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