Laboratory procedure of bacterial inhibition assay
BACTERIAL INHIBITION ASSAY By: MARY JEAN D. SOMCIO, RMT
PRINCIPLE:The Guthrie Bacterial InhibitionAssay for phenylalanine is a test inwhich a phenylalanine-freeminimal agar base is seeded withBacillus subtilis spores. Aninhibitor, β-2-thienylalanine, isadded to suppress growth of B.subtilis. The action of the inhibitoris blocked by the presence of blooddisk containing phenylalanine. Thesize and density of the growthzone is directly dependent on theamount of phenylalanine present.
MATERIALS: Overhead Projector Digital caliper 3mm Blood spotsHot Plate Stirrer Weighing Scale Erlenmeyer Flask Worklist Incubator Hand puncher
REAGENTS: Agar Plate Working Demains Reagent Bacillus subtilis Working Demains spores Salt Solution Inhibitor β-Lactamase Agar powder
PROCEDURE:A. Prepare Agar Plates1. Weigh PKU agar (PHE-free minimal agar base) into an Erlenmeyer’s flask. 2. Add distilled water and mix by using the magnetic stirrer.
PROCEDURE:A. Prepare Agar Plates3. Thaw the other components of the PHE agar -- B. Subtilis spores, inhibitor and β- Lactamase. 4. Bring working demains to room temperature.
PROCEDURE:A. Prepare Agar Plates5. Dissolve agar by boiling. Care MUST be taken when agar starts to boil to avoid spillage. 6. Add Working Demains Solution.
PROCEDURE:A. Prepare Agar Plates7. Heat until the solution turns red orange to golden brown. Remove from heat and equilibriate temperature between 55°C – 70°C. 8. Dilute with 50mL Distilled water the Bacillus subtilis spores.
PROCEDURE: A. Prepare Agar Plates 9. Add Bacillus subtilis spore suspension to the flask. 10. Add inhibitor (β-2-thienylalanine) and β-Lactamase working solution. Mix well with the use of the magnetic stirrer.
PROCEDURE:A. Prepare Agar Plates11. Pour approximately 150mL of agar into the plastic plates. See to it that there is no bubbles in the agar. 12. Check to see that plate is level so that the agar is of uniform thickness.
PROCEDURE:A. Prepare Agar Plates13. Allow agar to set for 30 minutes. 14. Store agar plates at 4°C in an upright position.
PROCEDURE:B. Test Proper (Day 1)1. Remove agar plates to be used for the day from the refrigerator at least an hour before planting to allow diffusion at room temperature. 2. Generate worklists.
PROCEDURE:B. Test Proper (Day 1)3. Punch standards and unknown samples using PE Multipuncher or manual puncher. 4. Plant the blood spots on the agar plates. Make sure to follow the exact sequence on the worklist.
PROCEDURE:B. Test Proper (Day 1)5. Place the plates in the incubator UPSIDE DOWN to prevent moisture from gathering on the agar surface. Incubate at 37°C for 16-18 hours.
PROCEDURE:B. Test Proper (Day 2)6. Remove plates from the incubator. Read the plates by first measuring the growth rings of the standards and then the samples. Compare the growth ring of each sample to the growth ring of 200µmol/L phenylalanine standard.If the growth ring is less than that of the 200µmol/L PHE standard, then no further action is required as long as there are colonies/growth present around and under the blood spot disc.
PROCEDURE: B. Test Proper (Day 2) Growth on the agar plates may vary. It may be:LP = Large growth on AB = Absence of growth or plate (>200µmol/L) patient might be on Antibiotic TAR = Target-shaped (abnormal growth)