Detection of Salmonella and Shigella in Food

Medium used

Selective agent and how it works

Differential agent and how it works

Typical cultural response for:  Salmonella / Shigella / coliforms / other important organisms.

Selective Enrichments

Selenite Cystine Broth

Incubated at 35 degree Celsius for 24 hours

- Sodium selenite inhibits gram positive bacteria and suppresses most gram negative enterics.

- Selenite Cystine Broth is formulated to allow the proliferation of Salmonella and while inhibiting the growth of competing non-Salmonella bacteria (BD, 2013).

- Enrichment medium for isolating Salmonella from other fecal organisms. It is used to detect Salmonella from diary product.

Tetrathionate Broth

Incubated at 35 degree Celsius for 24 hours

- Bile salts inhibit the growth of gram positive and gram negative microorganisms other than Salmonella spp.

- The use of iodine-iodide solution promotes the production of tetrathionate which inhibits the growth of coliforms and other enteric bacteria.

- The addition of brilliant green to the medium aids in the suppression of predominantly gram positive bacteria (IFU, 2016).

- Enrichment medium for isolating Salmonella

- Inhibit the growth of Shigella

- It is used to detect Salmonella in foods.

Selective and Differential plating

Selective agent and how it works

Differential agent and how it works

Typical cultural response for:  Salmonella / Shigella / coliforms / other important organisms.

MacConkey agar (MAC)

Incubated at 35 degree Celsius for 24 hours

-Bile salts and crystal violet

- They prevent the growth of gram positive bacteria.

- Gram positive bacteria cannot tolerate bile salts environment.

- Lactose

- It helps to differentiate lactose fermenting gram negative rods from non lactose fermenting gram negative rods.

- Lactose-fermenting organisms form pink colonies surrounded by a zone of bile salt precipitation. Color change is due to production of acid which changes the neutral red pH indicator from colorless to red. Acid production is also responsible for the formation of bile salt precipitation (IFU, 2016).

- Salmonella: colorless colonies (lactose -ve)

- Shigella: pale colored or colorless and transparent colonies (lactose -ve)

- E.coli: red colonies

- Proteus: colorless and transparent colonies

Hektoen Enteric agar  (HEK)

Incubated at 35 degree Celsius for 24 hours

- Bile salts, bromthymol blue and acid fuchsin

- They inhibit the growth of most gram positive organisms (HiMedia Laboratories, 2015).

- Lactose, salicin and sucrose

- They serve as fermentable source of carbohydrates to encourage the growth and differentiation of enteric bacteria (HiMedia Laboratories, 2015).

- Ferric ammonium citrate and sodium thiosulfate enable detection of hydrogen sulfide (HiMedia Laboratories, 2015).

- Salmonella: black colonies (reduce sulfur to hydrogen sulfide, producing a black precipitate.)

- Shigella: green colonies (non-lactose fermenter)

- E.coli: Salmon-orange colonies, may have blue precipitate

Salmonella-Shigella agar  (SS)

Incubated at 35 degree Celsius for 24 hours

- Bile salts, brilliant green, and citrates

- They inhibit the growth of gram positive bacteria and coliforms (Remel, 2010).

- Lactose

- Gram -ve bacilli which ferment lactose produce pink to red colonies. However, lactose-nonfermenters from transparent, colorless colonies (Remel, 2010).

- Sodium thiosulfate a sulfur source, and ferric ammonium citrate, an indicator are added to enable organisms which produce H2S to form black-centered colonies (Remel, 2010).

- Salmonella: colorless colonies with black centre (non- lactose feremnter, H2S +ve)

- Shigella: colorless colonies (non- lactose fermenter, H2S -ve)

- E.coli: pink to red colonies

Bismuth Sulfite Agar  (BS)

Incubated at 35 degree Celsius for 24 hours

- Bismuth sulfite and brilliant green are general inhibitors that hinder the growth of most bacteria while allowing salmonellae to grow (Dalynn, 2003).

- Ferrous sulfate utilization by Salmonella species results in H2S production and the formation of ferrous sulfide, an insoluble black precipitate (Dalynn, 2003).

- The insoluble black precipitate gives Salmonella colonies their characteristic brown-black coloration and also diffuses into the surrounding medium (Dalynn, 2003).

- Salmonella: dark gray to brown-black colonies with or without a metallic sheen.

- Shigella: inhibited to grow but some strains may grow as brownish-green colonies with depressed centre.

- E.coli: brown to green colonies

Xylose Lysine Desoxycholate agar (XLD)

Incubated at 35 degree Celsius for 24 hours

- Sodium desoxycholate

- It inhibits the growth of gram positive microorganisms (BD, 2007).

- Xylose

- It is incorporated into the medium since it is fermented by practically all enterics except for the Shigella and this property enables the differentiation of Shigella species (BD, 2007).

- Lysine

- It is included to enable the Salmonella group to be differentiated from the non- pathogens since without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from non-pathogenic species (BD, 2007).

- Sodium thiosulfate and ferric ammonium citrate as H2S indicator (BD, 2007).

- Salmonella: red colonies with black centre (H2S +ve)

- Shigella: pink to red colonies (H2S -ve)

- E.coli: yellow colonies

Biochemical ID

How is it inoculated?

Reagents involved

Interpretation: Pos vs Neg  colours etc.

Typical organism reactions:

Urea slant

- Using a heavy inoculum of growth from a pure culture, streak back and forth over the entire slant surface.

- The inoculated tube is incubated at 35 degree Celsius for 24 hours.

- A positive test consists of a color change red to hot pink or magenta, indicating a pH change to alkaline/basic.

- No reagents

- Salmonella: -ve (yellow, no color change)

- Shigella: -ve (yellow, no color change)

- E.coli: -ve (yellow, no color change)

- Proteus: +ve (pink color)

Triple Sugar Iron (TSI)

- To inoculate, carefully touch only the centre of an isolated colony on an enteric plated medium with a cool, sterile needle, stab into the medium in the butt of the tube, and then streak back and forth along the surface of the slant.

- Incubated at 35 degree Celsius for 24 hours.

- No reagents

- Salmonella: K/A (red/yellow), gas +ve, H2S +ve

- Shigella: K/A (red/yellow), gas -ve, H2S -ve

- E.coli: A/A (yello/yellow), gas +ve, H2S -ve

- Pseudomonas: K/K (red/red), gas -ve, HSS -ve

Lysine Iron Agar (LIA)

- Using an inoculating needle, stab the butt twice then streak the slant with growth from a pure culture.

- Incubated with loosened caps at 35 degree Celsius for 48 hours in an aerobic atmosphere.

- No reagents

- Salmonella:  K/K (purple/purple), + ve reaction for deamination of amino acids (aerobic alkaline rx.), -ve reaction for deamination of lysine (dark red slant), +ve reaction for decarboxylation of lysine (anaerobic alkaline rx.), +ve reaction for glucose fermentation (acid rx.), H2S +ve (black precipitate)

- Shigella: H2S -ve (no black precipitate), K/A (purple/ yellow)

Sulfide Indole Motility (SIM)

- Using an inoculating needle, inoculate the SIM Medium by stabbing the centre of the medium to a depth of 1/2 inch.

- Incubated at 35 degree Celsius for 24 hours.

- 5 drops of Kovacs reagent

- Salmonella: Indole -ve, motility +ve, H2S +ve

- Shigella: Indole -ve, motility -ve, H2S -ve

- E.coli: Indole +ve, motility +ve, H2S -ve

Methyl-Red / Voges-Proskauer broth (MRVP)

- Using a light inoculum, inoculate tubes of MR-VP media with pure cultures.

- Incubated at 35 degree Celsius for 48 hours.

- Methyl-Red (MR)test: 5 drops of methyl red indicator

- MR +ve if red color is immediately observed.

- Voges-Proskauer (VP) test: 15 drops of Voges-Proskauer Reagent A (5% alpha-naphthol)  and 5 drops of Voges-Proskauer Reagent B (40% KOH)

- VP +ve if red color is observed after 10-15 minutes.

- Salmonella: MR +ve (red color), VP -ve (yellow color)

- Shigella: MR +ve (red color), VP -ve (yellow color)

- E.coli: MR -ve (yellow color), VP +ve (red color)

Citrate Slant

- Inoculate slants with growth from a pure culture using a light inoculum.

- Abundant growth on the slant and a color change from green to blue in the medium indicates a positive test for growth using citrate.

- Incubated at 35 degree Celsius for 24 hours

- No reagents

- Salmonella: +ve (blue color)

- Shigella: -ve (green color)

- Klebsiella: +ve (blue color)

Phenol Red Mannitol

- Inoculate the medium by stabbing into the butt and streaking the surface of the slant.  

- The inoculum incubated at 35 degree Celsius for 24 hours.

- A positive test consists of a color change from red to yellow, indicating a pH change to acidic.

- No reagents

- Salmonella: Gas production, acid production; +ve reaction (yellow color)

- Shigella: acid production; +ve reaction (yellow color)

- E.coli: acid productions; +ve reaction (yellow color), gas production