Ex. 13: Urine Culture Technique and the Importance of Selective and Differential Media for Gram-Negative Rods Objectives ??

Vocabulary Coliforms Pure culture Types of culture media: – Selective – Enrichment – Differential UTI, Pyuria, Hematuria

Urinary Tract Infections (UTIs) Common? Normal genitourinary flora? Types of UTIs? Significant bacteriuria in “clean catch” Causative agents? Diagnosis?

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)

Table 12-1: Eosin Methylene Blue Agar (EMB) Results and Interpretation ResultInterpretationPresumptive ID Poor growth or no growth (P) Organism is inhibited by eosin and/or methylene blue Gram-positive Good growth (G) Organism is not inhibited by eosin and methylene blue Gram-negative Growth is “colorless” (no pink, purple, or metallic sheen) (C) Organism does not ferment lactose or sucrose. No reaction (NR) Noncoliform, such as P. aeruginosa Growth is pink and mucoid (Pi) Organism ferments lactose with little acid production (A) Coliform, such as Klebsiella pneumoniae “Dark” growth (purple to black, with or without green metallic sheen) (D) Organism ferments lactose and/or sucrose with much acid production (A) Coliform, such as E. coli

MacConkey Agar Contains peptones, lactose, bile salts and the dye crystal violet. Crystal violet and bile salts prevent growth of Gram+ and fastidious Gram – bacteria, such as Neisseria and Pasteurella. Lactose fermentation used to differentiate. 1 st solid differential media. Developed at the turn of the 20th century by Alfred MacConkey, M.D.

Materials needed per table:  Four “cystitis patient” urine specimens (either in urine specimen cup or already transferred to a tube for easier distribution) – Each student will process one sample!  Four EMB plates (one per sample)  Four calibrated 10 µl plastic loops (one per sample) For this exercise each student will process one patient sample. Day 1

Materials needed per table:  Your “cystitis patient” plate after 24 h of incubation.  EMB demonstration plate with various potential UTI organisms.  Gram staining materials Day 2