Experiment 6

Antimicrobial Susceptibility testing

Outline :
1. Study the effect of chemotherapeutics: bactericidal VS
bacteriostatic.
2. Differentiate between MIC and MBC
3. Study the principle of the Agar diffusion test and the E test

 Introduction:

-The principle drugs used in the treatment of infectious diseases fall into three categories
: Antibiotics,sulfonamide and chemotheraputics.collectively they referred to as
antimicrobics.

-Antibiotics are powerful medicines that fight certain infections and can save lives when
used properly. They either stop bacteria from reproducing or destroy them.

Before bacteria can multiply and cause symptoms, the immune system can typically kill
them. White blood cells (WBCs) attack harmful bacteria and, even if symptoms do
occur, the immune system can usually cope and fight off the infection. Sometimes,
however, the number of harmful bacteria is excessive, and the immune system cannot
fight them all. Antibiotics are useful in this scenario.

-Bacteriostatic antibiotics : limit the growth of bacteria by interfering with

bacterial protein production, DNA replication, or other aspects of bacterial
cellular metabolism.

- Bactericidal antibiotics kill bacteria and, ideally, nothing else.

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- Sulfonamide: antibacterial sulfonamides act as competitive inhibitors of
the enzyme dihydropteroate synthase (DHPS), an enzyme involved in folate synthesis.
Sulfonamides are therefore bacteriostatic and inhibit growth and multiplication of
bacteria

Figure 1 : Bacterial Metabolism and Antibiotic Efficacy

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 Figure 2: Static vs Cidal drug effect

Figure 3: Effective and non effective antibiotics and zone of inhibition

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-The term susceptible : means that the microorganism is inhibited by a concentration of

antimicrobial agent that can be attained in blood with the normally recommended dose

of the antimicrobial agent and implies that an infection caused by this microorganism

may be appropriately treated with the antimicrobial agent.

The term resistant: indicates that the microorganism is resistant to concentrations of the

antimicrobial agent that can be attained with normal doses and implies that an infection

caused by this microorganism could not be successfully treated with this antimicrobial

agent.

-Susceptibility testing : is performed on bacteria or fungi causing an individual's

infection after they have been recovered in a culture of the specimen.

-antimicrobial susceptibility tests: are performed by either disk diffusion or a dilution

method.
-Disk diffusion :

Figure 4: Disk diffusion method

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A standardized suspension of a particular microorganism is inoculated onto an agar

surface to which paper disks containing various antimicrobial agents are applied.

Following overnight incubation, any zone diameters of inhibition about the disks are

measured and the results are reported as indicating susceptibility or resistance of the

microorganism to each antimicrobial agent tested.

Figure 5: Disk diffusion method

**Additional link for Testing an Antibiotic Using a Disk Diffusion method:

https://youtu.be/sx1uDYSfINA

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- Dilution method:

Figure 6 : dilution method

- An alternative method is to dilute on a log2scale each antimicrobial agent in broth to

provide a range of concentrations and to inoculate each tube or, if a microplate is

used, each well containing the antimicrobial agent in broth with a standardized

suspension of the microorganism to be tested. The lowest concentration of

antimicrobial agent that inhibits the growth of the microorganism is the minimal

inhibitory concentration (MIC).

Additional link for Determination of MIC by Broth Dilution Method:

https://youtu.be/WAvRj1cRwXM

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Minimum inhibitory concentration (MIC) :
Is the lowest concentration of an antimicrobial (like an antifungal, antibiotic or

bacteriostatic) drug that will inhibit the visible growth of a microorganism after

overnight incubation.

MICs can be determined on plates of solid growth medium (called agar, shown in the

“Kirby-Bauer Disk Susceptibility Test” atom) or broth dilution methods (in liquid

growth media, shown in ) after a pure culture is isolated. For example, to identify the

MIC via broth dilution, identical doses of bacteria are cultured in wells of liquid media

containing progressively lower concentrations of the drug.

The minimum inhibitory concentration of the antibiotic is between the concentrations

of the last well in which no bacteria grew and the next lower dose, which allowed

bacterial growth. There are also several commercial methods available to experimentally

measure MIC values by disk diffusion or a dilution method.

The MIC and the zone diameter of inhibition are inversely correlated. In other words,

the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC

and the larger the zone of inhibition. Conversely, the more resistant the

microorganism, the higher the MIC and the smaller the zone of inhibition.
The Minimum Bactericidal Concentration (MBC) is the lowest concentration of

antibiotic required to kill an organism. .]Not as commonly seen as the Minimum

inhibitory concentration (MIC). It can be determined from broth dilution MIC tests by

subculturing to agar media without antibiotics.

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 Figure 7: MIC vs MBC

Sensetivity disks –antimicrobic impregnated disks -:the effictiveness of them based

on the size of the zone of inhibition ,and according to the table in figure 9 you decide
wether the bacteria is Resistant to this antibiotic , Intermediate or Sensitive.

-To Measure the Zone of Inhibition:

You can also measure directly across the zone of inhibition from edge to edge
crossing through the center of the antibiotic disk to directly measure the diameter.

Figure 8: Measure zone of inhibition

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Additional link for Measure the Zone of Inhibition:

https://youtu.be/-TZn3ie-iFk

Figure 8 :Evaluation for bacteria if susceptibility or resistance according to the zone

size

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 Figure 10 : Antibiotic sensitivity testing

 E test :
(Muller hinton agar is the media of choice for this test)
E test (Epsilometer test) is a way of determining antimicrobial sensitivity by
placing a strip impregnated with antimicrobials onto an agar plate. A strain
of bacterium or fungus will not grow near a concentration
of antibiotic or antifungal if it is sensitive. For some microbial and antimicrobial
combinations, the results can be used to determine a minimum inhibitory
concentration (MIC). Etest is a proprietary system manufactured by bioMérieux. It
is a laboratory test used in healthcare settings to help guide physicians by indicating
what concentration of antimicrobial could successfully be used to treat patients'
infections

Figure 11 : E test method

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Additional link of E test for antibiotic susceptibility:
https://youtu.be/K0nrFj8lFfA

Preparation of Mueller-Hinton plate

1. Allow a MH agar plate (one for each organism to be tested) to come to room
temperature. It is preferable to allow the plates to remain in the plastic sleeve while they
warm to minimize condensation.
2. If the surface of the agar has visible liquid present, set the plate inverted, ajar on its lid
to allow the excess liquid to drain from the agar surface and evaporate. Plates may be
placed in a 35°C incubator or in a laminar flow hood at room temperature until dry
(usually 10 to 30 minutes).
3. Appropriately label each MH agar plate for each organism to be tested. Preparation of
inoculum 1. Using a sterile inoculating loop or needle, touch four or five isolated
colonies of the organism to be tested. 2. Suspend the organism in 2 ml of sterile saline. 3.
Vortex the saline tube to create a smooth suspension.
4-Adjust the turbidity of this suspension to a 0.5 McFarland standard by adding more
organism if the suspension is too light or diluting with sterile saline if the suspension is
too heavy.
5. Use this suspension within 15 minutes of preparation.
Additional Notes :-
-Inoculum preparation Organisms to be tested must be in the log phase of growth in
order for results to be valid. It is recommended that subcultures of the organisms to be
tested be made the previous day.
-Never use extremes in inoculum density.
- Never use undiluted overnight broth cultures or other unstandardized inocula for
inoculating plates.
-If the organism is difficult to suspend directly into a smooth suspension, the growth
method of preparing the inoculums should be used.

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Inoculation of the MH plate
1. Dip a sterile swab into the inoculum tube.
2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure,
to remove excess fluid. The swab should not be dripping wet (Fig. 12).
3. Inoculate the dried surface of a MH agar plate by streaking the swab three times over
the entire agar surface; rotate the plate approximately 60 degrees each time to ensure an
even distribution of the inoculum (Fig. 13).
4. Rim the plate with the swab to pick up any excess liquid (Fig. 14). 5. Discard the
swab into an appropriate container.
6. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5
minutes, but no more than 15 minutes, for the surface of the agar plate to dry before
proceeding to the next step.

Figure.1 2. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the test plate. Step 2.
Rotate the swab against the side of the tube while applying pressure to remove excess liquid from the
swab prior to inoculating the plate.

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 Figure. 13. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton
agar plate. Step 3. (A) Inoculate the plate with the test organism by streaking the swab in a back-and-
forth motion very close together as you move across and down the plate. Rotate the plate 60° and repeat
this action. Rotate the plate once more and repeat the streaking action. This ensures an even distribution
of inoculum that will result in a confluent lawn of growth. (B) Diagram illustrating the pattern the swab
should follow as it is drawn across the plate.

Figure14. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton
agar plate. Step 4. After streaking the MuellerHinton agar plate as described in Step 3, rim the plate
with the swab by running the swab around the edge of the entire the plate to pick up any excessive
inoculum that may have been splashed near the edge

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Placement of the antibiotic disks
1. Place the appropriate antimicrobial-impregnated disks on the surface of the agar,
using either forceps to dispense each antimicrobial disk one at a time, or a multidisk
dispenser to dispense multiple disks at one time. (See steps a. through d. for the use of
the multi-disk dispenser or steps e. through g. for individual disk placement with
forceps.
a. To use a multidisk dispenser, place the inoculated MH agar plate on a flat surface and
remove the lid (Fig. 15).
b. Place the dispenser over the agar plate and firmly press the plunger once to dispense
the disks onto the surface of the plate.
c. Lift the dispenser off the plate and using forceps sterilized by either cleaning them
with an alcohol pad or flaming them with isopropyl alcohol, touch each disk on the plate
to ensure complete contact with the agar surface. This should be done before replacing
the petri dish lid as static electricity may cause the disks to relocate themselves on the
agar surface or adhere to the lid (Fig. 16).
d. Do not move a disk once it has contacted the agar surface even if the disk is not in the
proper location, because some of the drug begins to diffuse immediately upon contact
with the agar.
e. To add disks one at a time to the agar plate using forceps, place the MH plate on the
template (Fig. 17) provided in this procedure (Fig. 16a). Sterilize the forceps by cleaning
them with a sterile alcohol pad and allowing them to air dry or immersing the forceps in
alcohol then igniting.
f. Using the forceps carefully remove one disk from the cartridge (Fig. 16b).
g. Partially remove the lid of the petri dish. Place the disk on the plate over one of the
dark spots on the template and gently press the disk with the forceps to ensure complete
contact with the agar surface. Replace the lid to minimize exposure of the agar surface to
room air (Fig. 16c, d).
h. Continue to place one disk at a time onto the agar surface until all disks have been
placed as directed in steps f. and g. above. 2. Once all disks are in place, replace the lid,
invert the plates, and place them in a 35°C air incubator for 16 to 18 hours. When
testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against
vancomycin, incubate for a full 24 hours before reading

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 A B

Figure 15. Kirby-Bauer disk diffusion susceptibility test protocol, placementof antibiotic disks
using an automated disk dispenser. Step 1, a. through d. An automatic disk dispenser can be used to
place multiple disks simultaneously on a MH agar plate. (A) Set the dispenser over the plate.
(B) Place the palm of your hand on the top of the handle. (C) Press downfirmly and completely to
dispense the disks. The spring loaded handle will return to the original position when pressure is
removed.

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 A B

C D

Figure 16. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antibiotic disks using
forceps to manually place the disks. Step 1, e. through h. Antibiotic disks can be manually placed on
the MH agar plateif desired. (A) Place the Mueller-Hinton agar plate over the disk template. (B)
Remove one disk from the cartridge using forceps that have been sterilized. (C) Lift the lid of the plate
and place the disk overone of the positioning marks. (D) Press the disk with the forceps to ensure
complete contact with the agar surface. Replace the lid of the plate between disks to minimize exposure
to air-borne contaminants.

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