Kundrat Agar, Modified

Kundrat Agar, Modified

Kundrat Agar, Modified is recommended for the routine qualitative detection of residues of antibiotics, sulfonamides and other chemotherapeutic agents in meat and other food stuffs derived from animals.

Composition**

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 40.52 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Mix well and pour into sterile Petri plates or as desired.

Principle And Interpretation

Kundrat Agar, Modified is recommended for rapid or long term test defined for qualitative detection of residues of antibiotics, sulfonamides and other chemotherapeutic agents from meat and food samples. Cleaning agents, disinfectants and preservatives are not covered by this test. This method was developed by Kundrat (1,2) and is performed in the form of an agar diffusion test using a spore suspension of Bacillus stearothermophilus.

This medium contains peptone, gelatin, starch and glucose which provides nitrogen, carbon compounds and other essential growth nutrients to Bacillus stearothermophilus. The test organism ferments glucose and sucrose in the medium to form acid that causes bromocresol purple to change its colour from purple to yellow. Inhibitory action is seen as clearance around the inoculation zone and retains the original violet colour of the indicator.

Test procedure:

After autoclaving the medium, cool to 50-60°C. To each 200 ml of medium add contents of 1 ampoule (LA420) of Bacillus stearothermophilus spore suspension, mix and pour a volume of 5 ml into 90 mm Petri plates (according to the German DIN 10182 Part 1).

The filter paper discs with a diameter of 6 mm are soaked with liquid specimen or placed on organ (kidney, liver) or muscle sections. The discs are then gently pressed onto the surface of the culture medium. A maximum of 6 discs are placed on each plate. It is recommended to carry out preincubation of plates for 135 minutes at 65°C. Stacking of plates should be avoided so as to ensure even temperature exposure.

Either of the two methods are followed to carry out the test:

1. Rapid test, 45 minutes incubation: On placing the discs on the preincubated plates, incubate further for 45 minutes at 65°C without prediffusion.

   When performing rapid test, growth of the test organism is enhanced by preincubating the inoculated plates, the inhibition zones then appear more rapidly after application of the samples.

Results of rapid test can be recorded after 15-25 minutes incubation in the medium which is otherwise turbid as a result of spore growth. After the 45 minutes incubation, the inhibition zones become more distinct due to the change in colour of culture medium. Formation of inhibition zones is considered as a positive result.

2. Long term test, 3 hours incubation: The plates are not preincubated. On placing the filter paper discs on the plates, incubate further for 3 hours at 65°C without prediffusion.

   In case of long term test, only those inhibition zones greater than 10 mm is considered as positive. If a distinct colour change has not occurred after 45 minutes or 3 hours, it can be further incubated.

Quality Control

Appearance
Cream to yellow coloured with green tinge, homogeneous free flowing powder

Gelling
Firm, comparable with 1.0% Agar gel.

Colour and Clarity of prepared medium
Light purple coloured, clear to slightly opalescent gel forms in Petri plates

Reaction
Reaction of 4.04% w/v aqueous solution at 25°C. pH : 6.8±0.2

pH
6.60-7.00

Cultural Response

M1360A: Cultural characteristics observed after an incubation at 65°C for 18-24 hours.

Storage and Shelf Life

Store below 30°C in tightly closed container and prepared medium at 2-8°C.Use before expiry period on the label.

Reference

1. Kundrat W., 1968, Methoden zur Bestimmung von Antibiotika-Ruckstanden in tierischen Produkten. Z. Anal. Chem .;624-630.
2. Kundrat W., 1972:45- Minuten-Schnellmethode zum mikrobiologischen Nachweis von Hemmstoffen in tierischen Produkten.- Fleischwirtsch., 52; 485-487.
3. German DIN 10182 Part I.