Skip to main content
Biology LibreTexts

Lab 6: Antibiotic Susceptibility Testing

  • Page ID
  • \( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)

    clipboard_ed84101ca79506377949add8647f9cc57.pngIn microbiology, McFarland standards are used as a reference to produce solutions that contain approximately similar numbers of bacteria for use in standardized microbial testing (Kirby Bauer). This is done by matching the turbidity (cloudiness) of McFarland standard with that of the test solution. Therefore it is important to always mix the McFarland standard before beginning. The standard can then be compared visually to a suspension of bacteria in sterile saline. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the suspension is not turbid enough, more bacteria can be added.

    McFarland used today will be 0.5 = ~\(1.5 \times 10^8\) CFU/mL.


    clipboard_ea365aa35dc7b7df5d9bc82a2ca8e5531.pngMueller-Hinton Plate (MH Plate): A growth medium that is commonly used for antibiotic susceptibility testing and allows for even diffusion of the antibiotic in the medium. The Kirby-Bauer antimicrobial disk diffusion procedure is used with MH plates. The impregnated disk is placed on an agar surface, resulting in diffusion of the antimicrobial into the surrounding medium. Effectiveness of the antimicrobial can be shown by measuring the zone of inhibition (ZOI) for a pure culture of an organism.


    1. Working individually you will use a 0.5 McFarland Standard (1.5 x 108 CFU/mL) as a visual standard to make your own liquid standard from the plate provided to you (make sure to mix the standard tube before comparing turbidities).

    2. Use a cotton swab to take a small amount of your bacteria from your plate and gently swirl it into your tube of saline until the turbidity matches that of your McFarland Standard.

    Hold both tubes up to the light as you are swirling to best match the turbidity.


    3. Create a full lawn from your liquid standard sample on Mueller-Hinton (MH) plate by dipping a new cotton swab ONCE into your McFarland Standard you have made and then swabbing a single plate 4X in four different directions.


    4. Place your plate on the diagram on the handout.

    5. Dip your forceps into the ethanol, flame BRIEFLY, and wait for all the ethanol to evaporate. Repeat this step 3 times.

    In this procedure, it is the ethanol (not the heat) that disinfects the forceps. So, there is no need to hold the forceps in the flame for long periods of time.

    6. Aseptically add the six standard antibiotics, incubate at 37°C.

    7. Discard your saline tubes on the biohazard rack. DO NOT put them back with the unused saline!

    The antibiotic discs are labeled; no additional labeling for the discs is necessary.


    1. Measure the diameter of zones of inhibition is in mm. for each of your antibiotics and record your results below.

    2. If you can’t get the diameter because of an inadequate lawn or zones overlapping, measure the radius and multiply by 2.

    3. Compare your results to the chart provided to determine if your bacterial species is resistant or susceptible to the antibiotics we used.


    Bacterial Species:____________________________________________________________________________________________________________________________
    Antibiotic Species ZOI

    Resistant (R)/Susceptible (S)/Intermediate (I)

    Penicillin (p10)
    Chloramphenicol (C30)
    Trimethoprim (TMP5)
    Ciprofloxacin (CIP5)
    Streptomycin (S10)
    Augmentin (AMC30)

    You do not need to turn in this sheet but you will need it to answer questions on the worksheet on the next page.

    Lab 6: Antibiotic Susceptibility Testing is shared under a CC BY-SA license and was authored, remixed, and/or curated by Nazzy Pakpour & Sharon Horgan.

    • Was this article helpful?