MacConkey Sorbitol Agar is an adaptation of the formulation that was described by Rappaport and Henigh. It is selective and differential media for the detection of sorbitol-nonfermenting Escherichia coli serotype O157:H7 associated with hemorrhagic colitis. E. coli serotype O157:H7 is a human pathogen that is associated with hemorrhagic colitis which is caused by the actions of a Shiga-like toxins (SLT).
On the standard lactose-containing MacConkey Agar the strain is not different with other types of lactose fermenting E. coli. In contrast to most E. coli strains, E. coli O157:H7 ferments the sorbitol very slowly or does not ferment at all. This is why MacConkey Agar with Sorbitol permits the detection that there is E. coli O157:H7 in stool culture. 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.
Composition of Sorbitol MacConkey Agar
| Ingredients | Gm/L |
| Peptone | 17.0g |
| Proteose peptone | 3.0g |
| D-Sorbitol | 10.0g |
| Sodium Chloride | 5.0g |
| Bile Salts Mixture | 1.5g |
| Neutral Red | 0.03g |
| Crystal Violet | 0.001g |
| Agar | 13.5g |
Final pH 7.1 +/- 0.2 at 25ºC
Preparation of Sorbitol MacConkey Agar
- Suspend 50.03 grams of powder to 1000ml of distilled water. Mix thoroughly.
- Then, with frequent stirring, simmer for one minute, allowing the powder.
- Autoclave at 121°C for 15 minutes.
- Cool to 45-50degC , then pour into sterilized Petri plates.
Principle of Sorbitol MacConkey Agar
MacConkey Sorbitol Agar is only somewhat selective, because the amount of bile salts that blocks microorganisms with gram-positive grams are low in comparison with other media used for plating in the enteric. Peptone and proteose peptone provide essential nutrients, including carbonaceous and nitrogenous substances amino acids with long chains minerals, vitamins, as well as trace elements to aid in the development of organisms.
Crystal violet found in the medium blocks the growth of gram-positive bacteria particularly staphylococci and enterococci. Salts of sodium keep the osmotic balance. Neutral red is a sign. D-Sorbitol is a fermentable carbohydrate. The distinction of enteric microorganisms is accomplished through the combination of sorbitol with an indicator that is neutral in color.
The development of E.coli O157:H7 on MacConkey Agar with Sorbitol shows colored colonies, and the majority of the fecal organisms are fermenting sorbitol as pink and look. Pink to red colonies are formed based on the capacity of the isolate to ferment carbohydrate and sorbitol.
Intended Use
The recommended method for identifying and isolating of strains of Enteropathogenic Escherichia Coli that are associated with diarrhea in infants.
Type of specimen Used
This medium is employed for the isolating of Escherichia coli O157 (and other serotypes that are sorbitol-negative) from stool samples of patients who are suspected to have been infected by this pathogen, as well as for food, veterinary and environmental samples.
Technique
- Prepare the agar in accordance with the directions, then pour it into plates. If required, you need to dry out the surface on the agar.
- Innoculate the plates by incubating them with the solution of test substances (food or faeces) to create colonies that are separated.
- Incubate at 35degC for 24 hrs. Doyle and Schoeni 10 reported that 35-37degC is the ideal temperature for the growth of Escherichiacoli O157. At 44-45.5degC the growth rate is low even after 48 hours incubation.
The delay in reading plates past 24 hours is not recommended since the intensity of the sorbitol fermenting colonies decreases in contrast to non-fermenting colonies. Other Gram-negative organisms, such as Pseudomonas Proteus, Pseudomonas and Klebsiella species can thrive using Sorbitol MacConkey Agar however they can be distinguished from their appearance in the colonies.
A diagnostic reagent, Escherichia coli O157 test (DR0620) is readily available to ensure that immediate confirmation tests can be conducted on suspect colonies.
Result and Interpretation of Sorbitol MacConkey Agar
Following 18 to 24 hours incubation the plates will be able to show isolated colonies within areas of streaking and confluent growth in areas with high inoculation.
- Sorbitol fermenters: pink to red colonies, some surrounded by zones of precipitated bile
- Sorbitol non-fermenters: colorless colonies.
Storage and Shelf Life of Sorbitol MacConkey Agar
Storage temperature is between 10 and 30 degrees Celsius in a tightly sealed container. The prepared medium at 20-30 degrees Celsius. Utilize before the expiry date is listed in the bottle. After opening, the product must be stored dry after sealing the bottle tightly to avoid the formation of lumps as a result of the hygroscopic character of the product. Incorrect storage of the product can result in the formation of lumps. Store in a dry and ventilated location shielded from extreme temperatures and ignition sources. The container should be sealed tightly following use. Make sure to use it before the expiry date is in the container’s label.
Quality Control of Sorbitol MacConkey Agar
- Appearance Color: Light pink to yellow homogeneous free-flowing powder
- Gelling: Firm, comparable with 1.35% Agar gel.
- Colour and Clarity of prepared medium: A purplish red colour clear to lightly translucent gel forms inside Petri plates
- Reaction: Reaction of 5.0% w/v aqueous solution at 25°C. pH : 7.1±0.2
- pH: 6.90-7.30
- Cultural Response: Culture traits that are observed following an incubation period of 35-37°C for 18-24 hrs.
Unsupplemented
| Positive control: | Expected results |
| Escherichia coli O157:H7 Non-toxigenic NCTC12900 * | Good growth; 1-2mm straw colonies |
| Negative control: | |
| Escherichia coli ATCC® 25922 * | Good growth; 1-2mm pink colonies |
Supplemented with CT Supplement SR0172 and tested in accordance with ISO 11133;201412.
| Positive control: | Expected results |
| Escherichia coli O157:H7 Non-toxigenic NCTC12900 * WDCM00014 | Good growth; 1-2mm straw colonies |
| Negative controls: | |
| Escherichia coli ATCC® 25922 * WDCM00013 | No growth/pinpoint-0.25mm pink colonies |
| Escherichia coli ATCC® 8739* WDCM00012 | No growth/pinpoint-0.25mm pink colonies |
| Staphylococcus aureus ATCC®25923* WDCM00034 | No growth |
| Staphylococcus aureus ATCC®6538* WDCM00034 | No growth |
Uses of Sorbitol MacConkey Agar
- It is used to identify and detection of enteropathogenic Escherichia Coli O157:H7 which is the main serovar of hemorrhagic bowel disease (HC) as well as hemolytic uremic disorder (HUS).
- It assists in the differentiation of E. coli O157: H7 strain from other strains, including lactose-fermenting E. coli.
Precautions
While the majority of Escherichia Coli O157 strains display a common appearance on Sorbitol MacConkey however, there are some strains that are unusual. Sorbitol MacConkey agar should not be used only to identify VTEC varieties of Escherichia coli because some of the non-toxic strains cannot ferment sorbitol.
Sorbitol MacConkey Agar and Cefixime Tellurite Sorbitol MacConkey Agar are used for diagnostic use in vitro only by qualified microbiologists. They should not be used after the expiry date stated or if the product exhibits any indication of degrading.
Sterilise the equipment, specimens, and media in a timely manner after use.
Limitations of Sorbitol MacConkey Agar
- It has been observed that certain Enterobacteriaceae as well as Pseudomonas aeruginosa can be inhibited by MacConkey Agar when incubated in an environment with CO2-enriched.
- The long-term incubation time of the culture can cause colonies of E. E. coli serotype O157 losing their characteristic appearance of colorlessness and the color of colonies containing sorbitol could fade, making difficult to differentiate them from sorbitol-negative colonies.
- Longer incubation times can lead to the fading of pink-colored colonies that are sorbitol positive making it more difficult to interpret. In the event of prolonged incubation species that belong to E. coli O157:H7 can ferment sorbitol to produce pink-colored colonies.
- There are other kinds of facultatively anaerobic gram negative rods which do not ferment sorbitol.
- While the majority of non-O157:H7 coli produce sorbitol, around 6percent of isolates don’t. These atypical strains as well as other non-fermenting bacteria that eat sorbitol like Morganella and Hafnia look similar to O157 colonies and hence confirmation tests are possible.
- There are sorbitol-negative strains with serotypes different from O157:H7, which might or might not cause toxic compounds and clinical signs. MacConkey Agar with Sorbitol does not distinguish between toxin-producing and non-producing strains E. coli O157.
- MacConkey Sorbitol Agar, however is not to be solely used to determine pathogenic E.coli O157:H7 strains, as certain nontoxic strains also do not ferment the sorbitol. Biochemical testing, Gram staining and serological methods must be carried out to verify findings.
