Microbio Lab 5

MICROBIOLOGY LABORATORY 5 USTMED ’07 Sec C – AsM; Photos provided by JV.N. & MeaM.

Єtest Susceptibility Testing Procedure

SENSITIVITY TESTING Antibiotic Sensitivity Procedure

Testing

Using

The

1.

inoculation of the organism for testing using cotton swab by overlapping streaking using Mueller Hinton Agar Plate.

2.

Overlaying of Єtest strip on the previously inoculated culture media

3.

Reading of the strip and recording the results

Kirby-Bauer

Kirby Bauer method. The test, introduced by William Kirby and Alfred Bauer in 1966, consists of exposing a newly-seeded lawn of the bacterium to be tested, growing on a nutrient medium (Mueller-Hinton agar) to filter paper disks impregnated with various antibiotics. The culture is incubated for 16 to 18 hours and then examined for growth. If the organism is inhibited by one of the antibiotics, there will be a zone of inhibition around the disk, representing the area in which the organism was inhibited by that antibiotic. The diameter of the zone of inhibition around an antibiotic disk is an indication of the sensitivity of the tested microorganism to that antibiotic. The diameter of the zone, however, is also related to the rate of diffusion of the antibiotic in the medium. This fact must be kept in mind then interpreting the zone of inhibition of various antibiotics. MATERIALS: culture of the organisms in Mueller-Hinton agar plate with antibiotic sensitivity discs Ruler graduated in millimeters NOTE: 1. 2.

The complete procedure is in you(r) lab [lab u 2! Hahaha!] manual. Measure the zone of inhibition and record your results. Interpret the results based on the table provided

Disk Diffusion Method Procedure 1. dip the sterile swab into bacterial suspension compared to 0.5 MF standard then swab onto the surface of Mueller Hinton Agar using Overlapping technique.

2. 3.

Єtest – The Problem Solver in Antimicrobial Susceptibility Testing Etest is an antimicrobial gradient strip for the quantitative determination of susceptibility or resistance of microorganisms. It is a robust and simple technique, minimally affected by laboratory variations and can be used to test most microorganisms. An accurate and reproducible Minimum Inhibitory Concentration (MIC) is generated for reliable guidance of antimicrobial therapy.

Allow the organism to be absorbed by the medium. Place the appropriate antimicrobial (sensitivity) discs using the dispenser or a sterile forceps. Incubate for 24 hours at 37o Reading and Interpretation of the results. Measure the diameter of the Zone of Inhibition (area wherein there is no growth around the discs) using the millimeter of a ruler. Record your results and Interpret based on the table provided. Determine if the Antibiotic (organism) is Sensitive, Intermediate or Resistant. If there is overlapping in the zone of inhibition, you can just measure the radius and multiply the reading by 2 to get the diameter.

E. coli ATCC 25922. The isolate tested on this MuellerHinton agar plate is interpreted as susceptible (S) to all antimicrobial agents. Reading clockwise from the top, MZ, AN, AM, CZ, CTX, CXM, CF, GM, NN: the three discs in the center of the plate are SXT, FOX and TIM. Pseudomonas aeruginosa, a resistant strain. Growth on this Mueller-Hinton agar plate indicates that the isolate is resistant to six of 12 antimicrobial agents and susceptible to the remaing. The isolate is resistant to SXT, GM, ATM, TIM, TIC and MMZ. The isolate is susceptible to CIP, AN, NN, CA, IPM and PIP. [email protected] [email protected]

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