Escherichia coli 0157:H7

Escherichia coli 0157:H7 (E. coli) 0157:H7 (Gram Negative)

Media: Rainbow agar

Enrich sample with EC-Novobiocin broth
Incubate 35˚C 24 hrs
Do suitable serial dilution
Isolate in Rainbow agar, spread plate (3 replicates for each dilution)
Positive Escherichia coli 0157:H7 will form black colonies
From each plate choose a representative colony and inoculate into Phenol Red Sorbitol Broth (PRSB)
From each plate choose a representative colony and inoculate into Tryptose Broth
Incubate 35˚C 24 hrs


A positive result in PRSB will give yellow colour with gas produce. ( E.coli 0157:H7 don’t ferment Sorbitol, it’s PRSB negative)

From Tryptose Broth, Do an Indole test with Kovac reagent

A positive result in indole test will form a red layer in the alcohol layer

From Tryptose Broth, do a 0157 antigen test

Indole test
E. coli and Enterohaemorrhagic E. coli(EHEC) including 0157:H7 decompose tryptophan (comes from peptone) which will produce indole

Escherichia coli (EC)broth with Novobiocin (EC-Novobiocin)
Lactose enriches lactose fermentors like Coliform
Bile salts suppresses G+ and favours members of Enterobacteriaceae.
Novobiocin ( Wide spectrum antibiotics)suppresses G+ and a some G-, it generally select against non Novobiocin resistant bacteria and favours Enterobacteriaceae i.e. resistant against Novobiocin

PRSB
Sorbitol will differentiate E. coli 0157:H7 from other strains as 0157:H7 strain does not ferment Sorbitol. Sorbic acid causes phenol red to turn yellow.

Rainbow agar
Tellurite inhibits other micro organism and selects for E. coli 0157:H7
Novobiocin inhibits Tellurite reducing bacteria and Proteus spp.
E.coli 0157:H7 produces beta-galactosidase.
Indicator will turn black when there is beta-galactosidase activity

Tryptose broth
Provides a source of peptone/ tryptophan for strain 0157:H7, to facilitate Indole test