Salmonella-Shigella (SS) Agar: Composition, Principle, Colony Characteristics, and Limitations

A clinical microbiologist reviewing a stool culture protocol for a new district hospital is asked: "Can we just use SS agar for all suspected enteric pathogen cases?" The answer is "no".

Salmonella-Shigella agar is excellent at what it does: suppressing coliforms and recovering Salmonella from heavily contaminated fecal specimens. But the same high selectivity that makes it good for Salmonella makes it inhibitory to Shigella dysenteriae and unreliable for Shigella species in general. A laboratory using SS agar as its only enteric selective medium will miss some of the most clinically important cases of bacillary dysentery.

Salmonella-Shigella (SS) agar is used for selective isolation and differentiation of Salmonella and Shigella. It is used for the isolation, cultivation and differentiation of gram-negative enteric microorganisms from both clinical and non-clinical specimens such as from feces, urine, and suspected food items (fresh and canned foods). This medium is not recommended for the primary isolation of Shigella as some Shigella strains may not grow on SS agar due to a relatively high level of selectivity.

Other less inhibitory media used for the isolation, cultivation and differentiation of gram-negative enteric microorganisms are:

1. Desoxycholate (DCA) Agar,
2. MacConkey Agar,
3. Eosin Methylene Blue (EMB) Agar,
4. Xylose Lysine Deoxycholate (XLD Agar), and
5. Hektoen Enteric Agar

Composition of Salmonella-Shigella (SS) Agar and their function

Final pH: 7.0 ± 0.2 at 25°C

Principle

SS agar achieves its high selectivity through four synergistic inhibitory agents working simultaneously:

1. Bile salts: inhibit Gram-positive organisms and suppress some Gram-negatives.

2. Sodium citrate: inhibits Gram-positive organisms and suppresses most coliforms.

3. Sodium thiosulfate + ferric citrate (H₂S indicator system): the same system used in DCA: organisms producing H₂S from thiosulfate form black iron sulfide precipitate, giving Salmonella its characteristic black-centered colonies.

4. Brilliant green, the key selective agent that sets SS agar apart: Brilliant green is a triphenylmethane dye with potent inhibitory activity against most Gram-negative bacteria, including the majority of coliforms. At the low concentration used in SS agar (0.00033 g/L — just 0.33 mg per liter), it is bacteriostatic to most enterics while Salmonella species have inherent resistance. This allows Salmonella to grow where other organisms are suppressed.

Why Shigella is inhibited, the critical limitation: Shigella species are more susceptible to brilliant green than Salmonella. At the concentration incorporated in SS agar, brilliant green inhibits Shigella dysenteriae almost completely, and suppresses growth of S. flexneri and S. sonnei to a variable but significant degree. This is the direct cause of the paradox embedded in the medium's name: the medium named for Salmonella and Shigella is poorly suited for Shigella isolation.

SS agar combines brilliant green with a high bile salt content (roughly five times that of MacConkey agar). This combination makes it more inhibitory than MacConkey or DCA. The high selectivity maximizes suppression of competing flora in heavily contaminated specimens, but it comes at the cost of reduced Shigella recovery.

Differential mechanism: Neutral red is the pH indicator. Lactose fermenters produce acid, turning colonies red to pink. Non-fermenters (Salmonella, Shigella, Yersinia) produce colorless colonies. H₂S producing species (Salmonella, some Proteus) develop black centers.

Figure: Colony morphology of E.coli, Salmonella and Shigella in Salmonella-Shigella Agar

Preparation of the media

1. Suspend 60 g of the medium in one liter of deionized or distilled water.
2. Mix well.
3. Heat with frequent agitation and boil for one minute.
4. Sterilization in an autoclave is not necessary.
5. Pour into plates
6. Let the agar solidify and store it in the refrigerator (avoid freezing). Prepared culture media can be kept for at least a week in refrigeration. Note: Various commercial suppliers now supplies ready-to-use culture plates.

Culturing the sample

1. Allow the plates to warm to room temperature and the agar surface to dry before inoculating.
2. Heavily inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37°C 18-24 hours.
6. Examine colonial morphology.

Results

1. Lactose fermenter: If lactose fermentation occurs, the medium will turn red due to the acidic pH. e.g. Escherichia coli, Klebsiella pneumoniae gives red colonies.
2. Non-Lactose fermenter: Salmonella, Shigella, and other non-lactose fermenters appear as transparent or translucent colorless colonies. Colonies of Salmonella spp. may appear with or without black centers (depending on the species isolated).

Colony Characteristics on SS Agar

Proteus vs Salmonella on SS agar: Both produce colorless colonies with black centers. Key distinction: Proteus colonies are more glossy and translucent, often with a large central black dot; Salmonella colonies are more opaque. Always confirm with urease test (Proteus strongly positive) and TSI before reporting Salmonella.

When to Use SS Agar and When Not To

Use SS agar when:

• Specimens are heavily contaminated with coliform flora (e.g., formed stool, rectal swabs) and maximum suppression of background flora is needed
• Salmonella is the primary clinical concern (typhoid fever, non-typhoidal salmonellosis, food poisoning investigation)
• Used alongside a less inhibitory medium, SS agar is best used as one plate in a two-plate battery, not as the sole medium

Do not use SS agar as the sole medium when:

• Shigella dysentery is the primary concern, Shigella dysenteriae and most other Shigella strains are inhibited
• Specimens are from pediatric patients (children with bloody diarrhea, Shigella is a leading cause)
• Investigating outbreaks of bacillary dysentery

Recommended two-medium battery for enteric stool culture:

How to Remember

SS agar, high selectivity is both its strength and its flaw:

The key to understanding SS agar is the brilliant green dye. At the concentration used, brilliant green:

• Suppresses nearly all coliforms → clean background, easy to read
• Suppresses Shigella → misses the pathogen in the medium's own name
• Does not suppress Salmonella → the medium's real target

Memory anchor "SS agar should be called S agar": Despite being named Salmonella-Shigella agar, it is functionally a Salmonella-selective agar. Renaming it mentally as "S agar" is not just a memory trick, it prevents the clinical error of relying on SS agar alone when Shigella dysentery is suspected.

The enteric media selectivity spectrum:

Colony reading: two questions, three outcomes.

Precautions:

As SS Agar media contains components of animal origin (absence of transmissible pathogenic agents cannot be ruled out) so treat it as potentially infectious, and handle observing the usual universal blood precautions. Do not ingest, inhale, or allow the media to come into contact with skin.

Where Students Get Confused

1. The name promises more than the medium delivers. SS agar is named for both organisms but reliably recovers only Salmonella. Brilliant green inhibits most Shigella, and Shigella dysenteriae barely grows at all. If dysentery is the concern, SS agar alone will miss it. Use XLD or a less inhibitory medium alongside it.

2. Proteus can be mistaken for Salmonella. Both are non-lactose fermenters that produce colorless colonies with black centers from H₂S. Do not report Salmonella on colony appearance alone. Proteus is strongly urease positive and Salmonella is urease negative, so a urease test and TSI separate them before serology.

3. S. Paratyphi A has no black center. Most Salmonella produce the classic colorless colony with a black center. S. Paratyphi A is H₂S negative, so it is colorless without a black center and looks like Shigella or a plain non-fermenter. A black center is a useful clue when present, but its absence does not rule out Salmonella.

4. Coliforms are suppressed, not always absent. Brilliant green and bile salts inhibit coliforms but do not always kill them. On prolonged incubation, residual E. coli or Klebsiella may grow as pink colonies. Read plates at 18 to 24 hours; late-appearing pink colonies are usually breakthrough coliforms, not the target pathogen.

References

• Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
• Cheesbrough M. District Laboratory Practice in Tropical Countries, Part 2. 2nd ed. Cambridge: Cambridge University Press; 2006.
• World Health Organization. Guidelines for the Control of Shigellosis, Including Epidemics Due to Shigella dysenteriae Type 1. Geneva: WHO; 2005.
• Mahon CR, Lehman DC, Manuselis G. Textbook of Diagnostic Microbiology. 6th ed. St. Louis: Elsevier; 2018.