SS Agar, Modified

Intended Use:

SS Agar (Salmonella Shigella Agar) Modified is used for the selective isolation and differentiation of Salmonella and Shigella species from pathological specimens, suspected foodstuffs etc.

Composition**

Final pH (at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters. # Equivalent to Beef extract.

Directions

Suspend 57.02 grams in 1000 ml purified/distilled water. Heat to boiling with frequent agitation to dissolve the medium completely. DO NOT AUTOCLAVE OR OVERHEAT. Overheating may destroy the selectivity of the medium. Cool to about 45-50°C. Mix and pour into sterile Petri plates.

Principle And Interpretation

Salmonella and Shigella are gram-negative, facultatively anaerobic, non-sporulating rods in the family Enterobacteriaceae. They are widely distributed in animals, infecting mainly the stomach and the intestinal tissues. SS Agar is recommended as differential and selective medium for the isolation of Salmonella and Shigella species from pathological specimens (1) and suspected foodstuffs (2,3,4,5) and for microbial limit test (6). SS Agar is a moderately selective medium in which gram-positive bacteria are inhibited by bile salts, brilliant green and sodium citrate.

Peptone and HM Peptone B provide essential growth nutrients. Lactose is the fermentable carbohydrate. Brilliant green, bile salts and thiosulphate selectively inhibit gram-positive and coliform organisms. Sodium thiosulphate is reduced by certain species of enteric organisms to sulphite and H2S gas. This reductive enzymatic process is attributed to thiosulphate reductase. Production of H2S gas is detected as an insoluble black precipitate of ferrous sulphide, formed upon reaction of H2S with ferric ions or ferric citrate, indicated by black centered colonies.

The high selectivity of Salmonella Shigella Agar allows the use of large inocula directly from faeces, rectal swabs or other materials suspected of containing pathogenic enteric bacilli. On fermentation of lactose by few lactose-fermenting normal intestinal flora, acid is produced which is indicated by change of colour from yellow to red by the pH indicator neutral red. Thus these organisms grow as red-pigmented colonies. Lactose non-fermenting organisms grow as translucent colourless colonies with or without black centers. Salmonella species exhibit colourless colonies with black centers resulting from H2S production. Shigella species form colourless colonies, which do not produce H2S. While using samples suspected of being exposed to treatments that might have damaged the viability of microorganisms due to processing of food materials or samples from patients under antibiotic treatment etc., previous enrichment in Selenite cystine Broth Base (M025) or Tetrathionate Broth Base (M032) is necessary. Inoculate SS Agar plates with the enriched culture. After incubation the suspicious colonies should be subcultured on differential media to be identified biochemically or serologically.

Type of specimen

Clinical: faeces, rectal specimen; Suspected foodstuffs.

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (7,8). For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (2,3,4,5).

Warning and Precautions

In Vitro diagnostic use. For professional use only. 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 clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. The medium is highly selective and may be toxic to certain Salmonella or Shigella species. Hence it is recommended to useto inoculate plates of less inhibitory media parallel to SS Agar, such as Hektoen Enteric Agar (M467) or Deoxycholate Citrate Agar (M065) for easier isolation of Shigella species (9).

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 1.2% Agar gel.

Colour and Clarity of Prepared Medium Reddish orange coloured clear to slightly opalescent gel forms in Petri plates.

Reaction Reaction of 5.7% w/v aqueous solution at 25°C. pH: 7.2±0.2.

pH 7.00-7.40

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

* Corresponding WDCM numbers (#) Formerly known as Enterobacter aerogenes

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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
3. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
4. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
5. Williams S., (Ed.), 2005, Official Methods of Analysis of the Association of Official Analytical Chemists, 19th Ed., AOAC, Washington, D.C.
6. The United States Pharmacopoeia-National Formulatory (USP-NF), 2022.
7. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
8. 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.
9. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore.