Introduction

Shigella are Gram-negative, non-motile bacteria responsible for shigellosis, a severe intestinal infection characterized by diarrhea, fever, and stomach cramps. The disease spreads primarily through contaminated food, water, or poor hygiene practices.

The ISO 21567 standard provides a horizontal method for the detection of Shigella species in food and animal feed, including milk and dairy products.

Detection of Shigella in dairy products is extremely important because these bacteria have a very low infectious dose, meaning even small amounts of contamination can cause disease outbreaks.

Principle of the test method

The method is a qualitative culture-based detection method designed to determine whether Shigella is present or absent in a food sample.

Key stages include:

1. Pre-enrichment in a non-selective broth to allow recovery of injured bacteria.
2. Selective enrichment in Shigella broth to promote growth of Shigella while suppressing competing bacteria.
3. Isolation on selective agar plates.
4. Biochemical confirmation of suspect colonies.
5. Serological confirmation using antisera.

Materials & Equipment

Consumables

• Sterile sample containers or stomacher bags
• Sterile pipettes and pipette tips
• Buffered peptone water (pre-enrichment medium)
• Shigella enrichment broth (Shigella broth)
• Selective agar plates such as:
  • XLD agar
  • MacConkey agar
  • Hektoen Enteric agar
• Biochemical test media (TSI agar, urease reagents)
• Sterile inoculating loops

Equipment

• Incubator capable of 37 °C ±1 °C
• Stomacher or homogenizer
• Autoclave
• Colony counter
• Biosafety cabinet (recommended)

Step-by-Step Test Procedure

1. Sample Preparation

1. Weigh 25 g of solid food or measure 25 mL of milk or liquid dairy sample.
2. Place the sample aseptically into a sterile stomacher bag.
3. Add 225 mL of buffered peptone water.
4. Homogenize using a stomacher for 1–2 minutes.

This produces the initial suspension used for enrichment.

2. Pre-Enrichment

1. Incubate the homogenized suspension at 37 °C for 16–20 hours.
2. This stage allows injured or stressed Shigella cells to recover before selective enrichment.

3. Selective Enrichment in Shigella Broth

1. Transfer 1 mL of the pre-enrichment culture into 10 mL of Shigella broth.
2. Mix gently to ensure uniform distribution.
3. Incubate at 37 °C for 18–24 hours.

Purpose of Shigella broth:

• Enhances growth of Shigella species.
• Suppresses competing Gram-positive bacteria and many other enteric microorganisms.

Selective enrichment increases the probability of detecting Shigella when present in low numbers.

4. Isolation on Selective Agar

Using a sterile loop:

1. Streak the enrichment culture onto selective agar plates such as:
   • XLD agar
   • MacConkey agar
   • Hektoen Enteric agar
2. Incubate plates at 37 °C for 18–24 hours.

5. Colony Identification

Typical Shigella colony appearance:

XLD agar

• Red colonies with no black center

MacConkey agar

• Pale or colorless colonies (non-lactose fermenters)

Hektoen Enteric agar

• Green colonies without black precipitate

Suspected colonies should be selected for confirmation.

6. Confirmation Tests for Shigella

Triple Sugar Iron (TSI) Agar Test

Purpose

The TSI test determines the bacterium’s ability to ferment sugars (glucose, lactose, sucrose) and produce gas or hydrogen sulfide (H₂S). This helps distinguish Shigella from other enteric bacteria such as Salmonella.

Procedure

1. Pick a suspected colony from the selective agar plate.
2. Inoculate the TSI agar slant by stabbing the butt and streaking the slant.
3. Incubate at 37 °C for 18–24 hours.

Typical Result for Shigella

• Alkaline slant (red)
• Acid butt (yellow)
• No gas production
• No black precipitate (H₂S negative)

Interpretation

Shigella ferments glucose only, producing acid in the butt but not in the slant because lactose and sucrose are not fermented.

Urease Test

Purpose

The urease test determines whether the bacterium produces the enzyme urease, which breaks down urea into ammonia.

Procedure

1. Inoculate a urease medium with the suspect colony.
2. Incubate at 37 °C for up to 24 hours.

Expected Result for Shigella

• Negative result
• Medium remains yellow or unchanged

Interpretation

Many bacteria such as Proteus are urease positive, while Shigella is typically urease negative, helping to differentiate them.

Motility Test

Purpose

The motility test determines whether bacteria are capable of movement using flagella.

Procedure

1. Inoculate semi-solid motility medium by stabbing with a sterile needle.
2. Incubate at 37 °C for 18–24 hours.

Expected Result for Shigella

• Non-motile
• Growth remains along the stab line

Interpretation

This test is important because most Shigella species are non-motile, while many related bacteria (e.g., Salmonella) are motile.

Indole Test

Purpose

The indole test checks whether the bacterium can break down tryptophan into indole using the enzyme tryptophanase.

Procedure

1. Grow the bacterium in tryptone broth.
2. Add Kovac’s reagent after incubation.

Expected Result for Shigella

• Usually indole negative, although some species may be weakly positive.

Interpretation

Indole results help differentiate among members of the Enterobacteriaceae family.

Serological Confirmation (Slide Agglutination Test)

Purpose

Serological testing confirms the bacterium belongs to the genus Shigella and may identify its serogroup.

Procedure

1. Place a drop of saline on a clean slide.
2. Mix a small portion of the bacterial colony into the drop.
3. Add specific Shigella antisera.
4. Gently rock the slide and observe.

Expected Result

• Agglutination (clumping) indicates a positive reaction.

Interpretation

Agglutination occurs because antibodies in the antisera bind to antigens on the bacterial surface. This confirms the organism as Shigella.