Testing of Microbial Susceptibility to Chemotherapeutic Agents

Chemotherapeutic agents are chemical substances used for the treatment of infectious diseases or
diseases caused by the proliferation of malignant cells. Since 1935, a large number of chemotherapeutic
agents have been developed. Many of these compounds are prepared synthetically in the laboratory,
while others occur as by-products of the metabolic activity of bacteria or fungi. The latter group of
chemotherapeutic agents is called antibiotics.

The susceptibility of microorganisms to chemotherapeutic agents can be determined by either the tube
or agar dilution method or the paper-disk agar diffusion method of Kirby-Bauer.

Tube/Agar Dilution Method

By the tube/agar dilution method, one can determine the smallest amount of chemotherapeutic agent
required to inhibit the growth of the organism in vitro. For broth dilution methods, decreasing
concentrations of the chemotherapeutic agent(s) to be tested, usually prepared in twofold dilutions, are
placed in tubes of a broth medium that will support the growth of the test microorganism. The most
commonly-used broth for these tests is Mueller-Hinton supplemented with the cations magnesium and
calcium. For broth dilution susceptibility testing of staphylococci against methicillin, oxacillin, nafcillin or
cephalosporin, 2% sodium chloride must be added to the cation-supplemented Mueller-Hinton broth.

A standard inoculum of the microorganism [often 1.5 x 106 organisms per milliliter, 1:200 dilution of a
suspension of turbidity equal to a MacFarland standard 1.0] is added to an equal volume (often 1 ml) of
each concentration of antimicrobial agent and to a tube of the growth medium without antimicrobial
agent, which serves as a growth control. After sufficient incubation (usually for 16-18 hr), the tubes are
examined for turbidity, indicating growth of microorganisms. The lowest concentration of the agent
that inhibits growth of the organism is designated the minimum inhibitory concentration (MIC). If the
concentration of antimicrobial agent represented by the MIC can be readily achieved in the patient's
serum by normal routes of delivery, the organism is said to be susceptible to that agent. If the MIC is
above the achievable level or is within a range toxic to the host, the microorganism is said to be
resistant to the agent. Thus, susceptibility and resistance are functions of the site of the infection, the
microorganism and the antimicrobial agent being tested.

The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to
survive is said to be the minimum bactericidal concentration (MBC) or the minimum lethal
concentration (MLC).

Disc-Agar Diffusion Method

The single-disc method for susceptibility testing currently used is a slight modification of the procedure
developed by Bauer, Kirby, Sherris and Turck in 1965. When the susceptibility test is performed in
conformity with the standard procedure approved by the Clinical Laboratory Standards Institute or CLSI
(formerly known as National Committee for Clinical Laboratory Standards or NCCLS), one can correlate
the sizes of inhibition with the MIC of the drug for the microorganism under test. It is possible to
determine whether the microorganism is resistant or susceptible to the agent.
In this method, antibiotics are impregnated into paper discs and then placed on an inoculated Mueller-
Hinton agar plate. The plate is then incubated for 16-18 hours, and the diameter of the zone of
inhibition is measured to the nearest millimeter. Plates for testing the response of Staphylococcus
aureus to methicillin (oxacillin) should be incubated for 24 hours. The inhibition zone diameter that is
produced will indicate the susceptibility or resistance of the bacteria to the antibiotic. This can be
determined by using the CLSI zone diameter interpretative table.

In performing this test, it is important to take note of the following:

1. The size of the inoculum should be standardized, i.e., the turbidity of the log-phase inoculum should
be adjusted to equal that of the turbidity standard – 0.5 MacFarland (made up of 0.05 ml of 1.175%
BaCl2 and 9.95 ml of 1% H2SO4).

2. Mueller-Hinton agar is used as the test medium as this does not contain para-amino benzoic acid
(PABA). PABA inhibits the activity of sulfonamides being tested.

3. Mueller-Hinton plates used should not be older than three days from their day of preparation. This
is to ensure optimum hydration needed for the diffusion of the agents under test.

4. Cotton swab with the inoculum is streaked on the surface of the Mueller-Hinton plate three times,
rotating the plate 60o after each streaking. This procedure ensures that the whole surface has been
inoculated. Allow the culture to dry on the agar surface for 5 to 10 minutes at room temperature
before introducing the discs with the chemotherapeutic agents.

References

Finegold, S. and E. Baron. 2001. Balley and Scott's Diagnostic. Microbiology. 9th edition
The CV Mosby Company, Missouri.

Harley, J. and L. Prescott. 1993. Microbiology. 2nd edition. Brown Publishers, Dubuque.

Pelczar, M.E., Chan and N. Krieg. 1986. Microbiology. McGraw-Hill, Inc. N.Y.

Volk, W. and M. Wheeler. 1988. Basic Microbiology. 6th edition. Harper and Row, N.Y.