ANTIMICROBIAL

SUSCEPTIBILITY TEST:
Diffusion test procedure
GROUP 4

PURPOSE:
To show in-vitro sensitivity of
different bacteria to selected
antibiotics.

 MATERIALS:
1.
2.
3.
4.
5.
6.
7.

Stock cultures of different bacteria

Mueller Hinton Infusion Agar
Antibiotic disc
Sterile cotton swab
Sterile normal saline solution (NSS)
Forceps, inoculating wire loop, alcohol lamp
Barium sulfate comparison standard (0.5 ml
of 0.048M BaCl2 to 99.5 ml of 0.36N
H2SO4) McFarland Barium Sulfate no. 0.5

Mueller Hinton Infusion Agar

the most suitable medium for routine susceptibility testing for the following
reasons:

a) It gives satisfactory
growth of the majority
of rapidly growing
pathogens
b) It is not antagonistic to
the major antimicrobial
agents to be tested
c) It is isotonic with blood,
can be used to perform
susceptibility tests on
certain fastidious
organisms that cannot
grow on
unsupplemented
medium

d. It contains starch which absorbs

toxins from bacteria so that they
cannot interfere with antibiotics.
e. It is a loose agar which allows for
better diffusion of antibiotics which
results in a truer zone of inhibition.

Organism:
1. Escherichia coli
2. Staphylococcus aureus
3. Salmonella typhi
4. Shigella dysnteriae
5. Pseudomonas aeroginosa
6. Proteus vulgaris
7. Klebsiella pneumoniae
8. Salmonella paratyphi A
9. Enterobacter cloacae
10. Salmonella enteritidis

Antibiotic used:
1.
2.
3.
4.
5.

Ampicillin
Chloramphenicol
Gentamicin
Penicillin G
Trimenthoprimsulfamethoxazole
(SXT)

PROCEDURE
1. Using an inoculating
loop, obtain an
inoculum from the
stock culture and
suspend directly into
a small volume of
sterile NSS. Repeat
this produce until the
turbidity matches
that of the BaSO4
comparison
standard.

2. Dip in sterile nontoxic swab on an

applicator stick into the standardized
suspension. Remove excess fluid by
pressing and rotating the swab against the
side of the tube above the fluid level.
Streak the swab evenly in three directions
over the entire surface of the agar plate,
turning 60 degrees each time to obtain a
uniform inoculum. Make a final sweep to
the agar to the agar rim with the cotton
swab. Allow the plate to stand on flat
Surface 3-5 minutes (but not more than 15
minutes)

3. Within 15 minutes after the plates are

inoculated, apply antibiotic impregnated
disc to the surface of the inoculated plates
either with a mechanical dispenser or by
hand with sterile forceps. Gently press
down all disc onto the agar with forceps or
an inoculating needle to insure complete
contact with the agar surface. The special
arrangement of the disc should be such
that they are no closer than 15 mm to the
edge of the plate and far enough to
prevent overlapping zones of inhibition (at
least 20 mm apart)

4. Within 15 minutes after the disc are

applied, the plates are inverted and
placed in an incubator at 37 degrees
Celsius for 16 18 hours
5. At the end of the 16 18 hours
incubation, examine the plates and
measure to the nearest mm the zone
of complete inhibition.

READING AND INTERPRETATION:

The zone diameters for individual
antimicrobial agents are translated
into SUSCEPTIBLE, INTERMEDIATE, or
RESISTANT categories by referring to
an interpretative table. The end point
of all reading systems is complete
inhibition of growth, disregarding,
tiny colonies which can be detected
only by very close scrutiny.

 Susceptibility
- This category indicates that the antimicrobial
agent in question maybe an appropriate choice for
the infection caused by the bacterial isolate tested
Intermediate
- The antimicrobial agent may still be effective
against the tested isolate but usually at higher
concentration
Resistant
- This category indicates that the microbial agent
in question may not be an appropriate choice for
treating the infection caused by the bacterial
isolate tested

In clinically urgent situations,

preliminary readings can often be
obtained within 5 or 6 hours after
incubation but the plates should
always be reincubated, and final
report should be withheld until a full
16 18 hours have elapsed