Microbiological Analysis of Food and Water: Salmonella-Shigella Agar and MPN Method

Food and Water Microbiology

Salmonella-Shigella (S-S) Agar: Components and Purpose

Salmonella-Shigella (S-S) agar is a selective and differential medium used to isolate and identify Salmonella and Shigella species from clinical and environmental samples, such as food and water.

• Peptone: Provides essential nutrients for bacterial growth.

• Lactose: Differentiates lactose fermenters (coliforms) from non-fermenters (Salmonella and Shigella).

• Bile Salts: Inhibit growth of Gram-positive bacteria, allowing selective growth of Gram-negative enterics.

• Sodium Citrate: Further inhibits Gram-positive bacteria and some coliforms.

• Brilliant Green: Suppresses growth of most coliforms except Salmonella and Shigella.

• Neutral Red: pH indicator that turns red in acidic conditions (lactose fermentation).

• Sodium Thiosulfate and Ferric Citrate: Detect hydrogen sulfide (H2S) production; colonies producing H2S appear black.

Example: S-S agar is commonly used in water testing labs to screen for pathogenic enteric bacteria.

Colony Color Differentiation on S-S Agar

The color and appearance of colonies on S-S agar help distinguish between Salmonella, Shigella, and fecal coliforms.

• Salmonella: Colorless colonies with black centers (due to H2S production).

• Shigella: Colorless colonies without black centers (no H2S production).

• Fecal Coliforms (e.g., Escherichia coli): Pink/red colonies (lactose fermentation causes acid production, turning neutral red indicator).

Example: A water sample showing pink colonies on S-S agar indicates the presence of coliforms, not Salmonella or Shigella.

Phenol Red - Lactose Broth in the MPN Method

The Most Probable Number (MPN) method is used to estimate the concentration of viable coliforms in water samples. Phenol red - lactose broth is a differential medium used in this method.

• Phenol Red: pH indicator; turns yellow in acidic conditions (lactose fermentation).

• Lactose: Substrate for fermentation by coliforms.

• Purpose: Detects lactose fermentation by coliforms, indicated by color change and gas production.

Example: Tubes that turn yellow and produce gas after incubation indicate positive coliform presence.

Definition: Durham Tube

A Durham tube is a small, inverted glass tube placed inside a culture tube to detect gas production during fermentation.

• Function: Collects gas produced by bacteria fermenting lactose, visible as a bubble.

• Application: Used in MPN and other fermentation tests to confirm gas production.

Example: A Durham tube with a visible bubble after incubation confirms gas production by coliforms.

Use of Different Volumes in MPN Tubes

Different volumes of water sample are used to inoculate MPN tubes to increase the accuracy and statistical reliability of coliform detection.

• Reason: Allows detection of coliforms across a range of concentrations.

• Typical Volumes: 10 mL, 1 mL, and 0.1 mL are commonly used.

• Statistical Basis: Multiple dilutions help estimate the most probable number of organisms present.

Example: If only the largest volume tubes are positive, coliforms are present at low concentration.

Determining MPN Using a Reference Table

The MPN (Most Probable Number) is calculated by comparing the number of positive tubes at each dilution to a statistical reference table.

• Step 1: Record the number of positive tubes for each volume/dilution.

• Step 2: Use the MPN table to find the corresponding MPN value based on the pattern of positives.

• Step 3: Report the MPN per 100 mL of sample.

Example: If 3 tubes at 10 mL, 2 at 1 mL, and 1 at 0.1 mL are positive, the MPN table provides the estimated coliform count.

Sample MPN Table (Purpose: Statistical Estimation of Coliforms)

10 mL Positive	1 mL Positive	0.1 mL Positive	MPN/100 mL
3	3	3	110
3	2	1	15
2	1	0	3
1	0	0	0.3
0	0	0	0

Additional info: MPN tables are based on probability theory and provide statistical estimates rather than exact counts.