antibiotic assay

8/17/2019 Antibiotic Assay

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1 2ndYear Nutrition

Practical

Ms. Banan A. Atwah

1431 1432 H

ANTIBIOTIC ASSAY

Objectives:

To perform antibiotic susceptibility test using Kirby-Bauer disc diffusion

method (qualitative measure).

To learn how the zone of inhibition and its interpretation for a particular

antimicrobial agent. To determine E-test (semi-quantitative measure).

To determine Minimum Inhibitory Concentration (MIC) of an

antimicrobial agent against a test organism using tube dilution assay.

To determine Minimum Bactericidal Concentration (MBC) and both

are (quantitative measure).

Background: A susceptibility test may be performed for two main purposes:

a. To guide the clinician in selecting the best antimicrobial agent for

patient.

b. To accumulate epidemiological information on the resistance of

microorganism of public health importance in the community.

Antimicrobial susceptibility tests measures the ability of an antimicrobial

agent to inhibit the bacterial growth in vitro. Methods used to measure

inhibition are either agar diffusion or dilution assays.

Sensitivity and resistance are usually based on antimicrobial

concentrations achievable in blood or urine during treatment.

Disc diffusion method (Kirby-Bauer) is recommended as general

purpose method or (qualitative method).

Tube dilution method is used to determine (MIC) and (MBC)

(quantitative method).


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2 2ndYear Nutrition

Practical

Ms. Banan A. Atwah

1431 1432 H

A. Kirby-Baur (disc Diffusion):

Individual filter paper discs or rings consisting of 8 paper disc(MASTRING), each impregnated with specific antimicrobial agents,

are used for susceptibility testing of the common rapidly growing

aerobic and facultative pathogens.

Materials:

1. Antibiotic filter paper discs.

2.

Muller Hinton agar plates. 3. Muller-Hinton broth.

4. Sterile loops.

5. Sterile swabs.

6. Sterile forceps.

7. McFarland turbidity standard.

8. Cultures of bacteria.

Method: 1. Prepare a suspension of an overnight culture of test organism in Muller-

Hinton broth by touching 5-10 colonies with a sterile loop. 2. Compare the turbidity of suspension to 0.5 McFarland standard. The

high-density inoculums lead to overestimation of resistance and a low-density of sensitivity.

3. Label Muller-Hinton agar plates with specimen number. 4. Dipping a sterile swab into the inoculums. Excess inoculums are

removed by pressing and rotating the swab against the side of tubeabove the level of suspension.

5. Streak the swab all over the surface of the medium three times, rotatingthe plate through an angle of 60 after each application. Finally encirclearound the edge of the agar surface with the swab. Leave the agar to drya few minutes at room temperature.

6. Using a pair of sterile forceps, place an antimicrobial disc ring oninoculated plate make sure that the disc are completely in contact withsurface of the medium (6 different rings are available with one for

isolates from urine, one for gram-positive bacteria, one forPseudomonas , two for gram-negative bacteria, and a general one fortopical pathogen).


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3 2ndYear Nutrition

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Ms. Banan A. Atwah

1431 1432 H

7. Incubate the plates at 35-37 ºC within 30 minutes of their preparation

for 18 hours.8. After incubation period, using a ruler, measure the diameter of zones ofinhibition.

1. Fig: st l u s e R

Measure the zone of inhibition in (mm) and record your results in the worksheet provided.

Use standard zone size table for the K-B test, Table.1, and classify the testorganism as sensitive, intermediate or resistant to the panel ofantimicrobials used.

Sensitive = Zone of inhibition equal or ≥ standard zone.Resistant = Zone of inhibition equal or ≤ standard zone.Intermediate = Zone of inhibition between two values.

Figure 1: Kirby-Bauer (Disc Diffusion)


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4 2ndYear Nutrition

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1431 1432 H

Table.1: Standard Zone Size Table For K-B Test


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5 2ndYear Nutrition

Practical

Ms. Banan A. Atwah

1431 1432 H

B. Tube Dilution Assay (for MIC and MBC):

:n oitini f e D

1. Minimum inhibitory concentration (MIC):It is the lowest or minimum concentration of an antimicrobial agent thatinhibits the growth of an organism.

2. Minimum bactericidal concentration (MBC):It is the lowest concentration at which bacterial growth does not occur is

called the minimum bactericidal concentration.

1. Minimum inhibitory concentration (MIC):

: s l air et a M

1. Muller-Hinton broth

2.

Muller-Hinton agar. 3. Sterile loop. 4. Sterile test tubes and pipettes. 5. Variable concentration of antibiotics.6. 0.5 McFarland turbidity standard.7. Cultures of bacteria.

: d o ht e M

1.

Arrange 6 sterile test tubes in a rack and dispense 1 ml of sterile Muller-Hinton broth into each tube.

2. Prepare a stock solution of the antimicrobial agent about 64 µg/ml todilute the initial concentration which will be 32µg/ml.

3. Add 1 ml of stock solution of 64µg/ml to the first tube. Make two-folddilution of the antimicrobial agent by transferring 1 ml of the solutionfrom the solution from the first tube to the second one.

4. Continue to make serial dilution till the entire range is covered. The lasttube concentration is 2µg/ml and do not add to the 6th tube as it act as

growth control. 5. Adjust the turbidity of bacterial suspension overnight growth by 0.5

McFarland standard.


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6 2ndYear Nutrition

Practical

Ms. Banan A. Atwah

1431 1432 H

6. Add 1 ml of this suspension to each dilution and control tube.

7.

Incubate the tubes overnight at 37 ºC.

2 Fig.: st l u s e R

Compare with the control tube read all tubes for visible growth record the

result. The lowest concentration with no visible growth is the MIC for thetest organism.

: )Minimum bactericidal concentration (MBC.2

: d o ht e M

1. Take from the first tube that show no turbidity and inoculate it bystreaking in Muller-Hinton agar.

2. Incubate the tubes overnight at 37 ºC.

: st l u s e R Record the lowest concentration giving no growth on subculture as MBC.

Figure 2: MIC method


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7 2ndYear Nutrition

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1431 1432 H

C. (E- Test): Fig.3

Strips containing a gradient of antibiotic are placed on lawn of bacteria andincubated overnight. MIC is determined at point where zone of inhibitionintersects scale on strip.

Figure 3: E- Test


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8 2ndYear Nutrition

Practical

Ms. Banan A. Atwah

1431 1432 H

Kirby-Baur (disc Diffusion)

Your Interpretation

Diameter ofzone

inhibition (mm)

Disc Potency Antibiotic

antibiotic assay

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