Download Ex. 14: Selective Media for Isolating Gram

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
Result
Interpretation
Presumptive 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
1st solid differential media. Developed at the turn of
the 20th century by Alfred MacConkey, M.D.
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.
Day 1
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 2
Materials needed per table:
 Your “cystitis patient” plate after 24 h of incubation.
 EMB demonstration plate with various potential UTI organisms.
 Gram staining materials