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BACTERIAL INHIBITION ASSAY
      By: MARY JEAN D. SOMCIO, RMT
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.
MATERIALS:

                                      Overhead Projector


                    Digital caliper
                                                           3mm Blood spots



Hot 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:
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
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
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.
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.
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.
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.
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 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.
PROCEDURE:
A. Prepare Agar Plates
13. 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)
BE AWARE of
INFECTIONS
     &
 INFECTIOUS
  DISEASES

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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