Macconkey broth

From Jan 2021 to Sep 2021, 152 nonduplicate E.coli isolates were collected from clinical specimens (including urine, sputum, wound, and blood) of patients admitted to affiliated hospitals of the Abadan University of Medical Sciences. All isolates were accurately identified by performing standard methods [18 (link)]. Briefly, the specimens were inoculated in MacConkey broth (Merck, Darmstadt, Germany) and incubated at 37°C for 24 h. A loopful of broth culture was subsequently cultured on Eosin Methylene Blue (EMB; Biolife Italiana, Italy) and MacConkey agar. All grown lactose-fermenting colonies were identified via bacteriological tests (such as hemolytic activity on blood agar, motility test, and Gram staining result) and conventional biochemical tests including triple sugar iron agar, oxidase, catalase, production of lysine decarboxylase, citrate utilization test, Sulfur Indole Motility (SIM), Methyl Red & Vogues-Proskauer (MR-VP), and urease test [18 (link), 19 ]. Finally, purified isolates stored in trypticase soy broth (Merck, Darmstadt, Germany) containing 20% glycerol at -70°C until further use.

Overall 600 dairy products including 200 yoghurt, 200 doogh and 200 kashk samples were purchased from supermarkets and retailers in various parts of Iran at summer of 2012. All of these dairy products were made traditionally by native people and after collection were kept under refrigeration in plastic bags. Samples were transported under refrigeration (at 4-6°C) in thermal boxes containing ice packs. All samples were diluted in phosphate buffered saline (PBS, Merck, Germany). A 25 g portion of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for 2 min, using a Stomacher lab blender and incubated at 37°C for 24 h. A 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for 24 h. One loop of each tube was streaked on MacConkey agar (Merck, Germany). Such colonies were confirmed as E. coli using standard biochemical tests (e.g., Indole, Methyl red, Voges-Proskauer and Citrate utilization tests). Colonies were confirmed as E. coli by PCR [26 (link)]. E. coli isolates were stored in Tryptic Soy Broth (TSB, Merck, Germany) containing 20% glycerol at -70°C for further characterization.

Three milliliters of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for two minutes at normal speed, using a Stomacher lab blender and incubated at 37°C for 24 hours. A 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for 24 hours. One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli from each sample was streaked on a separate eosin methylene blue agar (EMB agar) plate (Merck, Germmany) and incubated at 37°C for 24 hours. A metallic green colony from each plate with a typical E. coli morphology was selected and examined by biochemical tests, including hydrogen sulfide, citrate, urease and indol based on the API test system (BioMerieux, Marcy L’Etoile, France) (23 ).

Twenty-five grams of faeces were enriched in 225 mL of MacConkey broth (EMD Millipore, Darmstadt, Germany) containing ertapenem (0.5 mg/L; Toronto Research Chemicals, Inc., Toronto, Canada), which was then hand homogenised before incubation at 37°C for 18 to 22 hours. Following incubation, 10 μL was streaked to two agar plates as an initial screening method for CRB, one MacConkey agar (EMD Millipore) plate containing ertapenem (1.0 mg/L; ME) and one modified-SUPERCARB (SC) agar plate. The ME agar was prepared as previously described by Nordmann, Girlich, and Poirel [21 (link)]; however, due to the unavailability of Drigalski agar, MacConkey agar instead was used in the SC agar. In addition, the concentration of ertapenem was raised from 0.25 mg/L to 0.5 mg/L, to reflect a change in interpretive criteria for ertapenem [10 ].

This cross sectional study was performed from October to December 2012. A total of 100 urine samples were collected from patients with UTIs. All of patients were younger than 3 years. All samples were collected from the hospitalized pediatrics of Baqiyatallah Governmental Hospital in Tehran, Iran. Midstream urine was collected in sterile condition to decrease potential bacterial, cellular and artifactual contamination. All samples were immediately transferred to the Biotechnology and Microbiology Research Center of the Islamic Azad University at 4°C. Totally, 3 mL of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for 2 min at normal speed, using a Stomacher lab blender and incubated at 37 °C for 24h. One milliliter sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37 °C for 24h. One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli was streaked on Eosin Methylene Blue agar (EMB agar) plate (Merck, Germany) and incubated at 37 °C for 24h. A metallic green colony from each plate with typical E. coli morphology was selected and examined by biochemical tests, including hydrogen sulfide, citrate, urease and indole.