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Microbiology Practical: A Guide To Becoming A Pro In The Lab

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Experiment Three: Antibiotic Susceptibility Testing of Bacteria

Abstract: Antibiotics are a very useful tool in treating illness caused by bacteria. Specific antibiotics act against specific bacteria in different ways to inhibit the growth of the bacteria. Spread plates with E.coli, E.cloacae and K.pneumoniae were prepared on Mueller- Hinton agar. An antibiotic dispenser was used to dispense six antibiotic discs onto each plate surface. The effectiveness of each antibiotic on the three bacteria was determined by measuring the radius of the ring of inhibited bacterial growth seen around each antibiotic disc. It was seen that E.coli was susceptible to Ciprofloxacin, E.cloacae was susceptible to Gentamycin and streptomycin and K.pneumoniae was susceptible to all antibiotics used, except for ampicillin.

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Introduction: Antibiotics are used to treat bacteria related illnesses. The correct antibiotic must be used in treatment, as antibiotics influence different bacteria in different ways. Antibiotic susceptibility of a species of bacteria can be tested by using the disc diffusion method. This method is an acceptable and accurate test when species- specific breakpoints are applied. (S. Ringertz et al, 1991). Antibiotic discs are placed on Mueller-Hinton growth plates of bacteria in order to view the area of inhibited growth, to determine the effectiveness of the antibiotic on that bacteria. Mueller-Hinton plates are used, as they ensure that toxic substances produced by the bacteria do not influence results. In this experiment, the zone of inhibited growth was measured using a ruler and the naked eye, however automated readings by camera and computer systems may be more accurate for standardisation. (E. A. Idelevich et al, 2016). Flourescence-based methods are also a useful tool in measuring antibiotic susceptibility of bacteria. One of these methods uses gradient potential differences across plasma membranes by the build-up of positively charged dyes within the membrane. (B. L Roth et al, 1996). This allows for the determination of how effective plasma membrane antibiotics are on bacteria.

Aim: To determine the susceptibility of E.coli, E.cloacae and K.pneumoniae to certain kinds of antibiotics.

Method: Three Mueller- Hinton agar plates were labelled with E.coli, E.cloacae and K.pneumoniae, respectively. 100µL of each of the respective overnight cultures was spread onto the Mueller-Hinton plates. The glass spreader used and the overnight growth tube openings were flamed before and after each use, and new pipette tips were used each time, to ensure sterility. This was then allowed to dry for 15 minutes. An automatic disc dispenser was then used to dispense 6 antibiotic discs onto the surface of the agar plates. This was then incubated overnight at 37⁰C and growth was observed the next morning.

Results and Discussion: the images below show the growth seen on each plate after overnight incubation.

The antibiotic susceptibility of each bacteria to the certain antibiotics used can be seen in the tables shown below.

  • E.coli
  • Antibiotic Class Antibiotic Abbreviation Concentration
  • (µg) Radius
  • (mm) R/I/S
  • Beta lactams Ampicillin AMP 10 0 R
  • Aminoglycosides Streptomycin STR 10 0 R
  • Tetracycline Oxytetracycline OXYTET 30 18 I
  • Glycopeptides Vancomycin VAN 30 7 R
  • Macrolides Erythromycin ERY 15 12 R
  • Quinolone Ciprofloxacin CIP 5 31 S
  • Table 3.1, showing effects of antibiotics on E.coli growth inhibition.
  • E.cloacae
  • Antibiotic Class Antibiotic Abbreviation Concentration
  • (µg) Radius
  • mm) R/I/S
  • Aminoglycosides Gentamycin GEN 30 22 S
  • Tetracyline Oxytetracycline OXYTET 30 12 R
  • Aminoglycosides Streptomycin STR 10 10 S
  • Glycopeptides Vancomycin VAN 30 9 R
  • Beta lactams Ampicillin AMP 10 0 R
  • Macrolides Erythromycin ERY 15 8 R
  • K.pneumoniae
  • Antibiotic Class Antibiotic Abbreviation Concentration
  • (µg) Radius
  • (mm) R/I/S
  • Aminoglycosides Gentamycin GEN 30 26 S
  • Tetracyline Oxytetracycline OXYTET 30 18 I
  • Aminoglycosides Streptomycin STR 10 15 S
  • Glycopeptides Vancomycin VAN 30 19 S
  • Beta lactams Ampicillin AMP 10 0 R
  • Macrolides Erythromycin ERY 15 23 S
  • Table 3.3, showing the effects of antibiotics on K.pneumoniae growth inhibition.
  • In the above tables, R=resistant, I= intermediate resistant and S= susceptible. The radius of the ring of inhibited growth gives an indication of whether the bacteria are susceptible, intermediate resistant or resistant to a specific antibiotic. The below key was used to determine this.

    Antibiotic Class Antibiotic Abbreviation Concentration

    • (µg) R I S
    • Quinolone Ciprofloxacin CIP 5 ≤15 16-20 ≥21
    • Aminoglycosides Gentamycin GEN 30 ≤12 13-14 ≥15
    • Tetracyline Oxytetracycline OXYTET 30 ≤14 15-18 ≥19
    • Aminoglycosides Streptomycin STR 10 ≤6 7-9 ≥10
    • Glycopeptides Vancomycin VAN 30 ≤14 15-16 ≥17
    • Beta lactams Ampicillin AMP 10 ≤16 – ≥17
    • Macrolides Erythromycin ERY 15 ≤13 13-22 ≥23

    We can see from the results that E.coli was susceptible to ciprofloxacin and intermediate resistant to oxytetracycline, but showed resistance to all other antibiotics used. E.cloacae was susceptible to gentamycin and streptomycin and resistant to all other antibiotics used. K.pneumoniae was susceptible to all antibiotics used, except for ampicillin, for which it was resistant and oxytetracycline, for which it was intermediate resistant. Therefore when treating E.coli, ciprofloxacin must be used, when treating E.cloacae related illnesses, gentamycin or streptomycin must be used, and when treating pneumonia, a variety of antibiotics can be used, but ampicillin must be avoided.

    Conclusion: Different bacteria are affected in various ways by different antibiotics that are used to inhibit their growth. E.coli and E.cloaca are resistant to many different antibiotics. Antibiotic resistance in bacteria is becoming a huge problem for practitioners, making treatment of bacterial illnesses very difficult. It is very important that the correct antibiotic be used for the correct bacterial infections, otherwise treatment will not be effective.

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