PART I

BASIC MICROBIOLOGY

ACTIVITY 11

ANTIMICROBIAL SENSITIVITY
TESTING
Antibiotics
 – biosynthesized chemicals.

 Biosynthesis – production of
substances by living organisms.

 Antibiotic substances are of no use in

the metabolism of microbes that
produce them.
Antibiotics
 By-products of microbial metabolism.
 Used by organisms to gain advantage
over their competitors.
 Substances produced by
microorganisms that kill or inhibit the
growth of other microorganisms.
Genera that produce the largest
number of antibiotics:
 Bacillus spp.
 Penicillium spp.
 Streptomyces spp.
Antibiotics

 Some are administered internally and

travel via the bloodstream to all parts of
the body.
 They kill and inhibit growth of
microorganisms at low concentration
to avoid undesirable damage to the
host.
 Low toxicity to body cells.
Antibiotics

 Should have high selective toxicity

 Selective Toxicity - Must kill or inhibit
the microbial pathogen without damaging
the host cell.
 Must undergo sensitivity testing
 Determination of the Level of
Antimicrobial Activity
 Dilution Susceptibility Tests
Used to determine the MIC and MLC
 Disk Diffusion Tests
Kirby-Bauer Method
Can test the resistance or sensitivity of an
organism to an array of antimicrobial agents.
Disinfectant vs. Antiseptic

 Disinfectant – antimicrobial agent applied

to inanimate objects.
 Antiseptics - antimicrobial agent applied to
living tissues.
Both are chemical agents; synthetic.
Both can be too harsh that they can damage
living tissues.
Chemotherapeutic Agents

 Antimicrobial chemicals used

therapeutically.
 Synthesized in the laboratory.
 Genetically engineered
Broad spectrum antibiotics

 Effective against many different

pathogenic bacteria.
 Has a widespread effect on Gram + and
Gram – bacteria.

 Physicians not only wish to know the

organisms causing an infection, but the
antibiotic that will control it.
Objectives

1. Perform the Kirby-Bauer method in testing

the effectivity of chemotherapeutic agents.
2. Interpret the Kirby-Bauer sensitivity test plate
using the zone of inhibition.
3. Identify the resistance and susceptibility of
the tested bacteria to different antibiotics.
4. Differentiate broad spectrum from narrow
spectrum drugs.
Procedure

 Bacterial Seeding
 Antibiotic Assay
Kirby-Bauer test (Disk Diffusion

A. Standardization of Inoculum
 0.5 Mac Farland Standard
• 1% BaCl2 - 0.5 ml
• 1% H2SO4 - 9.95 ml
 Equalize the turbidity of 0.5 Mc Farland with
the inoculum (Test organism)
 Inoculum has approx. 1.5 x 108 cfu/ml
B. Bacterial Seeding
 Mueller Hinton Agar (MHA)
 Cotton Pledget
 Standardized Inoculum
 Cotton pledget is dipped into the inoculum
and applied evenly onto MHA plate.
 Lawn of bacteria
A. Bacterial Seeding

1. Preparation of Inoculum
 McFarland 0.5 turbidity standard
When Barium is completely
suspended, the optical density of
the suspension is 0.5
Approx. number of bacterial cells
in the inoculum equalized with it
is:
1.5 x 108 cfu/ml
2. Culture Medium - Mueller-Hinton Agar
 Medium used for standardized antimicrobial disk diffusion
susceptibility testing.
 Formulation
 Acid hydrolysate of casein
• Supports the growth of microorganisms
 Beef extract
• Supply amino acids, N-compounds, minerals, vitamins
 Starch
• Energy source
MHA
 the best medium for routine susceptibility
tests because :
It has good reproducibility
It is low in sulfonamide, trimethoprim,
and tetracycline inhibitors
It gives satisfactory growth of most
bacterial pathogens.
3. Swab inoculum evenly onto MHA to make a
bacterial lawn
B. Antibiotic Assay

Placement of antibiotic disks on the lawn

of bacteria
Antibiotic disks - Small filter paper disk
impregnated with standardized amounts of
antibiotics.
 Antibiotic disks are pressed gently on the
surface of MHA seeded with test
organisms.
B. Antibiotic Assay

1. Placement of antibiotic disks on lawn of

bacteria
 C. Incubation for 24 hrs at 37°C
 D. Examination of plates for the
presence of clear rings.
C. READING AND MEASUREMENT
OF ZONES OF INHIBITION
 The zone of inhibition (arrow) is the point
at which no growth is visible to the
unaided eye.
D. INTERPRETATION OF ZONES OF
INHIBITION
    Inhibition zone diameter(mm)
Antibiotic Amount on disc Resistant Intermediate Sensitive
 
Ampicillinb 10 µg 11 or less 12-13 14 or more
Ampicillinc 10 µg 28 or less - 29 or more
Cephoxitin 30 µg 14 or less 15-17 18 or more
Cephalotin 30 µg 14 or less 15-17 18 or more
Chloramphenicol 30 µg 12 or less 13-17 18 or more
Clindamycin 2 µg 14 or less 15-16 17or more
Erytromycin 15 µg 13 or less 14-17 18 or more
Gentamycin 10 µg 12 or less 13-14 15 or more
Kanamycin 30 µg 13 or less 14-17 18 or more
Methicillinc 5 µg 9 or less 10-13 14 or more
Neomycin 30 µg 12 or less 13-16 17 or more
Nitrofurantoin 300 µg 14 or less 15-16 17 or more
Penicillin Gd 10 units 28 or less - 29 or more
Penicillin Ge 10 units 11 or less 12-21 22 or more
Polymyxin B 300 units 8 or less 9-11 12 or more
Streptomycin 10 µg 11 or less 12-14 15 or more
Tetracycline 30 µg 14 or less 15-18 19 or more
Trimethoprim- 1.25/23.75 µg 10 or less 11-15 16 or more
Sulfamethoxazole
(SXT)
Tobramycin 10 µg 12 or less 13-14 15 or more
Novobiocin 30µg 17 or less 18-21 22 or more