Urine Culture Technique and the Importance of Selective and Differential Media for Gram-Negative Rods Vocabulary Coliforms Pure culture Types of culture media: Selective Enrichment Differential UTI, Pyuria, Hematuria

EMB (Eosin Methylene Blue) Agar Contains peptones, lactose, sucrose, and the dyes eosin and methylene blue. Eosin dye inhibits growth of Gram+ bacteria. Methylene blue acts as indicator. Lactose and sucrose are nutrients (fermentable carbohydrates) The production of acid, upon lactose- or sucrose-fermentation, resulted in the two dyes interacting to produce brown to blue-black colonies. This formulation gives sharp and distinct differentiation between colonies of lactose- and non-lactose-fermenting organisms. However, it does not discriminate between which carbohydrate (lactose or sucrose) is being fermented. Yersinia enterocolitica, which ferments sucrose but not lactose, will produce the same blue-black colony as lactose-fermenters.(2-6) Gram-positive bacteria are inhibited by eosin

Urinary Tract Infections (UTIs) Common? Normal genitourinary flora? Types of UTIs? Significant bacteriuria in “clean catch” Causative agents? Diagnosis? Gram stains of urine specimens that have not been centrifuged can help determine the cause of a suspected urinary tract infection. If bacteria are present under an oil immersion lens (1,000x), the concentration is at least 104 or 105 bacteria per milliliter. Since the concentration of bacteria in most patients with symptomatic urinary tract infections is that high, the Gram stain will usually, but not always, be positive. See microbe library for picts: Authors Rebecca Buxton (Corresponding Author)Department of PathologyUniversity of UtahSalt Lake City, Utah 84132Email: rebecca.buxton@path.utah.edu +

For this exercise each student will process one patient sample. Day 1 Materials needed per student: Urine sample in urine specimen cup from suspected cystitis patient. Two EMB plates (one for the urine sample and one for a control organism) One calibrated 10 µl plastic loop for the unknown urine specimen One of the four control bacterial species assigned per table Materials needed per team of two: Bunsen burner, inoculating loop – use only for control species NOT for urine sample! For this exercise each student will process one patient sample.

Calibrated Loop Procedure for Patient Sample One patient sample per student Additionally: Regular Streak Plate technique for control (1 control per student)