ANTIBIOTIC ASSAY MEDIUM M – AOAC

Antibiotic Assay Medium M is formulated in accordance with AOAC for the microbiological assay of Lasalocid in feeds, using Bacillus subtillis (ATCC 6633) as the test organism. Prepare slant culture of Bacillus subtillis (ATCC 6633) on Assay Medium No. 1 and incubate for 16-24 hours at 37?C. Wash the growth with sterile distilled water and transfer it to surface of Assay Medium No. 32 and incubate at 37?C for 7 days. Wash the growth with sterile distilled water. Heat to 65?C for 30 minutes in water bath. Centrifuge, decant the supernatant and resuspend the cells. Repeat this for 3 minutes in water bath. Dilute suspension with sterile distilled water (1 + 50) to read 20%T on sprectrophotometer at 530 nm before use.
Use single inoculated agar layer. Optimum concentration of suspension of Bacillus subtillis is determined prior to assay to be added to Medium M to obtain inhibition zone of adequate size (17.5 ? 2.5 mm with 1.0 ?g/ml). For actual assay add appropriate amount of suspension to sterile, molten medium M (pH 6.0). Mix and add 6 ml to each plate. Prepare plates 2.5 3 hours before use. Weigh 1.0 g premix. Transfer to flask and add 100 ml methanol. Shake vigorously for 3 minutes and dilute with methanol. Dilute 4 ml of this to 100 ml methanol. Further dilute 3 ml with 22 ml methanol and water to 100 ml (1 ml= ca/?g lasalocid Na/ml 25% methanol). Prepare final concentration of feed to 0.0075%. For more details refer AOAC.
Using lasalocid, sodium obtain standard response line, assay solution. Place cylinders on each plate and alternatively fill with reference concentration and other standard concentration. Incubate at 35-36?C. Calculate zone diameters of L (Low concentration giving measurable zone) and H (Highest concentration) of standard response line and connect with straight line. This corrected reference point is used for sample calculations. Average the 9 readings of reference concentration and 9 readings of assay solution. If assay solution gives larger average than reference concentration, add difference between them to reference point on standard response line. If the assay solution gives lower average than reference concentration, subtract the difference from reference point. Using the corrected value of assay solution, amount of antibiotic is determined.

for microbiological assay of Lasalocid using Bacillus subtilis