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

Microbiological Analysis of Food and Water

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, particularly 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 detects acid production from lactose fermentation.

• Ferric Citrate and Sodium Thiosulfate: Detect hydrogen sulfide (H2S) production, which is characteristic of some Salmonella species.

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 (do not produce H2S).

• Fecal Coliforms (e.g., Escherichia coli): Pink/red colonies (due to lactose fermentation and acid production).

Example: A water sample yielding colorless colonies with black centers on S-S agar suggests the presence of Salmonella.

Phenol Red - Lactose Broth in the MPN Method

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

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

• Lactose: Substrate for coliforms; fermentation produces acid and gas.

• Application: Tubes showing yellow color and gas production are considered positive for coliforms.

Example: After incubation, tubes with yellow broth and gas in the Durham tube indicate coliform presence.

Definition: Durham Tube

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

• Purpose: Collects gas produced by bacteria, visible as a bubble.

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

Example: Presence of a bubble in the Durham tube indicates gas formation 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 reliability of coliform detection, especially when bacterial concentrations are low.

• Serial Dilutions: Allows estimation of bacterial numbers across a range of concentrations.

• Statistical Basis: Using multiple volumes increases the probability of detecting coliforms.

• Example: Typical MPN setup uses 10 mL, 1 mL, and 0.1 mL volumes in triplicate tubes.

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

Determining MPN Using a Reference Table

The MPN value is determined 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.

• Step 2: Use the MPN table to find the most probable number based on the pattern of positives.

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

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

Sample MPN Table (Purpose: Statistical Estimation of Coliforms)

10 mL	1 mL	0.1 mL	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 are standardized for water quality testing.