BIOL 2900 D

Course: Microbiology in Health and Disease Instructor: Bipin Patel

Laboratory Exercise

ANTIMICROBIAL SUSCEPTIBILITY TESTING

For some microorganisms, susceptibility to chemotherapeutic agents is predictable.

However, for many microorganisms (Pseudomonas, Staphylococcus aureus, and Gram-

negative enteric bacilli such as Escherichia coli, Serratia, Proteus, etc.) there is no

reliable way of predicting which antimicrobial agent will be effective in a given case. This

is especially true with the emergence of many antibiotic-resistant strains of bacteria.

Because of this, antibiotic susceptibility testing is often essential in order to determine

which antimicrobial agent to use against a specific strain of bacterium.

Several tests may be used to tell a physician which antimicrobial agent is most likely to

combat a specific pathogen:

A. Tube dilution tests

In this test, a series of culture tubes are prepared, each containing a liquid medium and a

different concentration of a chemotherapeutic agent. The tubes are then inoculated with

the test organism and incubated for 16-20 hours at 35C. After incubation, the tubes are

examined for turbidity (growth). The lowest concentration of chemotherapeutic agent

capable of preventing growth of the test organism is the minimum inhibitory

concentration (MIC).

B. The agar diffusion test (Bauer-Kirby test)

A procedure commonly used in clinical labs to determine antimicrobial susceptibility is

the Bauer-Kirby disc diffusion method. In this test, the in vitro response of bacteria to a

standardized antibiotic-containing disc has been correlated with the clinical response of

patients given that drug.

In the development of this method, a single high-potency disc of each chosen

chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of

disc were correlated with the minimum inhibitory concentrations of each antimicrobial

agent (as determined by the tube dilution test). The MIC for each agent was then

compared to the usually-attained blood level in the patient with adequate dosage.

Categories of "Resistant," "Intermediate," and "Sensitive" were then established.

One would require the following items to perform antimicrobial susceptibility testing:

Mueller Hinton Broth meeting CLSI standards

Mueller Hinton Agar plate meeting CLSI standards

0.5 McFarland standard

Pathogenic isolate to be tested

Sterile swabs

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Antimicrobial discs appropriate for the isolate being tested

A ruler

The basic steps for the Bauer-Kirby method of antimicrobial susceptibility testing are

given below.

1. The isolate from specimen is inoculated in Mueller-Hinton Broth. Since it is necessary

to start with known amount of bacteria, the broth is standardized by comparing its

turbidity against a standard of known density called McFarland Standard. Most common

bacterial types are adjusted to have density comparable to McFarland Standard of 0.5

which is equivalent to 1.5X108

Colony Forming Units (CFU)/mL of bacteria.

2. Each pair of students is provided with a Mueller Hinton broth tube containing a

bacterial isolate which has been already standardized against 0.5 McFarland

Standard.

3. Find out what the isolate is from your Instructor.

4. Using antimicrobial selection table, select out the antimicrobial discs you will be using,

based upon the organism to be tested, and source of the specimen.

5. Present a list of up to five antimicrobials your team would consider most appropriate,

and acquire filter paper discs soaked in antimicrobial solutions prepared according to

CLSI Standards.

6. Label plate with your initials and name of the isolate being tested.

7. Inoculate Mueller-Hinton agar plate with the standardized inoculum so as to cover

the entire agar surface with bacteria as shown below.

CORRECT INCORRECT

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8. Depending upon which organism is assigned to you, place applicable standardized

antibiotic-containing discs on the plate. Use a flamed and cooled pair of forceps to

remove one disc from each cartridge, place it at a reasonable distance from another

disc. Then flame and cool forceps, remove another disc and place it. Repeat this until

all discs are placed. Then with a flamed and cooled forceps, tamp each disc gently (do

not dig disc into agar with pressure).

9. Incubate the plate LID SIDE DOWN at 35C for 18-20 hours.

10. On next day, using a ruler, measure the diameter of any resulting zones of

inhibition in millimeters (mm) as shown below.

11. Using a standardized table, determine if the bacterium is susceptible,

intermediate, or resistant to each antimicrobial agent. The term intermediate generally

means that the drug may be used for infections in a particular body site, e.g., cystitis

because the drug becomes highly concentrated in the urine.

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12. Record your results below:

Organism Tested:

Specimen Source:

Antimicrobial Zone Size

(mm)

Interpretation Equivalent

MIC

Now answer the following questions:

a. Could ALL teams have been able to use the same antimicrobials?_

b. If not, why not?

c. Why would you need the CLSI Zone Interpretation Standard tables? Wouldnt

the antimicrobial with largest zone of inhibition be the best to choose?_Yes_No

d. If not, why not?

e. Why is it necessary to know the EXACT NAME of the organism, and not just Gram

Positive or negative?_

f. Why is it necessary to know the site of infection?

C. Automated tests

Computerized automated tests have been developed for antimicrobial susceptibility

testing. These tests measure the inhibitory effect of the antimicrobial agents in a liquid

medium by using light scattering to determine growth of the test organism. Results can

be obtained within a few hours. Labs performing very large numbers of susceptibility

tests frequently use the automated methods, however, the equipment may be quite

expensive, but accurate.

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