairy farm. The initial source of water was pipe-borne for all milk collecting and individual smallholder farming households. A water sample of 100 ml was collected from smallholder dairy farms. The samples were collected from directly from pipe (18), from narrow mouthed containers (46), and from wide mouthed containers (15) (e.g. barrels, plastic buckets and jugs). In order to simulate the actual water use practices in the area, no sterilization of the pipe faucet was carried out. The samples were collected in sterile capped plastic containers by following strict aseptic procedures. The water samples were kept in an ice-box and transported to Hawassa University, Veterinary Microbiology Laboratory within 6 h of collection for bacteriological analysis.
Bacteriological analysis
Enumeration of E. coli
Enumeration of total coliforms and E. coli in water samples were carried out using commercially available chromogenic medium, Brilliance™ E. coli/coliform selective agar (Oxoid, CM1046). The agar was prepared according to manufacturer instruction and the molten medium was kept at +50 °C in a water bath. After that, 1 ml of the water sample was pipetted into an empty sterile Petri dish after being thoroughly mixed and covered with approximately 15–20 ml of the molten medium. The plates were gently swirled thoroughly to mix the water sample and the medium and incubated for 24 h at 37 °C. After 24 h incubation, purple and pink colonies were counted as E. coli (Fig. 2) and expressed as colony forming units (CFU)/1 ml of water.
Bacterial isolation and presumptive identification
Isolation of bacteria was conducted by inoculating water sample simultaneously into sheep blood agar and MacConkey agar. After incubation for 24 h, individual bacterial colonies were observed and sub-cultured into new blood agars to get pure colonies. Growth of bacteria on MacConkey agar was characterized by its color on the media (e.g., whether fermenting lactose or not) (Fig. 3). The colononies from blood agar were stained with Gram stain and assessed whether positive or negative, for cellular morphology (rod or coccus) and cellular array (pair, cluster or chain) using a compound microscope. After this, different biochemical tests were done for presumptive identification of the bacterial isolates to genus or species level following standard methods (Quinn et al. 1999). Biochemical tests employed for the presumptive identification of the isolates were catalase test, culture on mannitol salt agar (Fig. 4), motility, indole, methyl red, Voges-Proskauer, citrate (IMViC) and triple sugar iron (TSI) reaction tests. Isolates E. coli were confirmed by growing on Eosin Methylene Blue (EMB) agar in which the isolates showed metallic green sheen appearance (Fig. 5).
Data analysis
The collected data was entered into Microsoft Excel and the difference in the proportion of E. coli positive samples among different sampling units was compared using Chi-sqaure test. Descriptive statistics (e.g. frequency, proportion) also calculated.