MacConkey Sorbitol Agar Base

Intended Use

Recommended as the selective medium for isolation and detection of Escherichia coli O157:H7 from food, animal feeding stuffs and clinical samples. The composition and performance criteria are in accordance with ISO 16654:2001& / Amd 2:2023.

Composition**

Final pH (at 25°C): 7.1±0.2

Supplement to be added after sterilization

TC Selective Supplement (FD147) - 2 vials: Potassium tellurite 1.250mg, Cefixime 0.025mg per vial.

**Formula adjusted, standardized to suit performance parameters $ - Equivalent to Enzymatic digest of casein ; # Equivalent to Enzymatic digest of animal tissues

Directions

Suspend 50.03 gram in 990 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. AVOID OVERHEATING. Cool to 45-50°C and aseptically add rehydrated contents of 2 vials of TC Selective Supplement (FD147). Mix well and pour into sterile Petri plates.

Principle And Interpretation

MacConkey Sorbitol Agar is recommended by ISO Committee (1) with a slight modification of MacConkey Sorbitol Agar formulated by Rappaport and Henigh (2). This medium is recommended for isolation of enteropathogenic Escherichia coli O157: H7, which ferments lactose but does not ferment sorbitol, hence produces colourless colonies. This organism has been recognized as a cause of hemorrhagic colitis (3). E.coli O157: H7 is a human pathogen associated with hemorrhagic colitis that results from the action of a shiga-like toxin (SLT) (4,5). MacConkey Sorbitol Agar however should not be solely used to detect pathogenic E.coli O157: H7 strains as some non-toxic strains will also not ferment sorbitol (6).

On standard MacConkey Agar containing lactose, this strain is indistinguishable from other lactose-fermenting E.coli. In MacConkey Sorbitol Agar Base, lactose is replaced by sorbitol. Unlike most E.coli strains, E.coli O157:H7 ferments sorbitol slowly or not at all (7,8). The growth of E.coli O157:H7 on MacConkey Agar with Sorbitol shows colourless colonies and most of the faecal flora ferment sorbitol and appear pink. MacConkey Agar with Sorbitol therefore permits ready recognition of E.coli O157:H7 (4,5,9).

Tryptone and HM peptone supply necessary nutrients like nitrogenous and carbonaceous compounds, minerals, vitamins and trace ingredients for the growth of organisms. Crystal violet and bile salt mixture present in the medium inhibit growth of gram-positive bacteria. The addition of cefixime and tellurite, as FD147 significantly reduces the number of sorbitol non-fermenters that are to be screened during the attempted isolation of E.coli O157:H7. Sodium chloride maintains osmotic equilibrium. Neutral red is an indicator. D-Sorbitol is the fermentable carbohydrate.

Type of specimen

Food samples

Specimen Collection and Handling

ISO 16654:2001&/Amd 2:2023 and ISO 11133:2014 /Amd. 2 : 2020 (E) (1,10)

a) Enrichment of the test portion homogenized in Modified Soyabean Bile Broth Base (M1286I) with incubation at 41.5±1 °C for 6 h and subsequently for a further 12 h to 18h.

b) Isolation onto MacConkey Sorbitol Agar Base (M298I). Incubate at 37±1°C for 21±3 h. Confirm the colonies by biochemical characterisation.

Warning and Precautions

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Further subculture must be carried out for confirmation.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Light yellow to pink homogeneous free flowing powder

Gelling: Firm, comparable with 1.35% Agar gel.

Colour and Clarity of prepared medium: Purplish red coloured clear to slightly opalescent gel forms in Petri plates

Reaction: Reaction of 5% w/v aqueous solution at 25°C. pH : 7.1±0.2

pH: 6.90-7.30

Cultural Response:

Productivity: Cultural characteristics observed with added TC Selective Supplement (FD147) , after an incubation at 36-38°C for 18-24 hours.

Selectivity: Cultural characteristics observed with added TC Selective Supplement (FD147) , after an incubation at 36-38°C for 18-24 hours.

Key : *Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (11,12).

Reference

1. Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Escherichia coli O157 AMENDMENT 2: Inclusion of performance testing of all culture media and reagents, ISO 16654:2001, Amd 2:2023.
2. Rappaport F. and Henigh E., 1952, J. Clin. Pathol., 6 : 361.
3. Karmali M. A., Petric M., Lim C. et al, 1985, J. Infect. Dis.,151:775.
4. Centre for Diseases Control, 1991, Morbid. Mortal, Weekly Rep 40:265.
5. March S. B. and Ratnam S., 1986, J. Clin. Microbiol., 23:869.
6. Pelczar M. J., Chan E. C. and Kreig M. R., 1986, Microbiology, 5th Ed., McGraw Hill Book Co., New York
7. Sanderson M. W., Gay J. M., Hancock D. D., Gay C. C., Fox L. K. and Besser T. E., 1955, J. Clin. Microbiol., 33: 2616.
8. Zadik J. M., Chapman P. A. and Siddons C. A., 1993, J. Med. Microbiol., 39:155.
9. Murray P. R., Baron J. H., Pfaller M. A., Tenover F. C. and Yolken R. H. (Ed.), 1999, Manual of Clinical Microbiology, 7th Ed. American Society for Microbiology, Washington, D. C.
10. Microbiology of food,animal feeding stuffs and water- Preparation, production,storage and performance testing of culture media, EN ISO 11133:2014 /Amd.2 2020 (E)
11. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
12. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.