The urine specimen was first inoculated on Cysteine-Lactose-Electrolyte-Deficient (CLED) agar using a sterile loop measuring 10 µl then later colonies from CLED agar were subjected to gram staining procedure and then sub-cultured on MacConkey and Blood agar plates (Oxoid, UK). The plates were incubated in the appropriate atmosphere at 37 ℃ for 24–48 h overnight. A colony count of ≥ 105 CFU/ml of urine was considered as significant bacteriuria. The identification of the culture-grown bacteria was based on the gram staining, colony characteristics, and biochemical reactions [35 ]. The Analytical Profile Index (API 20E) was used to support the bacterial identification process (bioMerieux, France).
Macconkey and blood agar plates
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
MacConkey and Blood agar plates are common laboratory culture media used for the isolation and differentiation of bacteria. MacConkey agar is used to distinguish between Gram-negative, lactose-fermenting and non-fermenting bacteria. Blood agar, on the other hand, is used to detect hemolytic activity of bacteria.
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3 protocols using macconkey and blood agar plates
1
Urine Culture and Bacterial Identification
2
Bacterial Isolation and Identification
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Conventional microbiological procedures were adopted for bacterial isolation from pus samples. Each sample was streaked simultaneously on MacConkey and Blood agar plates (Oxoid, United Kingdom) followed by incubation aerobically at 37°C for 24 hours.10 Plates were observed for bacterial colonies and the isolated colonies were further triple cloned. Isolates were identified by analytical profile index, API 20E system (bioMerieux, France) according to the manufacturer’s instructions (http://www.biomerieux-usa.com/clinical/api ). Thermo Scientific Genomic Purification Kit, Lithuania, was used for DNA extraction, according to the manufacturer’s instructions. Internal fragment of 1500 bp of 16S rDNA gene was amplified using universal primers, 27F-5’- AGA GTT TGA TCC TGG CTC AG -3’ and RD1-5’- AAG GAG GTG ATC CAG CC -3’ with Initial denaturation 95°C for 2 min, followed by 35 cycles of 30 sec. at 95°C, 30 sec. at 55°C and 2 min at 72°C. Final extension was set at 72°C for 10 min.11
3
Urine Culture Identification Protocol
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Urine samples collected from patients were inoculated on Cysteine Lactose Electrolyte Deficient agar, MacConkey, and Blood agar plates (Oxoid, Ltd., Hampshire, England) using a sterile calibrated wire loop measuring 1 microliter. The inoculated plates were incubated aerobically for 24 hours at 35–37°C, and the results were classified as significant or non–significant. Significant bacteriuria was defined as culture plates containing 105 colony-forming units (CFU)/mL of a single bacterial species. Bacterial identification was done presumptively based on the appearance of bacteria on culture media, microscopic examination, and the gram-reaction. Gram-negative bacteria were further identified using indole production, citrate utilization, H2S formation, gas formation, urea hydrolysis, lysine decarboxylation, lactose fermentation, and motility. The mannitol fermentation test, catalase, and coagulase tests were utilized to identify gram-positive bacteria.
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