Antimicrobial Inhibitor Test Agar pH 7.2

Intended Use

Antimicrobial Inhibitor Test Agar pH 7.2 is recommended for residual analysis of antimicrobial components in meat and organ samples, using Bacillus subtilis (ATCC 6633) as test organism.

Composition**

Final pH (at 25°C): 7.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 25.34 grams of dehydrated medium 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. Cool to 45-50°C. Mix 1 ml of Bacillus subtilis spore suspension per liter of sterile and cooled (45-50°C) medium. Add 50µg of trimethoprim per litre of medium. Mix thoroughly and pour into sterile Petri plates.

Principle And Interpretation

In addition to washing, treatments with antimicrobial compounds such as chlorine and organic acids are used to sanitize muscle foods. Various concentrations and the degree of effectiveness of the concentrations of these antimicrobial compounds have been reported. Antimicrobial Inhibitor Test Agar pH 7.2 is recommended for residual analysis of antimicrobial components in meat and organ samples, using Bacillus subtilis (ATCC 6633) as test organism by agar diffusion procedure and EEC Four-Plate-Test.

Agar Diffusion procedure: Small slices of the meat sample are placed on the seeded Test agar plates and incubated. Antimicrobial components, if present in the sample diffuses into the medium and inhibit the growth of test organisms causing growth free inhibition zone. This test is repeated with all the three Antimicrobial Inhibitor Test Agar with three different pH i.e. M1631 (pH 6.0), M1601 (pH 7.2) and M1632 (pH 8.0). Different antibiotic to be analysed such as penicillin, streptomycin and sulphonamide have different pH range for optimal activity for example penicillin G is active optimally at pH 6.0 and streptomycin at pH 8.0 and sulphonamide at pH 7.2. Trimethoprim (50µg) is added to increase the sensitivity of test agar for residual sulphonamide from meat/organ samples (1).

For EEC Four-Plate-Test method: Molten and cooled antimicrobial inhibitor test agar is seeded with test organisms, Bacillus subtilis spores suspension (2), mixed well and dispensed into Petri plates. In one half of the seeded plate, aseptically clump small piece (2-8mm) of meat or organ sample at proper distance and in second half of the plate, place test discs with standard antibiotic to be analyzed, as control. Use disc with 0.5µg sulphonamide as standard.

Incubate the plates for 18-24 hours at 30°C for Bacillus subtilis. After incubation measure the zone of inhibition. The inhibition zone between tissue section edge and growth limit of test organism is measured. The zone of at least 2 mm is regarded as positive and less than 2mm (12mm) is considered doubtful. The standard disc should display minimum 6 mm zone of inhibition.

Antimicrobial Inhibitor Test Agar pH 7.2 has peptone which serve as source for carbon, nitrogen and growth factors for the growth of organisms. Sodium chloride helps to maintain osmotic balance in the medium and phosphate salt offers buffering capacity to medium.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.3% Agar gel.

Colour and Clarity of prepared medium: Light amber coloured clear to slightly opalescent gel forms in Petri plates.

Reaction: Reaction of 2.53% w/v aqueous solution at 25°C. pH : 7.2

pH: 7.20

Cultural Response

M1601: Cultural response and zone of inhibition was observed using B. subtilis after an incubation at 30ºC for 18-24 hours

Storage and Shelf Life

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

Reference

1. Gudding R:1976 improved bacteriological method for the detection of sulphonamide residues info. Acta. Vet. Scand., 17 : 458 464 (1976).
2. Ferrini, A. M.; Mannoni, V., Aurdi P. Combined plate microbal assay (CPMA). Food additives and Contaminants, 23(1);16-24. 2006