SS Agar: Introduction, Composition, Principle, Procedure, Results- Interpretation, Uses,Limitations, and Keynotes

SS Agar: Introduction, Composition, Principle, Procedure, Results- Interpretation, Uses,Limitations, and Keynotes

Introduction of SS Agar

SS Agar is the short form of Salmonella-Shigella Agar and it is recommended for use as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical and non-clinical specimens (suspected foodstuffs).

Composition of SS Agar 

(Himedia)
Ingredients    Gms / Litre

  • Proteose peptone 5.0
  • Lactose 10.0
  • Bile salts mixture 8.5
  • Sodium citrate 8.5
  • Sodium thiosulphate 8.5
  • Ferric citrate 1.0
  • Brilliant green 0.00033
  • Neutral red 0.025
  • Agar 13.5
  • Distilled water: 1000 ml

Final pH ( at 25°C) 7.0±0.2

Principle of Salmonella-Shigella (SS) Agar

The basis for differentiation on Salmonella-Shigella (SS) agar depends on the fermentation of lactose and the absorption of neutral red as the bile salts precipitate in the acidic condition. Neutral red turns red in the presence of an acidic pH, thus showing fermentation has occurred. The inclusion of bile salts, sodium citrate, and brilliant green serves to inhibit gram-positive and coliform organisms. Salmonella, Shigella, and other non-lactose-fermenting organisms appear as transparent or translucent colorless colonies on SS Agar. Sodium thiosulfate is added to the medium as a hydrogen sulfide source, and ferric citrate is added as an indicator for hydrogen sulfide production.

Preparation of SS Agar

  1. Suspend 60 grams of the powder of Salmonella-Shigella (SS) agar in 1 liter of purified/distilled or deionized water.
  2. Mix thoroughly and heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
  3. Avoid overheating and do not autoclave.
  4.   Leave for cooling to 45-50°C.
  5. Mix well before dispensing.
  6. Pour into each plate and leave plates on the sterile surface until the agar has solidified.
  7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of SS Agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.
  • Prepared culture media can be kept for at least a week in refrigeration.

Test Requirements for SS Agar

  • Test specimens
  •  Salmonella-Shigella (SS) Agar plates
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains ( For negative control-Enterococcus faecalis ATCC 29212,  and Escherichia coli ATCC 25922 (Partial to complete inhibition)  while positive control-Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028, Shigella flexneri 12022 ATCC )

Test procedure of SS Agar

  1. Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
  2. Take Salmonella-Shigella (SS) Agar plates.
  3. 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.
  4. Incubate aerobically plates at 35 ± 2°C for 18-24 hours.

Result -Interpretation of SS Agar

If lactose fermentation occurs, the medium will turn red due to the acidic pH. Salmonella, Shigella, and other non-lactose fermenters appear as transparent or translucent colorless colonies on SS Agar. Colonies of Salmonella spp. may appear with or without black centers (depending on the species isolated).

Colony characteristics of SS agar

Typical colonial morphology on Salmonella-Shigella Agar after inoculation of Inoculum having 50-100 CFU is as follows:

  • Salmonella (good-luxuriant growth): Colorless, usually with a black center
  • Shigella flexneri (good growth): Colorless
  • E.coli (fair growth):  pink or red
  • Enterobacter/Klebsiella (fair growth) : Pink
  • Proteus(fair-good growth): Colorless, usually with a black center
  • Pseudomonas (fair growth): Irregular
  • Gram-positive bacteria (Staphylococcus, Micrococcus): No growth
  • Enterococcus faecalis: (none-poor growth): colorless

Uses of SS Agar

  1. SS agar is used as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical as well as non-clinical specimens.
  2. This medium is not recommended for the primary isolation of Shigella because of its inhibitory to most strains.
  3. SS agar was also developed to aid in the differentiation of lactose and non-lactose-fermenters from clinical specimens, suspected foodstuffs, and other such specimens.

Limitations of Salmonella-Shigella (SS) Agar

  1. 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.
  2. The presence of brilliant green in this medium makes it highly selective and has been shown to inhibit the growth of some Shigella species. Thus, a non-selective but differential medium such as MacConkey Agar or HE Agar should also be streaked to increase the recovery of fastidious, or low numbers of, gram-negative bacteria.
  3. The bile salts may crystallize over time and appear as small spider-like puff balls within the medium and even though they do not affect the performance of the medium.
  4. Some strains of Shigella, such as Shigella sonnei and Shigella dysenteriae serovar 1, may ferment lactose relatively slowly, and colonies change to lactose-fermenting after cultivation for 2 or more days.
  5. A few non-pathogenic organisms may also grow on Salmonella Shigella agar.

Keynotes on SS  Agar

  1. Other less inhibitory media used for the isolation, cultivation, and differentiation of gram-negative enteric bacteria are Desoxycholate Agar, MacConkey Agar, Eosin Methylene Blue (EMB) Agar, Xylose Lysine Deoxycholate (XLD Agar), and Hektoen Enteric Agar.
  2. Despite its name, Salmonella-Shigella (SS) agar is not suitable for isolating shigellae as it is inhibitory to most strains.
  3. The functions of SS agar ingredients are as follows-Lactose is the fermentable carbohydrate.  Beef extract and proteose peptone provide nitrogen, vitamins, and amino acids. Ferric citrate and Sodium Thiosulfate: Sodium thiosulfate is added to the medium as a hydrogen sulfide source, and ferric citrate is added as an indicator for hydrogen sulfide production. Sodium thiosulphate and Sodium citrate are selective agents, providing an alkaline pH to inhibit Gram-positive bacteria and suppress coliforms. Bile salts: Bile salts inhibit the growth of gram-positive bacteria.  Brilliant green and neutral Red both are pH indicators. Agar acts solidifying agent and sodium chloride is the source of electrolytes whereas water is the source of hydrogen and oxygen.

Precautions for SS Agar

  • 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 handled 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.

Related Videos

#Shigella flexneri growth on various media ( SS agar, MacConkey medium, and XLD agar),  its biochemical tests ( TSI, SIM, Urease, and Citrate), and serotyping-

TSI Test-R/Y, no H2S, no evidence of gas formation

SIM Test- H2S-Neg, Indole-Neg, Motility-Non motile

Citrate Utilization Test-Negative

Urea hydrolyzation Test -Negative

Growth on  Salmonella-Shigella (SS) Agar: Colorless colonies

ON MacConkey agar– Non-Lactose fermenter( NLF) colonies

XLD agar: Colorless colonies

Catalase test-Positive

According to serotyping- Came under serogroup B, which is why the organism is Shigella flexneri.

#Shigella antisera for serotyping are shown below-

  1. Polyvalent A: Shigella dysenteriae
  2. P.  B: Shigella flexneri
  3. P.  C: Shigella boydii

Further Readings

  1. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/SSAgar.htm
  2. http://himedialabs.com/TD/M108D.pdf
  3. file:///C:/Users/jebo/Downloads/107667.pdf
  4. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
  5. https://www.tmmedia.in/sites/default/files/TM386_Salmonella_Shigella_Agar_ss_Agar_TD.pdf
  6. https://www.neogen.com/solutions/microbiology/salmonella-shigella-agar/
  7. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  8. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  9. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  10. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  11. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  12.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  13. file:///C:/Users/jebo/Downloads/1064_en_1.pdf
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