Laboratory procedure of bacterial inhibition assay
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This document describes the procedure for performing a bacterial inhibition assay to detect phenylalanine levels. The assay uses Bacillus subtilis spores seeded into an agar plate. An inhibitor is added to suppress bacterial growth unless phenylalanine is present. Blood samples are punched into the plates, and any growth of bacteria around the samples indicates the presence of phenylalanine. The procedure involves preparing the agar plates, generating worklists, planting the samples, incubating the plates overnight, and measuring any growth rings to interpret phenylalanine levels. Precautions are taken to avoid contamination and allow for accurate diffusion and reading of results.
Laboratory procedure of bacterial inhibition assay
- 1. BACTERIAL INHIBITION ASSAY By: MARY JEAN D. SOMCIO, RMT
- 2. PRINCIPLE: The Guthrie Bacterial Inhibition Assay for phenylalanine is a test in which a phenylalanine-free minimal agar base is seeded with Bacillus subtilis spores. An inhibitor, β-2-thienylalanine, is added to suppress growth of B. subtilis. The action of the inhibitor is blocked by the presence of blood disk containing phenylalanine. The size and density of the growth zone is directly dependent on the amount of phenylalanine present.
- 3. MATERIALS: Overhead Projector Digital caliper 3mm Blood spots Hot Plate Stirrer Weighing Scale Erlenmeyer Flask Worklist Incubator Hand puncher
- 4. REAGENTS: Agar Plate Working Demains Reagent Bacillus subtilis Working Demains spores Salt Solution Inhibitor β-Lactamase Agar powder
- 5. PROCEDURE: Working Demains Preparation Salt Solution (1Liter Preparation) (1Liter Preparation) REAGENT WEIGHT (GRAMS) REAGENT WEIGHT (grams) Magnessium Sulfate 10 Glucose 100 Manganese Chloride 1 Di-Potassium 150 Hydrogen Phosphate 60% Ferric Chloride Solution 0.69 mL Potassium Di- 50 Hydrogen Phosphate 22 mL Ammonium Chloride 25 Ammonium Nitrate 5 Sodium Sulfate 5 L-Glutamic Acid 5 L-Asparagine 5 L- Alanine 2.5
- 6. PROCEDURE: A. Prepare Agar Plates # OF AGAR DISTILLED WORKING BACILLUS INHIBITOR β- PLATES (g) H2O (mL) DEMAINS (mL) SUBTILIS (mL) (µL) LACTAMASE (µL) 1 1.5 140 16 2 100 100 2 3 280 32 4 200 200 3 4.5 420 48 6 300 300 4 6 560 64 8 400 400 5 7.5 700 80 10 500 500 6 9 840 96 12 600 600 7 10.5 980 112 14 700 700 8 12 1120 128 16 800 800 9 13.5 1260 144 18 900 900 10 15 1400 160 20 1000 1000
- 7. PROCEDURE: A. Prepare Agar Plates 1. Weigh PKU agar (PHE-free minimal agar base) into an Erlenmeyer’s flask. 2. Add distilled water and mix by using the magnetic stirrer.
- 8. PROCEDURE: A. Prepare Agar Plates 3. Thaw the other components of the PHE agar -- B. Subtilis spores, inhibitor and β- Lactamase. 4. Bring working demains to room temperature.
- 9. PROCEDURE: A. Prepare Agar Plates 5. Dissolve agar by boiling. Care MUST be taken when agar starts to boil to avoid spillage. 6. Add Working Demains Solution.
- 10. PROCEDURE: A. Prepare Agar Plates 7. 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.
- 11. 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.
- 12. PROCEDURE: A. Prepare Agar Plates 11. 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.
- 13. PROCEDURE: A. Prepare Agar Plates 13. Allow agar to set for 30 minutes. 14. Store agar plates at 4°C in an upright position.
- 14. 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.
- 15. 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.
- 16. 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.
- 17. 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.
- 18. 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)
- 19. BE AWARE of INFECTIONS & INFECTIOUS DISEASES