Salmonella-Shigella (SS) Agar

By Acharya Tankeshwar •  Updated: 05/04/22 •  4 min read

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

Despite its name, Salmonella-Shigella (SS) agar is not suitable for isolating shigellae as it is inhibitory to most strains.

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

  1. Lactose: fermentable carbohydrate
  2. Beef extract, proteose peptone: provides the nitrogen, vitamins, and amino acids in SS Agar
  3. Ferric citrate: Sodium Thiosulfate is also a sulfur source, and acts with Ferric Citrate as an indicator to detect hydrogen sulfide production.
  4. Sodium thiosulphate &Sodium citrate: selective agents, providing an alkaline pH to inhibit Gram-positive organisms and suppress coliforms
  5. Bile salts : The bile salts inhibit growth of gram-positive microorganisms
  6. Brilliant Green/Neutral Red: pH indicator.
  7. Agar: Solidifying agent.

Final pH: 7.0 +/- 0.2 at 25ºC


The presence of bile salts mixture and dyes (brilliant green) inhibits the growth of gram-positive species to a varying degree. Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium. Organisms which ferment lactose produce acid which, in the presence of the neutral red indicator, results in the formation of red/pink colonies. Lactose non-fermenters form colorless colonies. The latter group contains the majority of the intestinal pathogens, including Salmonella and Shigella.

The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers.

E coli Shigella Salmonella
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 autoclave is not necessary.
  5. Pour into plates
  6. Let the agar solidify and store 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.


  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).

Typical colonial morphology on Salmonella-Shigella Agar is as follows:

Name of the organismsColony characteristics in Salmonella-Shigella agar
E.coliSlight growth, pink or red
Enterobacter/KlebsiellaSlight growth, pink
ProteusColorless colonies, usually with black center
SalmonellaColorless colonies, usually with black center
PseudomonasIrregular, slight growth
Gram-positive bacteriaNo growth


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.


Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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18 responses to “Eosin Methylene Blue Agar (EMB Agar)”

  1. Anonymous says:

    Good day,
    Please I need a help,I am working on water sample. What agar can I use in place of membrane feacal coliform agar in order to isolate feacal coliform.

    • Tankeshwar Acharya says:

      In our laboratory, we first incubate the water sample in MacConkey broth (please refer to MPN method) if result comes positive, we streak it (from MacConkey broth) to EMB Agar and incubate the plate at 44.5°C.

  2. ELVIS KICHANA says:

    please, i am a water microbiologist and would like to know how to distinguish E.coli from Total coliform on EMB Agar……..

  3. Necxus Andrew says:

    i carried out a research titled “the common bacteria associated with the hands of primary pupils” here in a local government in Nigeria using two primary schools as a study area. the sample was collected according to Age, Gender, and Schools. the bacteria isolated includes: staphylococcus aureus, staphylococcus epidermidis, pseudomonas sp, and Escherichia coli. am confuse on how to arrange the tables in respect to Age, school, organisms isolated and the type of statistical method/ tool to use. pls help me with the table using even a sample of the table.thanks.

  4. anonymus says:

    am grateful for a great deal of insight from you guys

  5. anonymus says:

    how can I carry a swab on door handle and subsequent plating on EMB agar for identification and enumeration?

  6. Ella Pilot says:

    My name is Ella Pilot and I’m doing a science fair project looking at bacteria in the soil of my school’s playground to see if it’s fecal coliform. We believe it may be because of the smell that comes during the spring run-off. We had a pond near right behind the school, that got filled in when houses were built over top, and now we get the water run-off from the houses on two of the sides of our playground.
    Could I send you the pictures of the colonies I prepared in EMB agar? I need help identifying the bacteria colonies.

    Thanks for taking the time to read this and reply.

    Ella Pilot

  7. akenji says:

    great site. i am a clinical biologist and i use your site for quick revision and more.

    • tankeshwar says:

      Thank you so much Akenji. In your convenience, please share how it can be made more useful to visitors.

  8. Jessica says:

    Hello i am in microbiology right now as a sophomore in high school and I am having trouble identifying what bacteria grew on the EMB agar plate.. It is gram negative and grew purple with clear in some area around the edges any clue?

    • tankeshwar says:

      EMB agar is a selective media(only Gram-negative bacteria grow in it). Lactose fermenter (such as Escherichia coli, Klebsiella spp, Citrobacter spp, Enterobacter spp), give purple color (colorless colonies by Non-lactose fermenter), but you have to use panel of biochemical tests to identify the isolate you have.

  9. lizy says:

    does emb assists on the growth of fungus

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