Sorbitol MacConkey Agar (SMAC): Introduction, Composition, Principle, Procedure, Results Interpretation, Uses and Limitations

achaurasiya

6 years ago

Sorbitol MacConkey Agar (SMAC) is recommended for isolation of E. coli O157:H7. Rappaport and Henig first described the formulation of the medium, and it was later confirmed by March and Ratham who reported MacConkey

Introduction of Sorbitol MacConkey Agar (SMAC)

Sorbitol MacConkey Agar (SMAC) is recommended for the isolation of Escherichia coli O157:H7. Rappaport and Henig first described the formulation of the medium, and it was later confirmed by March and Ratham who reported MacConkey Agar with Sorbitol to have a sensitivity of 100% and a specificity of 85%. This SMAC medium has proved to be an inexpensive, rapid, simple yet reliable means for the detection of E. coli O157:H7. It is a selective and differential medium for the detection of enterohemorrhagic E. coli O157:H7.
E. coli O157:H7 is an enteric pathogen that typically causes hemorrhagic colitis and bloody diarrheal illnesses. It may be followed by hemolytic uremic syndrome, especially in young children.

Composition of Sorbitol MacConkey Agar (SMAC)

(Himedia)

Ingredients Gms / Litre

Peptone 17.0
Proteose peptone 3.0
D-Sorbitol 10.0
Bile salts mixture 1.0
Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Agar 13.5
Distilled Water: 1000 ml

Final pH ( at 25°C) 7.1±0.2

Principle of Sorbitol MacConkey Agar (SMAC)

Sorbitol MacConkey Agar is also known as MacConkey agar with sorbitol a selective and differential medium for the detection of E. coli O157:H7. Bile salt and crystal violet present in the medium are selective agents which inhibit the growth of gram-positive bacteria ( e.g. enterococci, micrococci, staphylococci, etc). D-Sorbitol is a fermentable carbohydrate and also helps in the differentiation of enteric microorganisms is achieved by the combination of sorbitol and the neutral red indicator. Peptone and proteose peptone supply necessary nutrients like nitrogenous and carbonaceous compounds, long-chain amino acids, minerals, vitamins, and trace ingredients for the growth of organisms. Sodium chloride (NaCl) maintains osmotic equilibrium. Neutral red is an indicator. The growth of E.coli O157:H7 on MacConkey Agar with Sorbitol shows colorless colonies and most of the fecal flora ferment sorbitol and appear pink. Colorless or pink to red colonies are produced depending upon the ability of the isolate to ferment the carbohydrate sorbitol as shown in the above picture and below video.

Preparation of Sorbitol MacConkey Agar (SMAC)

Suspend 50.03 grams of the powder of Sorbitol MacConkey Agar in 1 liter purified/distilled or deionized water.
Mix thoroughly and heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Avoid overheating and do not autoclave.
Leave for cooling to 45-50°C.
Mix well before dispensing.
Pour into each plate and leave plates on the sterile surface until the agar has solidified.
Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Sorbitol MacConkey Agar (SMAC)

Store in sleeve wrapping until just prior to use at 2-8ºC and away from direct light.
Plates from opened stacks of 10 plates can be used for one week when stored in a clean area at 2 to 8° C.
Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for Sorbitol MacConkey Agar (SMAC)

Test specimens
Sorbitol MacConkey Agar (SMAC) plates
Inoculating loop
Bunsen burner
Incubator
Control strains ( Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Escherichia coli O157:H7 NCTC 29900, Shigella flexneri 12022 ATCC )

Test procedure of Sorbitol MacConkey Agar (SMAC)

Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
Take Sorbitol MacConkey Agar plates.
Heavily inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop.
Incubate aerobically plates at 35 ± 2°C for 18-24 hours.

Result Interpretation of Sorbitol MacConkey Agar (SMAC)

Sorbitol fermenters: Pink to red colonies is due to fermentation of sorbitol, some surrounded by zones of precipitated bile. e.g. Escherichia coli ATCC 25922
Sorbitol non-fermenters: Colorless colonies are due to the non- fermentation of sorbitol. e.g. Escherichia coli O157:H7

Colony characteristics of Sorbitol MacConkey Agar (SMAC)

Salmonella Typhi ATCC 6539: Pink
Shigella flexneri ATCC 12022: Colorless
Escherichia coli ATCC 25922: Pink
Escherichia coli serotype O11 and O55: Colorless
Escherichia coli O157:H7 NCTC 29900: Colorless

Enterococcus faecalis ATCC 29212: Partial to complete inhibition

Staphylococcus aureus ATCC 25923: No growth

Uses of Sorbitol MacConkey Agar (SMAC)

SMAC is used as a selective and differential medium for the detection of enterohemorrhagic Escherichia coli O157:H7.
It also aids in the differentiation of the E. coli O157: H7 from other strains, especially lactose fermenters E. coli.
This medium is very useful for the presumptive identification of Escherichia coli O157:H7 from clinical samples as well as non-clinical samples ( food and dairy samples).

Limitations of Sorbitol MacConkey Agar (SMAC)

Colony morphology is only presumptive identification and hence biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
SMAC however should not be solely used to detect pathogenic E.coli O157: H7 strains as some strains will also not ferment sorbitol. e.g. Escherichia coli serotype O11 and O55.
It has been reported that some Enterobacteriaceae and Pseudomonas aeruginosa are inhibited on SMAC when incubated in a CO₂ enriched atmosphere.
Prolonged incubation of the bacterial culture may result in colonies of Escherichia coli serotype O157:H7 losing their characteristic colorless appearance and also the color of sorbitol-positive colonies can fade, making them hard to distinguish from sorbitol-negative colonies and thus there is a chance of a false-positive result.
Prolonged incubation can result in fading of pink-colored sorbitol-positive colonies making interpretation more difficult and whereas upon longer incubation, some strains of Escherichia coli O157:H7 can ferment sorbitol and produce pink-colored colonies, such scenario is responsible for false negative.
There are also other species of facultative anaerobic gram-negative rods that do not ferment sorbitol.
There exist sorbitol negative strains of serotypes other than O157:H7 which may or may not produce toxins and clinical symptoms and hence SMAC does not differentiate between toxin-producing and non-producing strains of Escherichia coli O157.

Key Notes on Sorbitol MacConkey Agar (SMAC)

SMAC is only slightly selective, since the concentration of bile salts, which inhibits gram-positive microorganisms, is low in comparison with other enteric culture media like MacConkey agar, DCA, XLD agar, SS agar, etc.
The addition of cefixime and tellurite is a more selective and differential medium designed to inhibit Proteus mirabilis, non-O157 E. coli strains, and other sorbitol-nonfermenting strains that need to be screened during the attempted isolation of E. coli O157:H7.
Sorbitol MacConkey Agar with 5-Bromo-4-chloro-3-indolyl-b-D-glucuronide (BCIG) is a very useful medium, a product of Oxoid able to reduce the number of false suspect E. coli O157 colonies by 36%.
Colorless colonies of SMAC suspected Escherichia coli O157 should be confirmed with the Escherichia coli O157 Latex Test.
SMAC is recommended medium for the presumptive identification of Escherichia coli O157:H7.
Sorbitol MacConkey agar is a modification of MacConkey agar in which lactose is replaced by sorbitol.
Foods implicated in human illness related to E. coli O157 include meats, dairy products, vegetables, salads, apple juice, and water.

Precautions for Sorbitol MacConkey Agar (SMAC)

This product may contain components of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not guarantee the absence of transmissible pathogenic agents and thus, it is recommended that these products be treated as potentially infectious, and handle observing the usual universal blood precautions. Do not ingest, inhale, or allow to come into contact with skin.
This product is for in vitro diagnostic use only and it is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to “standard precautions.