LABORATORY METHODS IN ANTIBIOTIC SELECTION
Antibiotic Sensitivity Testing
Bacteriostatic Activity:
. Level of antimicrobial activity that inhibits the
growth of an organism. This is determined in
vitro by testing a standardized concentration of
organism against a series of antimicrobial
dilution. The lowest concentration that inhibits
the growth of the organism is referred to as the
Minimum Inhibitory Concentration (MIC)
Bactericidal Activity:
. Level of antimicrobial activity that kills the test
organism. This is determined in vitro by exposing
a standardized concentration of organisms to a
series of antimicrobial dilutions. The lowest
concentration that kills 99.9% of the population
is referred to as the Minimum Bactericidal
Concentration (MBC)
Actually bacteria develops resistance to live when they
just need the antibacterial substances and we have to
do some susceptibility assays in order to understand
resistance pattern of the bacteria to the antibiotic , and
there are some different methods. The one of the
Manuel method is disk diffusion method. the other is
and the automatized ones also are based on the broth
dilution assays and the last one is epsilometer (E) test.
These have different properties rom each other. We
use some guidelines in order to just have the
standardized protocol because everyone has to do
standardized protocols every laboratory all over the
world in order to get the proper results.
First, initially we were using Clinical and laboratory
standards institute (CLSI), but now we use European
committee on the anti microbial susceptibility testing
(EUCAST). Thus, EUCAST guidelines we actually have no
the MIC value.
MIC means minimal inhibitor concentration of the drug
to the bacteria and with these tables actually we’re
standardized the assays, we help the result for the
related the MIC values, so bacteria static activity
means level of antimicrobial activity that inhibits
growth of the inhibits. Not kills. Inhibition related with
MIC, so the lobes concentration that inhibits growth of
the organism refers this minimum inhibitory
concentration. For pharmacological approach this MIC
value also use. In order to develop and antibacterial
substance. The other is the Killing concentration. This
bactericidal concentration is the concentration that the
bacteria have the lowest concentration for the
inhibiting and the killing is the most the ones at the
lowest concentration kill approximately a hundred
percentage, all bacteria over the kill.
 Uses of Antibiotic Sensetivity Testing
 Antibiotic Sensitivity Test: A laboratory test
which determines how effective antibiotic
therapy is against a bacterial infection.
 Antibiotic sensitivity testing will control the use
of antibiotic in clinical practice.
 Testing will assist the clinicians in the choice of
drugs for the treatment of infection

The MIC value is obtained with broth dilution

If the infection these bacteria you should use
assays, so the broth delusion assay actually is an
antibacterials. For example, upper throat
assay that we have a broth medium, broth
respiratory tract infection is the mostly detected
distilled. It will be written on the slides. Muller
infection and caused by virus, and nearly all of them
Hinton broth are we just put the same amount of
are prescribed with antibacterials, so this is a big
bacteria into the tube which we arrange the
medical mistake. You should just know how
number of bacteria using Mc Farland standards, so
differentiate viral infections and bacterial from each
the number of the bacteria that in put in every
other, and you mustn’t use if there is no indication
tube is standard tend to power 8 in number, so
antibacterial. Because these are antibacterials kill
after first we just put the two fold dilution of the
also microbiome at the same time. Also, it causes
antibacterial into the tubes. Two fold lessing
some resistance problems, so these controlling is
amount, so in order to obtain MIC value the
important insensitive test. Of course you should
halving full concentration will give as at the end of
first identify relive the pathogen related the
24 hours we obtain concentration that stops,
infection isolate the pathogen after we will take the
inhibits the bacteria. But this concentration it is
pathogen for the resistant assays. So at the first 24
the first tube that we observe distoping of the
hours you isolate the pathogen related the
concentration of the antimicrobial. In these tube,
infection. The other 24 hours you will have
there are some bacteria that are I cannot detect,
antibacterial susceptibility assay results. So it totally
so this is the first step for this assay at 24 hours
takes 48 hours to this procedure. So the antibiotic
we obtain the MIC value. If no have some
susceptibility assays identification of the bacteria
inoculations from the tube that you do not have
take in the colony, and you will have the methods
any growing inside with naked eye, You will just
that we use.
inoculate on the plate, and after 24 hours the
concentration that gives no growth means that
this concentration has killed the bacteria. At the
first step the MIC concentration is obtained a 24
Components of Antibiotic Sensitivity
hours and after another 24 hours the minimal Testing
bactericidal concentration is over. So this is the
most important part of this assays because mostly 1. The identification of relevant pathogens in
this diffusion is the disks on the solute plate. MIC exudates and any body fluids collected from
inside the disk also prepares in these assays. The patients.
automatized system based on the MIC assay, so 2. Sensitivity tests done to determine the degree
this is the very important part of antibiotic of sensitivity or resistance of pathogens
susceptibility assay. By these methods we obtain isolated from patient to an appropriate range of
the affective antibiotic and we use the most antimicrobial drugs.
affective one (of course we will compare the 3. Assay of the concentration of an administered
spectrum activities: white ? spectrum, deviated
drug in the blood or fluid of patient required to
spectrum, rough spectrum must be choose
control the schedule of dosage.
because you have to just at the same time take
care very mostly care the resistance issue because
resistance issue is so important, and you must just
choose the antibiotics very properly and at the
same time of course the first rue for the
indication.).
Uses of Antibiotic Sensitivity Testing
 Help to guide Physician choosing antibiotics
 The accumulated results on different pathogens
their sensitivity will guide the physician in
choosing empirical treatment in serious patients
before the individual’s laboratory result are
analyzed in the Microbiology laboratory
 Reveals the changing trends in the local isolated
 Helps the local pattern of antibiotic prescribing
Testing for Antibiotic Sensitivity
. The method includes several steps including
obtaining a bacterial sample; identifying the type of
bacteria in the bacterial sample; selecting a set of
antibiotics based on the identify of the bacteria in
the bacterial sample; obtaining a control sample
from the bacterial sample.
Why Need Continues for Testing for
Antibiotic Sensitivity
. Bacteria have the ability to develop resistance
following repeated or subclinical (insufficient) doses,
so more advanced antibiotics and synthetic
antimicrobials are continually required to overcome
them
Testing Antibiotic Susceptibility
. Antibiotic sensitivity test: A laboratory test which
determine how effective antibiotic therapy is against
a bacterial infections.
Why are we waiting for 48 hours? If it is a bacterial
infection, you cannot stop not arranging the anti
microbial treatment problems. So you have to choose
an anti bacterial and it will be just related with
bacteriological statistics. I mean we first treat you
should have to know for systemic infection first the
microorganism, and you will for this period just give a
proper antibacterials, but after giving the sensibility
results you will just pass through the proper one. The
rules work like that. So, the resistance is a very mostly
important problem all over the world because some
bacterias came the superbugs. It means they have
lots of resistants. You can’t find anything to kill them.
So, resistance for national, international or local may
be for a little bit, but it is a world by problem. So, we
need antibiotic susceptibility assays, and what will we
do to take the bacteria sample, and now we will work
with the methods.
Culture and Susceptibility Testing
. Disk diffusion (Kirby Bauer)
. Serial dilution (Macro and Micro)
— Automated (Vitek, MicroScan)
. Antimicrobial gradient method (E-Test)
Methods are this diffusion assay, broth dilution (serial
dilution) assay and antibacterial gradient method
(epsilometer Test). Of course we use in laboratory
disk diffusion (Kirby Bauer Method), but the most
developed ones in our country use automatized
related the broth assay. We have Manuel system in
hand as well because we may use in some certain
cases, and E-test is also an important test because it
gives MIC value at the same time. It is a solid-phase
assay, so serial method will give MIC value
antimicrobial gradient assay give MIC value.
1-) DISK DIFFUSION (KIRBY BAUER)
 The in vitro of bacterial cultures with antibiotics
to determine susceptibility of bacteria to
antibiotic therapy. Bauer- Kirby Test
Kirby- Bauer Methods a Commonly
Used Method in Basic Laboratory
 Kirby- Bauer antibiotic testing (KB testing or disk
diffusion antibiotic sensitivity testing) is a test
which uses antibiotic-impregnated wafers to test
whether particular bacterial are susceptible to
specific antibiotics
How to Perform Kirby- Bauer Testing
 The basics are easy.
 The bacterium is swabbed on the agar and the
antibiotic discs are placed on top.
 The antibiotic diffuses from the disc into the agar
in decreasing amounts the further it is away from
the disc.
 If the organism is killed or inhibited by the
concentration of the antibiotic, there will be NO
growth in the immediate area around the disc:
This is called the Zone of the inhibition
(A/N= Mc Farland; 1% BaCl, and 1%
H2SO4)
Mc Farland Standard
The first, diffusion in vitro testing of the bacterial
sensitivity with this by Kirby Bauer assay, and this disk
have the antibiotic in impregnated proper cell. They
are for arranged for certain bacteria, and this assay
actually you just first isolate the bacteria, you have
the bacteria in hand, so you have the Muller Hinton
Agar plates, and you will inoculate the bacteria to the
plate up to Mc Farland standards. Mc Farland
Standard is used for prepared by some chemicals
(Prof. will show) and they will resemble they growth
pattern of the bacteria in appearance, so this blurred
region gives us the certain amount of the bacteria.
You prepare the amount of the bacteria up to the Mc
Farland standard and it is 0.5. You just prepared the
bacteria inoculation up to the 0.5 Mc Farland
standard, so you will inoculate the bacteria and plates
the disk related to that kind of bacteria and they are
just obtained from the guideline and disk are
conversely prepare, so the disk will be placed in the
proper centimeters from each other and you will take
the plates to inhibitors in 37 degree centigrade and
you wait up to 24 hours at the and there will be
growth, but some antibacterial works (kills the
bacteria). It is called zone of inhibition. They will just
inhibit the growing of the bacteria. Bacteria will be
just prepared with the Mc Farland plated on the plate
and the disks will be just placed like that and after
this 18-24 hours there are some inhibition zones
around the disks. So, they pick the inoculum and we
will just prepare with related to Mc Farland 0.5 optic
density. These are preparing 1 percentage BaCl and 1
percentage H2SO4 and at the end there are BaSO4
precipitates and this optic density will give you as you
see as increasing the density. We use this 0.5 Mc
Farland in order to prepare the amount of the
bacteria and It will be just equal to tend to power 8
times of bacteria. So, we will inoculate the bacteria in
place. We placed the disks in certain distances at the
plate. Placed on the agars surface between 2 or 3
centimeters between each other and nearly 2
centimeters from the plate edge.So the bacteria
growt will be obtained at the end. Some antibiotics
killed the bacteria some not.
Streaking the Inoculum
 Kirby- Bauer (or Bauer-Kirby) disk diffusion antibiotic
susceptibility testing applies a defined inoculum (Compared
to Mcfarland 0.5 OD standard) streaked as a lawn onto a
large Mueller-Hinton agar or Blood agar plate (In 3 directions
to ensure confluence) I

Making Proper inoculum

 Swab a Mueller-Hinton plate with each of the bacteria. Dip a
sterile swab into the broth and express any excess moisture
by pressing the swab against the side of the tube

Bacterias are Inoculated as Lawn Culture

 Method of inoculation-good results are obtained by placing
a standard loopful of inoculum suspension on the plate and
then spreading it with a dry sterile swab.

Disk Diffusion Method

 After completely swabbing the plate, turn it 90 degrees and
repeat the swabbing process. (It is not necessary to re-
moisten the swab.). Run the swab around the circumference
of the plate before discarding it in the discard bag.

Placing theSize
Zone Antibiotic Disks
Differ on Sensitivity Pattern
 ItThen, usingdetermined
has been a dispenserthat
suchzones
as the
ofone pictured,
inhibition of aantibiotic-
certain
impregnated disks are placed onto the agar surface. As the
diameter (varies for antibiotic and to a lesser extent, bacterial Resistant
bacteriacorrelate
species) on the lawn
withgrow, they’re
sensitivity orinhibited
resistancetoto
varying
the antibiotic
degrees
tested. by the antibiotic diffusion from the disk

For example, which antibiotic is resistance to the bacteria?

(Answer: C) because This plate is Mueller- Hinton Agar. We
inoculate all the plate with bacteria. We are working with the
bacteria. Our interesting part is bacteria and as the bacteria is
inoculated and the antibiotic is placed after 24 hours the bacteria
 So this plate tell me the clockwise direction
which antibiotics are resistants for this
bacteria? (Answer: Nearly nine and five)
approximately most susceptible you cannot
decide by just looking. You should measure.
Which is the most susceptible? (Answer:
You cannot decide by just looking, but
naked eye when the zone diameter is more,
it is more susceptible).

The area of Inhibition is Look at the Charts for Establishing

Masured with Scale the Zones of Sensitivity
 Record the results for everyone  The zone sizes are looked up on a
on your table in the table below standardized chart to give a result of
sensitive, resistant or intermediate

Interpretation
 Place the metric ruler across the zone of inhibition,
at the widest diameter, and measure from one
edge of the to the other edge. HOLDING THE
PLATE UP TO THE LIGHT MIGHT HELP.
 The disc diameter will actually be part of that
number. If there is NO zone at all, report it as 0—
 2-) BROTH DILUTION TEST
Serial Dilution (Macro and Micro)
 Agar and Broth dilution methods for Minimum
Inhibitory Concentration (MIC) determination.
 Serial dilutions of an antibiotic are prepared in
a nutrient medium and then inoculated with a
standardized concentration of the test
bacterium.
 After overnight incubation, the lowest
concentration of antibiotic that is able to
inhibit the growth of the bacteria is referred to
as the Minimum Inhibitory Concentration
(MIC)
What is Minimum Inhibitory
Concentration
 Minimum Inhibitory Concentration (MIC), in
microbiology, is the lowest concentration of an
antimicrobial that will inhibit the visible
growth of a microorganism after overnight
incubation.
 Minimum inhibitory concentration are
important in diagnostic laboratories to confirm
resistance of microorganisms to an
antimicrobial agent and also to monitor the
activity of new antimicrobial agents.
NOTE: By the way, we use a positive and negative control. One tube has no antibiotic
inside that is positive control. One tube has no bacteria inside that is negative control.
Testing Minimum Inhibitory
Concentration
 In alternative measure of susceptibility is to
determine the Minimum Inhibitory
Concentration (MIC) and the Minimum
Bactericidal Concentration (MBC) of a drug. A
series of broths are mixed with serially diluted
antibiotic solutions and a standard inoculum is
applied. After incubation, the MIC is the first
broth in which growth of the organism has been.
The second one is broth dilution assay. Broth dilution
assay as I’ve described some serial of tubes or
microplace, and we just put standard amount of
media. Media means Mueller-Hinton growth into the
tubes. This is standard. For example, one milliliter or
a hundred micrometer at the micro plate. After you
will put equal amount of bacteria. And the number of
bacteria is arranged with Mc Farland. Mc Farland
Standard use in order to have the equal amount of
bacteria. We just use at optic density and measure
with spectrum photometer in order to measure
them. (A/N: Prof said it is very detail.). So, equal
amount of bacteria, what is the serial dilution that
we put into tubes? Serial dilution of what? To fold
decreasing or to fold increasing? (Answer: Bacteria,
Medium and antibiotic) at the end.
For example, this tube this is a serial dilution for Mc
Farland put the equal amount of bacteria inside, and
what is the MIC value? (Answer: 6.25 because tube
that do not the others just demonstrate the growing of
the bacteria) the first that naked eye cannot see any
growing is the MIC value. Can you say bacterial
concentration at that step at the end of 24 hours?
(Answer: No because You should take inoculation to
the solid-phase and wait for another 24 hours. In order
to understand Minimum Bactericidal Concentration
(MBC) value.). It’s very important to understand
because currently we use main principle for
automatized systems as well. We give MIC value to the
report in the record of the patient.
The other example: What is the MIC value?
(Answer: 0.125 çünkü üremenin olmadı gözle
görülen ilk tüp 1 bölü 8). And we also take
passages from the tubes and inoculate to the
place. What is the bactericidal concentration?
(Answer: 0.25).

NOTE: If a bacteria has near concentrations for

MIC and MBC that means it is potent antibiotic. If
the MIC value is close to bactericidal value, it
should be just 4 times the ratio just if it is the 4
fold. This is the potent group, but it is near to the
concentration this is a potent antibacterial.

For the second line what is the MIC value?

(Answer:2).

The MIC value is 4

NOTE: For Micro dilution it will begin little amounts and you just
understand that clear wells are MIC value.
The lowest concentration is
MIC value because every
concentrations are
susceptible
 3. ANTIMICROBIAL GRADIENT
METHOD ( E-TEST)
What is E-Test
 E test is an antimicrobial gradient technique in
which 15 reference MIC dilutions of an
antibiotic have been repackaged with
innovative dry chemistry technology onto a
plastic strip.
 The predefined gradient provides precise and
accurate assessment of antimicrobial activity
against both fastidious and non-fastidious
microorganism.

The last one is E-test gradient assay. It means that

this is solid-phase assay. You have a plate, but at
the same time you have a plastic strip. That the
mid concentration is I mean 2 fold decreasing
concentration impregnated on the strip. So, you
have the result actually this is a solid-phase you
inoculate the bacteria, and place the MIC having
concentration strips of different antibiotics to the
plate, and placing down like this. At the end, when
the growth inhibition zones cuts the MIC will be
just the cutting edge. It is like ellipse because the
amount of antibiotic top of the strip is more than
bottom. Concentration is decreasing gradually.
Stripin üstünde daha çok antibiyotik var giderek
azalan konsantrasyon stripin üstündeki yarıklara
işaretler konuluyor. En başta en yüksek
konsantrasyon var ve aşağıya doğru azalarak
ilerliyor ve MIC’de kesiliyor. Bu yüzden elips
şeklinde. So, this test was last and you will have
just have the MIC results with the cutting edge of
the MIC. So it is just use for special group of
resistance because it is a little bit expensive test.
Antibiotic Assays
The amount of antibiotic in the blood or other
body fluids of patients measured for following
reasons
1. To ensure epadequate therapeutic
concentration are reached in serious infection
with parental administration
2. To avoid toxic concentration
3. To study pharmacokinetics of antibiotic
Limitation of Antibiotic Sensitivity
Usage
Both microbiologist and clinician should however
bear in mind that the response therapy in vivo may
not always reflect the results of testing the
sensitivity of patient’s pathogen in vitro

To identify the clinical response with identified

concentration

At present automated methods are popularly

used as the testing protocols are technically
demanding.
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