Salmonella and shigella on ss agar

Обновлено: 28.03.2024

Salmonella Shigella (SS) Agar is moderately selective and differential medium for the isolation, cultivation and differentiation of Salmonella spp. and some strains of Shigella spp. SS Agar is a modification of the Desoxycholate Citrate Agar. It is recommended for testing clinical specimens and food testing for the presence of Salmonella spp. and some Shigella spp.

Composition of Salmonella Shigella Agar

IngredientsGms / Litre
Beef Extract5.00
Enzymatic Digest of Casein2.50
Enzymatic Digest of Animal Tissue2.50
Lactose10.00
Bile Salts8.50
Sodium Citrate8.50
Sodium Thiosulfate8.50
Ferric Citrate1.00
Brilliant Green0.00033
Neutral Red0.025
Agar13.50

Distilled Water = 1000 ml

pH ( at 25°C) 7.0 ± 0.2

Principle of Salmonella Shigella Agar

The inclusion of Bile Salts, Sodium Citrate and Brilliant Green serve to inhibit gram-positive, coliform organisms and inhibit swarming Proteus spp., while allowing Salmonella spp. to grow. Beef Extract, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue provide sources of nitrogen, carbon, and vitamins required for organism growth. Lactose is the carbohydrate present in Salmonella Shigella Agar. Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide by the production of colonies with black centers. Neutral red turns red in the presence of an acidic pH, thus showing fermentation has occurred.

Uses of Salmonella Shigella Agar

  1. It is used as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical and non-clinical specimens.
  2. This medium is not recommended for the primary isolation of Shigella.
  3. It was also developed to aid in the differentiation of lactose and non-lactose-fermenters from clinical specimens, suspected foods, and other such samples.

Preparation of Salmonella Shigella Agar

  1. Suspend 60.0 grams of Salmonella Shigella Agar in 1000 ml distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Do not autoclave.
  4. Mix well and pour into sterile Petri plates.

Result Interpretation on Salmonella Shigella Agar

Result Interpretation on Salmonella Shigella Agar

Salmonella will not ferment lactose, but produce hydrogen sulfide (H2S) gas. The resulting bacterial colonies will appear colorless with black centers.

Shigella do not ferment lactose or produce hydrogen sulfide gas, so the resulting colonies will be colorless.

Coliform bacteria such as E. coli will ferment the lactose in the media, resulting in bacterial growth with a pink color. They do not produce any hydrogen sulfide.

Enterobacter and Klebsiella appears larger than E. coli, mucoid, pale, opaque cream to pink.

Quality Control on Salmonella Shigella Agar

Positive

Salmonella enteriditis ATCC 13076= Colorless colonies with black center

Salmonella typhi ATCC 6539 = Colorless colonies with black center

Salmonella typhimurium ATCC 14028= Colorless colonies with black center

Shigella flexneri ATCC 12022 = Colorless colonies

Negative

Enterococcus faecalis ATCC 19433 = Inhibited

Escherichia coli ATCC 25922 = Inhibited

Enterobacter aerogenes ATCC 13048 = partially inhibited. Cream-pink

Limitations of Salmonella Shigella Agar

  1. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
  2. The incorporation of brilliant green into this medium makes it highly selective, and has been shown to inhibit the growth of some Shigella
  3. The bile salts may crystallize over time. They appear as small spider-like puff balls within the medium and do not affect the performance of the medium.
  4. Some strains of Shigella, such as sonneiand S. 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 grow on Salmonella Shigella

References

  1. SS Agar. Hardy Diagnostics.
  2. Salmonella-Shigella Collin County Community College District.
  3. Salmonella Shigella Agar (SS Agar). Oxoid Limited. Thermo Fisher Scientific Inc.
  4. SS Agar – Salmonella Shigella Agar (Medium). Microbiology Made Easy.
  5. Salmonella-Shigella Agar Culture Media. My Bio Source.
  6. SS agar. BioMerieux.
  7. Salmonella Shigella Remel.
  8. Salmonella Shigella Agar. Becton, Dickinson and Company.
  9. Salmonella Shigella Agar (7152). Acumedia Manufacturers, Inc.
  10. Salmonella Shigella Agar (SS Agar). Laboratorios Conda, S.A.
  11. SS Agar (Salmonella Shigella Agar) Plate. HiMedia Laboratories Pvt. Ltd.
  12. 85640 SS-Agar (Salmonella Shigella Agar). Sigma-Aldrich, Inc.

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3 thoughts on “Salmonella Shigella (SS) Agar- Composition, Principle, Uses, Preparation and Result Interpretation”

thank you very much, i would like to know much about. 1: Incubation period, 2: how long will it take for the result to come out. 3: prepared SSA will stay for how long?.

I would also like to know. I used to use XLD for Salmonella analyses for pharmacy industry, the incubation was 24 – 36 hours, and could last for 4 weeks (i have validated it) and now will change to SSA. is it the same?

Thank you very much. This was helpful. Are there any species of Salmonella that grow as white colonies on SS media

Salmonella-Shigella (SS) Agar: Introduction, Composition, Principle, Procedure, Results Interpretation, Uses and Limitations

Salmonella-Shigella (SS) Agar 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).

Introduction of Salmonella-Shigella (SS) Agar

Salmonella-Shigella (SS) Agar 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 Salmonella-Shigella (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 serve 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 Salmonella-Shigella (SS) Agar

  1. Suspend 60 grams of the powder of Salmonella-Shigella (SS) agar in 1 liter 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 Salmonella-Shigella (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), contamination.
  • The product is light and temperature-sensitive; protect from light, excessive heat, moisture, and freezing.
  • Prepared culture media can be kept for at least a week in refrigeration.

Test Requirements for Salmonella-Shigella (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 Salmonella-Shigella (SS) Agar

Result Interpretation of Salmonella-Shigella (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 Salmonella-Shigella (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 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 which appears 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.

Key Notes 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. 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: The 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 Salmonella-Shigella (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 handle 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.

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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 relatively high level of selectivity.

    ,,
  1. Eosin Methylene Blue (EMB) Agar,
  2. Xylose Lysine Deoxycholate (XLD Agar), and

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

Table of Contents

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

Principle

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

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).

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.

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Certain bacterial species liberate sulfur from sulfur-containing amino acids or other compounds in the form of H₂S. This ability of these bacteria can be used as an important characteristic of their identification.

Table of Contents

Principle

Bacterial species capable of producing H₂S release sulfide from cysteine or thiosulfate present in the medium by their enzymatic action. Bacteria that produce cysteine desulfhydrase are able to remove the sulfhydryl and amino groups from cysteine, yielding hydrogen sulfide, ammonia, and pyruvic acid. Hydrogen sulfide is also produced by the reduction of thiosulfate in anaerobic respiration by the enzyme thiosulfate reductase.

Thus formed H₂S gas, which is colorless, combines with H₂S indicators (iron, bismuth or lead) present in the medium producing insoluble, heavy metal sulfides that appear as a black precipitate.

Media for the detection of Hydrogen Sulfide (H₂S)

As various types of media are available for the detection of H₂S production with varying degrees of sensitivity, microbiologists can choose a specific detection system based on their needs and characteristics of the test isolate. For example, lead acetate, the most sensitive indicator, should be used whenever bacteria that produce only trace amounts of H₂S are tested.

Note: When incorporated in culture media, lead acetate may inhibit the growth of many fastidious bacteria so while testing, instead of incorporating it into the media, a lead acetate impregnated filter paper should be draped under the cap of a culture tube.

Hydrogen sulfide production test

SIM is more sensitive in the detection of H2S than either TSI or KIA, because of its semisolid nature, its lack of interfering carbohydrates, and the use of peptonized iron as an indicator.

As H₂S detected in one medium may not be detected in another, it is necessary to know the test system used when interpreting identification charts. In diagnostic microbiology, SIM, KIA, or TSI tubes are commonly used for the detection of H₂S production. Among these three biochemical test media, TSI is the least sensitive. It is believed that sucrose present in this test medium suppresses the production of hydrogen sulfide. SIM is more sensitive than TSI and KIA. Lack of carbohydrates to suppress H₂S formation, and the use of peptonized iron as the indicator makes SIM a better test medium for the detection of H₂S production.

With all H₂S detection systems, the endpoint is an insoluble, heavy metal sulfide, which produces a black precipitate in the medium or on the filter paper strip. Because hydrogen ions must be available for H₂S formation, the blackening is first seen in test media in which acid formation is maximal, that is, along the inoculating line, within the deeps of slanted agar media, or in the centers of colonies growing on agar surfaces.

Procedure

  1. Tube Media
    1. Warm medium to room temperature and examine for cracks. Do not use if cracks appear.
    2. Using a sterile inoculating needle, touch the center of a well-isolated colony.
    3. Stab to within 3 to 5 mm from the bottom of the tube.
    4. Withdraw the needle.
    5. For KIA or TSI, streak the entire surface of the agar slant.
    6. Optional for fastidious organisms: add a strip of lead acetate paper to top of tube and hold in place with the cap of the tube so that it extends 1 in. into the tube.
    7. Place cap loosely on tube. Do not tighten the cap to allow for release of gas in the tube.
    8. Incubate aerobically at 35 to 37°C for 18 to 24 h.
    9. Observe for black precipitate indicating hydrogen sulfide production.
    10. If desired, extend incubation only to detect H2S production. Campylobacters may take 3 days for production of H2S.
    1. Streak plate so as to obtain isolated colonies.
    2. Incubate aerobically at 35 to 37°C
    3. Observe for blackened colonies

    Result and Interpretation

    • Positive reactions
      • H2S production in tube media: black color throughout the medium, a black ring at the junction of the butt and slant, or any black precipitate in the butt. Blackening usually begins at the line of inoculation.
      • H2S production in plate media: black colonies surrounded by a brownish-black zone or metallic sheen
      • Lead acetate paper: brownish-black coloration of the paper strip
      • H2S production in tube media: no blackening in tube
      • H2S production in plate media: no blackening and no metallic-sheen colonies
      • Lead acetate paper: no change in color of the strip

      Hydrogen sulfide-positive organisms

      1. Citrobacter freundii
      2. Salmonella species
      3. Proteus mirabilis
      4. Proteus vulgaris
      5. Edwardsiella tarda

      Limitations

      1. H2S production may be inhibited on TSI for organisms that utilize sucrose and suppress the enzyme mechanism that results in the production of H2S.
      2. Lead acetate is toxic to bacteria and may inhibit the growth of some bacteria.

      References and further reading

        Seventh Edition.
      1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.

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      Hektoen Enteric (HE) Agar was introduced in 1968 by Sylvia King and William I. Metzger. They formulated HE Agar medium while working at the Hektoen Institute in Chicago, to increase the recovery Salmonella and Shigella from clinical specimens. It is a selective as well as a differential culture medium.

      They enriched the media with extra amounts of carbohydrates and peptones to offset the inhibitory effects of the bile salts. The two dyes added to the media, bromthymol blue and acid fuchsin, have lower toxicity than other dyes, thus pathogen recovery was improved. HE agar is currently used as both direct and indirect plating medium for fecal specimens to enhance the recovery of species of Salmonella and Shigella from heavy numbers of mixed normal fecal flora.

      Purpose

      Hektoen enteric agar is currently used as a direct and indirect plating medium to recover gastrointestinal pathogens, such as Salmonella and Shigella, from food, water, and fecal samples suspected of containing these organisms.

      • Selective medium: It inhibits most nonpathogenic (normal flora) enteric organisms and assists in selective recovery of Salmonella and Shigellafrom feces.
      • Differential medium: It allows microbiologists to note visual differences in colony morphology and quickly eliminate nonpathogenic gram-negative rods from pathogenic gram-negative rods with minimal additional testing.

      HE agar can be used for the primary plating of fecal specimens. It may also be used to subculture the overnight growth from enrichment broths (such as gram-negative broth or selenite broth) inoculated with fecal specimens suspected of containing low numbers of Salmonella. Direct inoculation of colonies from agar plates may produce sufficient growth of organisms that would otherwise be inhibited in a more dilute inoculum from diarrheal feces or broth culture.

      Principle

      Hektoen enteric agar is a selective as well as differential media for the isolation and differentiation of enteric pathogens from clinical specimens.

      The presence of the bile salts and dyes inhibits most gram-positive organisms allowing only gram-negative rods to grow on HE agar. The high concentration of bile salts partially or fully inhibits most of the nonpathogenic coliform flora of the intestinal tract. Since the enteric pathogens, Salmonella and Shigella can tolerate these inhibitory substances they generally grow faster and larger than the coliforms.

      Ingredients for Hektoen enteric agar and their roles/functions:-

      1. Protease, peptone, and yeast extracts: Animal peptones and yeast extract provide the nutritive base.
      2. Sodium chloride
      3. Bile salts: The high bile salt concentration inhibits growth of all gram positive bacteria and retards the growth of many strains of coliforms.
      4. Sodium thiosulfate (sulfur source) and ferric ammonium citrate (to detect production of H2S gas)

      Hektoen enteric agar colonies

      Stool culture on Hektoen enteric agar: mixed flora including Escherichia coli (red arrow), Salmonella (blue arrow), and Proteus vulgaris (yellow arrow).

      The production of H2S by certain enteric gram-negative rods, such as Salmonella, can be detected on HE agar due to the addition of thiosulfate and ferric ammonium citrate to the formula. Salmonella produces bacterial enzymes that cause a sulfide molecule to be released from the thiosulfate present in the media. This sulfide molecule then couples with a hydrogen ion to form H2S gas. The H2S gas reacts with the ferric ammonium citrate, forming a precipitate, resulting in colonies that are black or have a black center.

      1. Carbohydrates: Lactose, Saccharose and Salicin
      1. Dyes used: Bromthymol blue and Acid fuchsin
      • Nonpathogenic coliforms (if they are able to grow in the presence of the bile salts) will produce orange-yellow colonies due to the production of acid from at least one of the carbohydrates. This acid causes the bromthymol blue indicator to change from its neutral green color to an orange-yellow color. The bile salts may precipitate out of the media and appear as a hazy zone around the colonies.
      • If a lactose- and sucrose-negative organism utilizes salicin, salmon-pink to orange-yellow colonies will be present.
      • The inability of Salmonella and Shigella to produce acid from the utilization of lactose, sucrose, or salicin results in colonies that are translucent, light green, or greenish-blue and allows them to be quickly differentiated from nonpathogenic organisms.

      Since HE agar is primarily a screening agar, additional testing is required to confirm or rule out Salmonella or Shigella. Several options are available for confirmatory testing ranging from commercial identification kits to tubed biochemical (e.g., TSI agar, urea agar, lysine decarboxylase) to serological typing of somatic and capsular antigens.

      yellow-orange colonies of Proteus vulgaris in Hektoen Enteric Agar


      Yellow-orange colonies of Proteus vulgaris in Hektoen Enteric Agar

      1. Rapid lactose fermenters (such as E.coli) are moderately inhibited and produce bright-orange to salmon pink colonies.
      2. Salmonella colonies are blue-green typically with black centers from hydrogen sulphide gas.
      3. Shigella appear greener than Salmonella, with the color fading to the periphery of the colony.
      4. Proteus strains are somewhat inhibited; colonies that develop are small transparent and more glistening or watery in appearance than species of Salmonella or Shigella.

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