The Effect of Antibiotics on Bacteria t is valuable for the physician to have laboratory data indicating whether a bacterium is susceptible or resistant to various anti- biotics. This data, together with knowledge of possible side effects, patient allergies, and other information, will be used in prescribing the antibiotic. The laboratory test should be rapid, accurate, and relatively inexpensive to perform. In most laboratories, the effect of antibiotics on bacteria is determined by the Standardized Disk Susceptibility Test as outlined in the Federal Register. The procedure also is referred to as the Kirby-Bauer method, after its developers. Bacteria are swabbed on Mueller-Hinton agar, which gives reproducible results and does not inhibit sulfonamides. Paper disks containing known amounts of the antibiotic are added, and after incubation, the plates are observed for clear zones of inhibition surrounding the disks. By referring to a standardized table, the effect of the antimicrobial drug on the organism may be ascertained. The physician may be reasonably certain that if the stated dose of antibiotic exists in the tissues, then bacterial inhibition will take place. pecial Materials • Broth cultures of two selected bacterial species • Plates of Mueller-Hinton agar, or materials for their preparation • Sterile swabs and beakers of disinfectant • Antibiotic disks • Forceps in beakers of 70% ethyl alcohol • Millimeter rulers rocedure 1. Obtain or prepare two plates of Mueller-Hinton agar. Label them on the bottom side with your name, the date, and the name of the procedure, and designate one plate for each of two organisms to be tested. The depth of the agar should be about 4 millimeters. Obtain broth cultures of the two organisms and two sterile swabs. 2. Dip a swab into a broth culture of bacteria, press it to the inside of the tube to express the excess fluid, and inoculate one of the Mueller-Hinton plates by making a lawn of bacteria. The swabbing should be performed at three P S THE EFFECT OF ANTIBIOTICS ON BACTERIA 14 135 14 I PURPOSE: to evaluate the sensitivity of bacterial species to antibiotics.
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The Effect ofAntibiotics on Bacteria
t is valuable for the physician to have laboratory data indicatingwhether a bacterium is susceptible or resistant to various anti-biotics. This data, together with knowledge of possible side
effects, patient allergies, and other information, will be used in prescribingthe antibiotic. The laboratory test should be rapid, accurate, and relativelyinexpensive to perform.
In most laboratories, the effect of antibiotics on bacteria is determinedby the Standardized Disk Susceptibility Test as outlined in the FederalRegister. The procedure also is referred to as the Kirby-Bauer method,after its developers. Bacteria are swabbed on Mueller-Hinton agar, whichgives reproducible results and does not inhibit sulfonamides. Paper diskscontaining known amounts of the antibiotic are added, and after incubation,the plates are observed for clear zones of inhibition surrounding the disks.By referring to a standardized table, the effect of the antimicrobial drugon the organism may be ascertained. The physician may be reasonablycertain that if the stated dose of antibiotic exists in the tissues, then bacterialinhibition will take place.
pecial Materials
• Broth cultures of two selected bacterial species• Plates of Mueller-Hinton agar, or materials for their preparation• Sterile swabs and beakers of disinfectant• Antibiotic disks• Forceps in beakers of 70% ethyl alcohol• Millimeter rulers
rocedure
1. Obtain or prepare two plates of Mueller-Hinton agar. Label them on thebottom side with your name, the date, and the name of the procedure, anddesignate one plate for each of two organisms to be tested. The depth of theagar should be about 4 millimeters. Obtain broth cultures of the two organismsand two sterile swabs.
2. Dip a swab into a broth culture of bacteria, press it to the inside of the tubeto express the excess fluid, and inoculate one of the Mueller-Hinton platesby making a lawn of bacteria. The swabbing should be performed at three
P
S
T H E E F F E C T O F A N T I B I O T I C S O N B A C T E R I A 14 135
14
I PURPOSE: to evaluate thesensitivity of bacterial speciesto antibiotics.
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different angles, and a final sweep should be made around the rim of theplate as shown in Figure 8.2 in Exercise 8. Discard the swab in the beakerof disinfectant after use. Inoculate the second plate with the second or-ganism in like manner.
3. Allow the plates to dry at room temperature for about 10 minutes so that theexcess moisture is absorbed.
4. Using a sterile flamed forceps or an automatic dispenser, apply a series ofantibiotic disks to the surfaces of the plates as directed by the instructor. Thedisks should be about 3 cm from each other to prevent overlapping of anti-biotic, and at least 2 cm from the edge of the plate. If the forceps is used, itshould be flamed lightly after applying each disk. Light pressure may be applied to the disks using a forceps or inoculating loop to ensure firmcontact.
5. Incubate the plates in the inverted position at 37° C for 16 to 18 hours, andthen refrigerate them until observed. The short incubation time is neces-sary because bacteria may overcome the inhibition, and bacterial over-growth may occur during extended periods.
6. Examine the plates for the presence of clear zones of inhibition sur-rounding the disks, as shown in Figure 14.1. These zones represent areaswhere bacterial growth was inhibited by the antibiotic. With a millimeterruler, measure the diameter of the zone, including the disk. Measure-ments should be made to the nearest millimeter. Use the bottom of theplate for your measurements. The determinations should be entered inTable 14.2 in the Results section.
7. Referring to Table 14.1, determine whether the organism was susceptibleto the antibiotic (S), showed intermediate susceptibility (I), or was resistant(R). Enter these determinations in Table 14.2 next to the diameters. Also,note whether tiny colonies appear within the zone of inhibition. Diagramsof the plates may be entered in the spaces provided.
136 14 T H E E F F E C T O F A N T I B I O T I C S O N B A C T E R I A
!After swabbing plates,always place the swabs intodisinfectant to avoidcontaminating nearbymaterials.
Quick ProcedureAntibiotic Susceptibility Test
1. Inoculate a plate of testmedium with bacteria.
2. Place antibiotic disks onthe surface.
3. Incubate.
4. Observe and measurezones of inhibition.
5. Determine S, I, or Rfrom table.
1 2 3 4 5 6 7 8 9 10
Disk
Zone of inhibition
Bacterial lawn
F I G U R E 1 4 . 1Measurement of zones of inhibition on antibiotic susceptibilityplates.
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T H E E F F E C T O F A N T I B I O T I C S O N B A C T E R I A 14 137
continued*Courtesy BD Biosciences.
TABLE
Diameter of Zone of Inhibition and Interpretation*