TN Agar

Principle And Interpretation

Members of the genus Vibrio are defined as gram-negative, asporogenous rods that are straight or have a single rigid curve. Three species of Vibrio, namely Vibrio cholerae, Vibrio parahaemolyticus and Vibrio mimicus are well-documented human pathogens.

V. cholerae, the type species of the genus Vibrio is the causative agent of cholera outbreaks and epidemics.

V. cholerae can be differentiated from other Vibrio species except V. mimicus, because of its obligate requirement for sodium ions (Na+).TN Agar is formulated as per APHA (1) for cultivation of Vibrio species from foods.

Casein enzymic hydrolysate provides nitrogenous, carbonaceous compounds, sulphur, vitamin B complex and other essential growth nutrients. Sodium chloride improves the selectivity of the medium.

Weigh 25 grams of food sample such as seafood or vegetable and blend or cut into small pieces into two jars. To one jar add 225 ml Alkaline Peptone Water (M618) and to another jar add 225 ml Gelatin Phosphate Salt Broth. Incubate at 35 ± 2°C for 6 to 8 hours. Transfer a loopful from surface growth of each broth culture to two plated media, i.e. TCBS Agar (M189) and Gelatin Phosphate Salt Agar (M921), and incubate at 35 ± 2°C for 18-24 hours. Subculture 3-4 colonies from each plating medium to TN Agar. Growth from TN Agar is further confirmed by inoculating Kligler Iron Agar slants (M078) and stabbing the butt. After incubation, V. cholerae cultures will have an alkaline (red) slant and an acid (yellow) butt, no gas, and no blackening (H2S production) in the butt. Presumptive test for suspected strains of V. cholerae from TN Agar is carried out by using string test (2).