Technical Specification Sheet

Harlequin® Sorbitol MacConkey Agar (SMAC) (NCM1007)

Intended Use
Sorbitol MacConkey Agar (SMAC) is for the isolation of Escherichia coli O157:H7 and is not intended for
use in the diagnosis of disease or other conditions in humans.

Description
Pathogenicity of Escherichia coli O157:H7, the primary serovar associated with haemorrhagic colitis
(HC) and haemolytic uraemic syndrome (HUS), is linked to the production of verocytotoxins (VT1 and
VT2). However, it should be noted that not all strains of O157 produce verocytotoxins, and that strains
from other serovars can be toxin producers (e.g. O26, O103, O111, O113, O145). E. coli O157 has
been associated epidemiologically with food poisoning outbreaks involving beef burgers and cold cooked
meats.
This medium is a modification of Sorbitol MacConkey Agar (SMAC). The addition of the chromogenic
substrate BCIG (5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide) improves the specificity of the medium. E.
coli O157:H7 is typically sorbitol negative and ß-glucuronidase negative producing pale translucent
colonies on this medium. The majority of other E. coli strains are ß-glucuronidase positive and sorbitol
positive (pink/red colonies). A small percentage of E. coli are ß-glucuronidase positive and sorbitol
negative and thus appear as blue/green colonies on this medium. Consequently, this medium can
distinguish between non-O157 sorbitol negative E. coli and the genuine toxigenic E. coli O157:H7. This
reduces the number of unnecessary confirmation tests that are performed. The medium can be made
Effective Date: 1/4/2019

more selective by the addition of Cefixime Tellurite supplement (X161) to prepare CT-SMAC. Most
workers recommend the use of CT-supplemented medium alongside unsupplemented medium to
ensure maximum isolation of E. coli O157. This medium can also be useful for the detection of other
VTEC producing E. coli in conjunction with specifically targeted IMS beads ( ).

Typical Formulation
Peptone 20.0 g/L
Sorbitol 10.0 g/L
Bile Salt No.3 1.5 g/L
Sodium Chloride 5.0 g/L
BCIG 0.1 g/L
Neutral Red 0.03 g/L
Crystal Violet 0.001 g/L
Agar 12.0 g/L

Supplement
X161 CT supplement 1 vial per 500ml
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Final pH: 7.1 ± 0.2 at 25°C

Formula may be adjusted and/or supplemented as required to meet performance specifications.

Precaution
1. Refer to SDS

Preparation
1. Suspend 48.6 grams of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 45-50°C, add 2 vials of X161 CT supplement and pour plates.
 Technical Specification Sheet

Test Procedure
From O157 Broth (NCM0196), surface streak for single colonies.
Incubate at 37°C aerobically for 18-24 hr.
Examine plates for sorbitol negative, ß-glucuronide negative colonies. Confirm as O157:H7 by
serology, (commercial kits or antiserum available).

Quality Control Specifications

Dehydrated Appearance: Prepared medium is pale red with a light violet tinge.

Minimum QC:
Escherichia coli O157:H7 NCTC 12900 (non-toxigenic)
Escherichia coli NCIMB 50034
Enterococcus faecalis NCIMB50030 (inhibition)

Results
Organism Colony Size (mm) Color

E. coli O157:H7 sorbitol -ve, ß-glucuronide -ve 2.5 - 4.0 Translucent

E. coli sorbitol +ve, ß-glucuronide +ve 2.5 - 4.0 Pink/red or purple center
E. coli sorbitol ve, ß-glucuronide +ve 2.5 - 5.0 Green or translucent
with green center
Effective Date: 1/4/2019

Note - Sorbitol positive toxigenic E. coli O157:H7 have been isolated and appear as sorbitol positive and ß-
glucuronide positive on this medium.

Expiration
Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not
free flowing or appearance has changed from the original color. Expiry applies to medium in its intact
container when stored as directed.

Limitations of the Procedures

Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this
medium.

Storage
Store dehydrated culture media at 2-30°C away from direct sunlight. Once opened and recapped, place
container in a low humidity environment at the same storage temperature. Protect from moisture and light
by keeping container tightly closed.
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References
1. Okrend, A.J.G., Rose, B.E., and Lattuada, C.P. (1990) Use of 5-Bromo-4-Chloro-3-Indoxyl- ß-D-
Glucuronide in MacConkey Sorbitol Agar to Aid in the Isolation of Escherichia coli O157:H7 from
Ground Beef. J.Food Protection 53 (11) 941-943