This document discusses antimicrobial sensitivity testing. It provides information on: 1) Antimicrobial resistance has led to increased morbidity, mortality, and healthcare costs. Inappropriate antibiotic usage by physicians and the public fuels resistance. 2) Sensitivity testing determines how effective antibiotic therapy is against bacterial infections. It assists clinicians in choosing effective drugs and controlling antibiotic use. 3) Common methods include diffusion tests like Kirby-Bauer that use discs impregnated with antibiotics to measure inhibition zones, and dilution methods that determine the minimum inhibitory concentration. Both qualitative and quantitative techniques exist.

1. Antimicrobial Sensitivity
Testing
Ms. Megha Chaudhary
Department Of Medical Microbiology
BIU, Bareilly

2. Resistance to antimicrobial agents (AMR) has resulted in
morbidity and mortality from treatment failures and increased
health care costs.
Appropriate antimicrobial drug use has unquestionable benefit,
but physicians and the public frequently use these agents
inappropriately.
The availability of antibiotics over the counter, despite
regulations to the contrary, also fuel inappropriate usage of
antimicrobial drugs in India.

3.  Antibiotics are very commonly used substances to eradicate bacterial
infections by bacteriostatic or even bactericid effect.
 They act at a very specific stage (target), although other less important
or secondary interactions can occur.
 We studied the interaction of three antibiotic families (beta-lactamins,
aminosides, rifampicin) with bacterial cell.
 Penicillin disturbs the cell wall synthesis and more accurately the
glycopeptide (or murein) formation, a substance giving rigidity or
shape to bacteria.
 The study of the action mechanism of these antibiotics enables us
to show the action specificity of these products in the bacteria.

5. a) Sensitive: Ordinary recommended dose achieve a systemic
concentration which is > M.I.C of the antimicrobial agent tested
against the isolate.
(b) Intermediate sensitive: Isolate is amenable to treatment with
a larger dose of the antimicrobial agent, so that concentration at
the site of infection is above the breakpoint value to tackle with
high M.I.C value of the isolate.
(c) Resistant: M.I.C value of the isolate is so high that the required
concentration is unachievable at the site of infection.
IMPORTANT TERMS

6. Antibiotic sensitivity test: A laboratory test
which determines how effective antibiotic therapy is
against a bacterial infections.
Antibiotic sensitivity testing will control the use of
Antibiotics in clinical practice.
Testing will assist the clinicians in the choice of drugs
for the treatment of infections.

7. Diffusion Dilution
Diffusion
& Dilution
Kirby-Bauer
Method
Stokes
Method
Tube
dilution
Agar
dilution
E- Test
Qualitative Methods Quantitative Methods
ANTIMICROBIAL SUSCEPTIBILITY MANNUAL METHODS AUTOMATED METHODS

8. DIFFUSION METHOD
A filter disc containing measured quantity of drugs is put on a solid
medium that has been seeded with the test bacteria and after
overnight incubation, plate is read for appearance of zone of
inhibition around Ab discs and reported as sensitive/ intermediate /
resistant.

10. (A) KIRBY BAUR METHOD
Principle: Production of inhibitory zone of a recommended size as
recommended in Kirby Bauer Chart in a lawn culture of an
isolate around an antimicrobial agent impregnated filter paper
disc indicates sensitivity.
Medium: Nature of the medium affects the result of susceptibility
test. A totally defined medium which support reproducible
result is yet to be formulated with satisfaction. Factors related
to the media affecting the result:
i. Quality of agar — affects diffusibility of antimicrobials.
ii. Quality of protein hydrolysate — Varies from batch to batch
affecting the result.
iii. Final pH of the stored medium — affects the action of
antimicrobial.

11. iv. Cationic (Mg++ and Ca++) concentration — affects the result.
v. Presence of thymidine and thymine at higher concentration
antagonizes the action of antimicrobials (sulfonamides) which
are inhibitors of folate metabolism.
vi. Hydration, unless optimum, might influence the diffusibility of
antimicrobials.
Considering the above facts, it has been recommended by
CLSI, the international agency for quality control of
antimicrobial susceptibility test, to use ‘Mueller-Hinton Agar’
in Kirby-Bauer method for non-fastidious bacteria.

12. To prepare the inoculum from the primary culture plate, a loop
is touched on the tops of each of 3–5 colonies, of similar
appearance, of the organism to be tested.
Procedure

13. Growth is transfered to a tube of peptone water.

14. Preparation of inoculum:
Bacteria is picked up by a loop
touching the tip of similar looking 5
colonies.
A suspension is to be made in
peptone water.
The opacity is adjusted to
Macfarland opacity tube 0.5.
This will produce a
concentration of 1.5 x 107/ml
bacteria.
Macfarland 0.5 capacity tube is
prepared by mixing 0.5 ml
concentrated H2SO4 with 99.5 ml of
saturated solution of BaCl2.

15. Plates are inoculated by dipping a sterile swab into the
inoculum.
Excess inoculum is removed by pressing and rotating the
swab firmly against the side of the tube above the level of the
liquid.

16. Streak the swab all over the surface of the medium three
times, rotating the plate through an angle of 60⁰ after each
application.
Finally, pass the swab round the edge of the agar surface.

17. Leave the inoculum to dry for a few minutes at room
temperature with the lid closed.
The antibiotic discs may be placed on the inoculated
plates using
sterile forceps.
a template.
a sterile needle tip.
antibiotic disc dispenser.

18. Media recommended for non fastidious bacteria:
(a) N. Gonorrhoeae: G.C. Agar supplemented with 10% (final
concentration) saponin lysed horse/sheep blood.
(b) Fastidious anaerobes: Wilkin-Chalgren Agar (Oxoid)
supplemented with 5% (final concentration) saponin lysed
horse/sheep blood.
(c) Other fastidious bacteria: Isosensitest agar (oxoid)
supplemented with 5% saponin lysed horse/sheep blood and
10 mg/L nicotinamide adenine dinucleotide (NAD).

20. Using a ruler
On the under surface of the
plate containing transparent
medium.
Using a pair of calipers
On the plate containing
opaque medium.

21. SENSITIVE
A pathogen reported as sensitive
suggests that the infection.
It has caused is likely to respond
to treatment if the antibiotic
to which it is susceptible is used
in normal recommended doses.

22. A pathogen reported as MS suggests
that the infection it has caused is
likely to respond to treatment.
If the antibiotic is used in larger than
normal doses or when the antibiotic
is concentrated at the site of infection.
INTERMEDIATE / MODERATELY SENSITIVE

23. A pathogen reported as
resistant suggests that the
infection.
It has caused will not respond
to treatment with that antibiotic
irrespective of dose or site of
infection.
RESISTANT

24. ADVANTAGE :
1.Easy to substitute one disk for another.
2.Dependent on a commercial provider for the drug
profiles available.
3.Easier to spot contamination and low-level resistance.
DISADVANTAGE:
1. Use only with rapidly growing organism.
2. MBC can not be done using agar diffusion techniques.