Antimicrobial sensitivity test

Background

Chemotherapeutic agents are chemical substances used to treat infectious diseases. Their mode of action is to interfere with microbial metabolism, thereby producing a bacteriostatic or bactericidal effect on the microorganisms, without producing a like effect in host cells. Chemotherapeutic agents act on a number of cellular targets. Their mechanisms of action include inhibition of cell-wall synthesis, inhibition of protein synthesis, inhibition of nucleic acid synthesis, disruption of the cell membrane, and inhibition of folic acid synthesis. These drugs can be separated into two categories:   

1. Antibiotics are synthesized and secreted by some true bacteria, actinomycetes, and fungi that destroy or inhibit the growth of other microorganisms. Today, some antibiotics are laboratory synthesized or modified; however, their origins are living cells.                                                      
2. Synthetic drugs are synthesized in the laboratory

Principle

The available chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a limited spectrum of activity, effective against only one group of microorganisms. Others exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibilities of many pathogenic microorganisms are known, but it is sometimes necessary to test several agents to determine the drug of choice. A standardized diffusion procedure with filter paper discs on agar, known as the Kirby-Bauer method, is frequently used to determine the drug susceptibility of microorganisms isolated from infectious processes. This method allows the rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition those results from diffusion of the agent into the medium surrounding the disc. In this procedure, filter-paper discs of uniform size are impregnated with specified concentrations of different antibiotics and then placed on the surface of an agar plate that has been seeded with the organism to be tested.

Materials required

Test culture: 0.85% saline suspensions adjusted to an absorbance of 0.1 at 600 nanometer (nm) or equilibrated to a 0.5 McFarland Standard, Mueller-Hinton agar plates, antibiotic discs, forceps, Bunsen burner, sterile cotton swaps, 70% ethanol, millimetre ruler.

Protocol

1.  Place MHA plates right-side-up in an incubator heated to 37°C for 10 to 20 minutes, allowing the plates to warm up.
2. Label the bottom of each of the agar plates with the name of the test organism or strain to be inoculated.
3.  Using aseptic technique, inoculate all agar plates with their respective test organisms as
4.  Follows; Dip a sterile cotton swab into a well-mixed saline test culture and remove excess inoculum by pressing the saturated swab against the inner wall of the culture tube.
5.  Using the swab, streak the entire agar surface horizontally, vertically, and around the outer edge of the plate to ensure a heavy growth over the entire surface.
6.   Allow all culture plates to dry for about 5 minutes.
7.  Distribute the individual antibiotic discs (maximum of 5 discs in 15mm plates) at equal distances (~2.5 cm) with forceps dipped in alcohol and flamed.
8.  Gently press each disc down with the wooden end of a cotton swab or with sterile forceps to ensure that the discs adhere to the surface of the agar. Note: Do not press the discs into the agar.
9.   Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C.
10.   Following incubation, examine all plate cultures for the presence or absence of a zone of inhibition surrounding each disc.
11.  Using a ruler graduated in millimeters, carefully measure each zone of inhibition to the nearest millimeter. Tabulate the results.
12.   Compare your results with Table and determine the susceptibility of each test organism to the chemotherapeutic agent.

	Measurements in millimeter
Antibiotic disc	Ampicillin (A) 10µg	Erythromycin (E) 10µg
Test culture	R	S(16mm)

S = sensitive; R = resistant; I = intermediate

Result

The test culture was resistant to ampicillin, clindamycin, nystatin, intermediate to amoxicillin and sensitive to streptomycin.