ANTIBIOTIC SUSCEPTIBILITY TESTS

Antibiotic susceptibility tests were created due to the development of resistant strains, which aids in the
identification of an effective drug.

MOST WIDELY USED TESTING METHODS

 Broth Microdilution/rapid automated instrument methods

MANUAL METHODS
 Disk diffusion
 Gradient diffusion methods

Macrobroth or Tube-Dilution Method

 2-fold dilutions of antibiotic
 Faster (uses only smaller quantities)
 Bacterial suspension: 1-5x105 CFU/mL
o Advantage: Generation of quantitative result (ie. the MIC)
o Disadvantage: Tedious process (it's small, accuracy of pipetting skills is needed; errors may
come in)
 Also convenient - no need to wash
 Lowest concentration - minimal inhibitory concentration (MIC)

Broth Dilution Method

 Must be standardized with Mcfarlan (for uniformed density)
o MBC - minimum bactericidal (kills) count

How to calculate MIC (dilutions)

 ELISA READER

E-TEST
 Determines the susceptibility
 Etest - thin plastic test strips impregnated with a dried antibiotic concentration and with a
concentration scale
 2-3, expensive
 Best suited to MIC for only 1/2 drugs needed
 Systematic biases

Easy-to-use MIC-TECH
 Just apply the strip to an inoculated agar plate either manually and incubate
 After incubation, an ellipse will appear

Disk Diffusion Susceptibility Test

 Simple and practical
 Bacterial inoculum of approx. 1-2x108 CFU/mL
 Zone diameter of each drug are interpreted using the criteria = CLSI Standard
 Results of test are qualitative
 Highest concentration is closest to the disk
  Zone of Inhibition (measured to the nearest mm) is used to determine the effectiveness of the
antimicrobial agent
o Affecting size of zone of inhibition (general)
 Molecular weight of the antimicrobial agent
 Type of media
 Concentration of the bacterial culture
 Too many variances - a standard was made: KIRBY BAUER METHOD

COMPLETE FACTORS AFFECTING SIZE OF INHIBITION

 Inoculum density
 larger zones with light inoculum, vice versa
 Timing of disc application
 After application of disc, plate is kept for a long time at RT, small zones may
form
 Temperature of Incubation
 larger zones with <35 degrees Celsius
 Incubation time
 16-18 hours
 Depth of agar
 thin media = large ZOI
 Plate size
 Proper spacing of discs
 2.5 cm
 Potency of antibiotic discs
 deterioration in content leads to reduced size
 Composition of medium
 affects rate of growth, diffusion of antibiotics and activity of antibiotics
 Acidic pH of medium
 Tetracycline, novobiocin, methicillin zones are larger (interfere with
susceptibility and to yield good growth
 Alkaline pH of medium
 Aminoglycosides, erythromycin zones are larger
 Reading of zones

Kirby-Bauer Method
 Standardized method to ensure reliable results that takes all factors into consideration
 Agar used: Mueller-Hinton agar
o pH 7.2-7.4; poured to 4mm of depth in 100 or 155 diameter plates
 Depth is essential in diffusion of AM agent
o Inoculated with a specific concentration of bacteria; measured through turbidity as
compared to McFarland standards

Mueller-Hinton Agar
 Few properties make it excellent for antibiotic use
 Starch is known to absorb toxins released from bacteria (cannot interfere with antibiotics)
 A loose agar - allows better diffusion of the antibiotics than most other plates
 Non-selective, non-differential medium
Beef Extract
 Contains beef extract and acid hydrolysate of casein
o Nutrients, vitamins, amino, acids, sulfur, other essential nutrients

Stokes Method for Antibiotic Sensitivity Testing

 In stokes controlled . . . a control organism is inoculated in part of plate and the test organism is
plated on the remainder

Insert drawing

 Disks are placed at the interface of the zones of inhibition as compared.

Fig. Antifungal Assay Mycelial Expansion

Agar Plug Diffusion Method

 Used to highlight the antagonism between microbe
 Substances diffuse from the plug to the medium
o Uses puncher of different sizes (for agar plug and well)
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Agar Well
 Punch a hole
o To evaluate antimicrobial activity of plants or microbial extracts
 Used for solid or sticky stuff
 A hole with a dm of 6 to 8 mm is punched aseptically with a sterile cork

Cross Streak Method

 Test actinobacteria vs other bacteria
 Used to rapidly screen microbes for antagonism
 Microbial strain of interest is seeded by a single streak in the center of the agar plate

Thin Layer Chromatography

 In 1946, Woodall and Levi
 Uses a longer and shorter wavelength
 Showing migration and class of compound
o Direct bioautography
 Most applied method
 TLC-direct - useful for rapid chemical and biological screening of plant extracts

Agar Overlay Assay

 Immersion bioautography

Time Kill Test

 Appropriate for determining the bactericidal and fungicidal effect
 What dose you can kill these bacteria/fungi
o Brine Shrimp Lethality Assay (uses paramecium to check)

ATP Bioluminescence Assay

  Luminometer - detection of microbes
 Adhesive triphosphate
 Used in food testing

Flow Cytofluorometric Method

 Rapid detection of damaged cells depends on appropriate dye staining