ANTIMICROBIAL SUSCEPTIBILITY TESTING

Objectives:
 Explain the rationale in performing Antimicrobial Susceptibility Testing (AST)
 List the steps required to perform disk diffusion test
 Name the variables that must be controlled when the test is performed and how it would affect the results

Indications for Performing AST

 To determine the sensitivity or resistance of pathogenic bacteria to various antimicrobial compounds in order to assist the physician
in selecting treatment options for the patient.
 In vitro testing- done before drugs/antibiotics are administered to patients to determine antimicrobial activity to measure
susceptibility of a given microorganism to known concentration of drugs
o Make sure that the antibiotics given to the patient will have a therapeutic success (it can kill bacteria that caused infection to the
patient).
o Important to conduct the test only to the bacteria that caused the infection because if performed in the wrong organism, it can
contribute to the development of antimicrobial resistance.

Methods:
1. Broth dilution- quantitative; concentration of the antibiotic required to inhibit the growth of bacteria
o Gives MIC and MBC- reference methods for AST
 Minimum Inhibitory Concentration (MIC)- Lowest concentration of antibiotic that inhibits the visible growth of an organism
in vitro
 Minimum Bactericidal Concentration (MBC)- Lowest concentration of the drug that kills 99.99% of bacteria
o Quantitative: Broth or Agar Dilution (Broth: liquid media; Agar: plated media)
o MIC Procedure:
 Create Serial Two Fold Dilution of the antibiotic are inoculated in the standard suspension of organism and incubate for 16-18
hours or as long as 24 hours in 35°C
 The final bacteria concentration for the broth or agar dilution technique is 5 x 105 CFU/mL
 Observe 2 things: turbidity or absence of turbidity
 After overnight incubation at 35°C, MIC is determined visually by the lowest concentration of drug that inhibits the growth
demonstrated by the absence of turbidity
 Absence of turbidity = microorganism is susceptible to the antibiotic and indicates the concentration that inhibits its growth
 Example: As low as 2 µg/mL, the bacteria is inhibited
 After performing MIC, proceed to MBC
o MBC Procedure:
 Inoculate from MIC cultures into the antimicrobial-free media by subculturing the tubes
with no bacterial growth in agar plates
 Check the agar plates for presence/absence of growth/colonies
 Example: As low as 8 µg/mL, it kills 99.9% of the bacteria
2. Disk Diffusion- qualitative; gives interpretation of Sensitive or Resistant
o Kirby Bauer Disk Diffusion Method (since 1966)
o Determine the presence/absence of growth around the disc which is an indirect measure of the ability of the antibiotic to inhibit the
microorganism
o Check on the plate: clearing around the disk = clear zones- Zone of Inhibition
 Zone of inhibition (ZOI)- diameter of clear zones indicating no bacterial growth which are being measured
 Absence/presence of growth around the disk is an indirect measure of the ability of the antibiotic to inhibit the organism
o 6mm impregnated disk- antibiotic impregnated on to the disk will diffuse into the agar and this clearing indicates bacterial growth
and are being measured
o Materials:
 Broth suspension of bacteria  Antibiotic impregnated discs
 Trypticase soy agar (replace) or Mueller Hinton agar  Sterile cotton swab
(culture medium of choice)  Forceps
o Test Procedure Overview:
 Once isolated colonies are available from an organism that has been identified as a potential pathogen, it is necessary to proceed
as follows to perform the susceptibility test.
1. Select colonies 5. Add antimicrobial disks
2. Prepare inoculum suspension 6. Incubate plate
3. Standardize inoculum suspension 7. Measure inhibition zones
4. Inoculate plate 8. Interpret results
o Step 1: Selecting colonies

It is important that in selecting colonies for the test it should be in isolated pure colony.
 In the 4 quadrants of colony growth, obtain colonies from 4th quadrant to make sure what
is tested is the potential pathogen that causes infection to the patient.
 Loop or cotton swab to pick well-isolated colonies
 If you do not have well-isolated colonies, subculture the organism to a fresh plate
o Step 2: Inoculum preparation (most critical step)
 Most critical step because we have to make sure that the number of bacteria present in the suspension is the correct cell density
required for the test.
 Two methods:
 Direct colony suspension- use of saline; colonies not older than 18-24 hours
 Log phase growth- use of broth; growth occurs after 2-8 hours of incubation
 Log phase growth
 4 phases of bacterial growth
 Lag phase- little or no growth of bacteria is observed because they are preparing to divide
 Log/Exponential phase- 2-8 hrs after incubation, maximum rate of bacterial growth is observed and the most susceptible
antimicrobials due to high bacterial metabolic activity
 Plateau/Stationary phase- number of bacteria alive= dead; due to limited nutrients in the environment
 Death/Degradation phase- increase in number of dead bacteria
 Preparation of Bacterial Suspension for AST:
1. Using sterile wire loop, touch isolated colonies of the same colony morphology of the organism to be tested
2. Suspend the organism in 2 mL of saline
3. Vortex to create a smooth suspension
4. Compare side by side and adjust the turbidity to 0.5 McFarland Standard
o Step 3: Inoculum Standardization
 Positioned side by side with standard, against a white card containing horizontal black lines
 Turbidity to be observed by looking to the black lines through the suspension
 Too dense= too many bacteria- difficult to see the lines through the inoculum
 Comparison be done under adequate lighting
 Lighter than McFarland standard- add more bacteria (colonies)
 Denser than McFarland standard- add Sterile NSS to dilute it
 If too light for direct colony suspension- add more colonies into the broth/saline
 If too light for log phase method- reincubate the suspension
 0.5 McFarland Turbidity standards
 Use to standardize approximate number of bacteria in liquid suspension by visually comparing turbidity of test suspension with
McFarland standard
 Required cell density of the bacterial preparation: 1.5x108 CFU/mL
 If not available commercially, it can be prepared in the lab
 Add 0.5 mL of 1.175% of BaCl2 + 99.5 mL of 1% H2SO4 (stirring constantly)
 If measured spectrophotometrically = absorbance should be 0.08-0.13 at 625 nanometers in order to meet the expected
bacterial density of the suspension
 After preparing suspension, dispense to small tubes of 4-6 mL, sealed tightly and stored in the dark at room temperature for
6 months
 Provides an optical density comparable to the density of a bacterial suspension approx. 1.5 X 10 8 CFU/mL
 Inoculum suspensions should be used within 15 minutes of preparation on Mueller-Hinton Agar using Overlapping Streak
Method
 Mueller-Hinton Agar (MH)
 Unsupplemented M-H Agar: Testing Non-Fastidious Organisms- not require special nutrients to grow
 Beef infusion 300 g/l  Starch 1.5 g/l
 Casamino Acid 17.5 g/l  Agar 17 g/l
 Beef infusion and casamino acid will provide nitrogen, vitamins, carbon and amino acids
 Starch is added to absorb toxic metabolites that may be produced
 Agar is the solidifying agent
 Supplemented M-H Agar: Testing Fastidious Organisms- require special or added nutrients to grow
 All of the above plus:
 5% defibrinated sheep or horse blood,  2% sodium chloride
 1% growth supplement
 Mueller-Hinton Agar with 5% Sheep Blood
 Testing streptococci
 Incubated 5-7% CO2
 Haemophilus Test Medium of Mueller-Hinton Medium base supplemented with X (Hematin) & V (NAD) Factors
 Haemophilus influenzae  Haemophilus parainfluenzae
  GC (Gonococcal) Agar- N. gonorrheae
 MH Agar with 2% NaCl- Methicillin resistant S. aureus (MRSA)
 Factors that have to be standardized:
 Standard Agar Depth: 4-6 mm (4mm)
 <4 mm (too thin): false susceptible results; >6 mm (too thick): false resistant results
 150 mm plate- dispense approximately 60 mL agar
 100 mm plate- dispense approximately 25 mL agar
 Cation Content
 Ca2+ and Mg2+ content too high: false resistance for tetracyclines and aminoglycosides
 Ca2+ and Mg2+ content too low: false sensitive results for tetracyclines and aminoglycoside
 pH of the Mueller Hinton Agar: pH 7.2 – 7.4
 pH < 7.2: False resistant results
 pH > 7.4: False sensitive results
 Preparing Agars: using powder (Plated medium)
1. Weigh
2. Dissolve Agar powder in distilled water
3. Sterilize using autoclave
4. Dispense it to empty sterile petri dishes
 Antibiotic Cartridges
 6mm paper disc is impregnated with known concentration of an antimicrobial compound
 Once opened, store in a sealed container (refrigerator temperature) with a dessicant for not longer than 1 week (2-8°C)- working
supply
 Freeze (-20°C) for up to 1 month in a dessicator- long term storage
 Allowed to warm to room temp before use for about 1-2 hours
 If used cold- create condensation and moisture = affect the interpretation of results and ZOI
o Step 4: Inoculation of the Mueller-Hinton Agar
1. Dip a sterile swab into the inoculum tube.
2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid (so as to avoid
over-inoculation). The swab should not be dripping wet
3. Inoculate the dried surface of a MH agar plate by streaking the swab 3 times over the entire agar surface; rotate the plate
approximately 60 degrees each time to ensure an even distribution of the inoculum
4. Rim the plate with the swab to pick up any excess liquid.
5. Discard the swab into an appropriate container.
6. Allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate
to dry before proceeding to the next step.
o Step 5: Antibiotic Disc Placement (Individual/Manual)
 Dispense antibiotics manually using sterile forceps making sure the forceps is allowed to cool down prior to using
 When dispensing antibiotics to the inoculated MH agar plate, the entire surface of the antibiotic touches the plate and should be
placed flat on the surface and tap with light pressure to ensure adhesion on the plate
 Once the disk is on the plate, it should not be moved/transferred because diffusion around the disk will be observed very rapidly
and the reading of the ZOI will be affected
 Disk dispenser may be used to ensure even distribution of antibiotics on the plate.
 How many disk should be placed in the inoculated plate:
 150 mm plate- max of 12 antibiotic discs  100 mm plate- max of 5 antibiotic discs
 Placement of Discs
 15 mm from the edge and 20 mm equidistant from each other = to ensure no overlapping of the zone of inhibition
 Technical Considerations (Summary)
 Inoculation of bacterial/inoculum suspension (adjusted) to McFarland standard (MHA plate)
 Incubate/allow the plate to sit at room temperature (3-5 mins) not more than 15 minutes
 Application of antimicrobial disks on the inoculated MHA plate
 Invert plates and incubation after disk application.
 Use of non-expired disks (allowed to warm at RT 1-2 hrs)
 Press each disk down firmly to ensure complete, level contact with the agar.
 Do not relocate a disk once it has touched the agar surface
 Technical Errors:
 False Resistant
 Heavy inoculum  Delay in disc application
 Thick medium  Increase Ca/Mg (aminoglycosides) P. aeruginosa
 False Sensitive
 Light inoculum
 Thin medium
  Factors Affecting The Test:
1. Standard agar depth (4mm)
2. pH of the agar (7.2-7.4)
3. Cation content
4. Age of the inoculum use within 15 minutes after preparation
5. Cell density of the inoculum (1.5 x 108 CFU/mL)
6. Antibiotics discs (6mm) used:
 150 mm plate- max 12,  15 mm distance from the edge
 100 mm plate- max 5  20 mm equidistant from other antibiotics
7. Place antibiotics within 15 minutes of inoculated MH
 Ensure complete, level contact with agar
o Step 6: Incubation
 After dispensing the antibiotics on the inoculated Mueller-Hinton Agar, incubate for 16-18 hours in 35-37°C
 Check for a confluent lawn of growth
 The entire surface of MH agar is inoculated with the test suspension making sure there is only single colony type present
 Check for purity of growth
 Factors Affecting The Test:
 Incubating the Plate
 Invert and incubate plates with agar side up
 Temp, Atmosphere, and Duration of Incubation (for non-fastidious bacteria)
 Ambient air at 35C for 16 – 18 hours
o Step 7: Measure inhibition zones
 Measure the diameters of clear zones around the disk indicating no bacterial growth
 Determines the sensitivity of resistance of certain pathogenic bacteria to different antimicrobials
using antibiotic-impregnated disc
 Done by a Vernier Caliper or Ruler
 Round up to the nearest mm
 In there’s overlapping the zone of inhibition, then measure the radius and multiply it by 2
 To measure zones from the back of the plate using reflected light:
 Hold the plate a few inches above a black nonreflecting surface
 Measure to the nearest millimeter using a Vernier caliper/ruler
 If ZOI for MH with 5% sheep blood is measured, remove lid and measure the ZOI at the top
 How To Use Vernier Caliper
 Use cm and outside jaws and Vernier scale- movable one
 Outer Jaw- outer diameter; Inner Jaw- inner diameter
 Find the last value on the main scale before the zero line on the Vernier scale.
 Main scale (stationary); vernier scale (movable); increments of 0.1 cm
 Reading in cm, multiplied by 10 since reporting is in mm (ex: 2cm= 20mm)
 There are kinds of Mueller-Hinton agar that a change of color is observed after
incubation.
 Usually observed in Pseudomonas aeruginosa which produces pigments and
creates a color change on the medium itself.
o Step 8: Interpret results
 Double zone: Measure the innermost zone
 On repeat test: Measure the colony free zone
 Repeat test with single colony or subculture of single colony from primary culture
plate.
 Zones with swarming (Proteus mirabiis): measure the obvious zone. Ignore the
swarm even if it covers the zone
 Turbidity comes from the swarming of the microorganism because of the flagella
 Feathered zone around CAZ disk: Measure the point at which you can see an
obvious demarcation between growth and no growth
 Results for the zone of inhibition is compared to the antibiograms
 Antibiograms: Antibiotic susceptibility patterns
 The number value will tell if the organism is susceptible/intermediate/resistant to the organism.
 Each organism has its own panel of antibiotics to use
 Sample Antimicrobial Interpretative Chart
 Determination of which antibiotic has to be used/reported is identified by the study
performed by the Clinical Laboratory Standards Institute (CLSI)
 Group A antibiotics- primary antibiotics that has to be tested and reported
 Group B antibiotics- antibiotics tested in the lab and are reported selectively
  Example:
 Organisms: Proteus mirabilis (Gram-Negative Enteric Organism)
 Group A (Primary Test & Report): Gentamicin and Ampicilin
 Group B (Primary Test, Repost Selectively): Ceftriaxone and Levofloxacin; Tetracycline and Chloramphenicol
 Interpretation of Results:
 Sensitive/susceptible (S)- microorganism can be killed by the given antimicrobial with a high likelihood of therapeutic success.
 Intermediate (I): bacteria are inhibited in vitro by a concentration of this drug that is associated with an uncertain therapeutic
effect.
 Resistant (R): bacteria are inhibited in vitro by a concentration of this drug that is associated with a high likelihood of
therapeutic failure.
 Interpretation: “The organism is susceptible/resistant to the antibiotic”
 “The antibiotic is susceptible/resistant to the organism” (unacceptable interpretation)

Mechanism of Resistance to Antimicrobials:

 Enzymatic degradation or modification of antimicrobial agent
 Decreased uptake of the antimicrobial agent
 Altered antimicrobial target

Semi and full automated MIC Testing

 Kirby Bauer Test- traditional, simplest, practical method
 E-Test (Semi-Quantitative)
o Has the combined convenience of disk diffusion with the ability to generate MIC data.
o Uses plastic strips
 BIOMIC
o Combines the ease of conventional disk diffusion with video digital analysis to automate interpretation of ZOI

Antimicrobial Susceptibility Testing Kirby Bauer Disk Diffusion Method

 Purpose: To determine the sensitivity or resistance of pathogenic bacteria to various antimicrobial compounds
 Principle:
o A standardized suspension of organism is inoculated onto Mueller Hinton agar. Paper disks impregnated with specific antibiotic
concentrations are placed onto the agar.
o After 18-24 hours of incubation the diameters of the zones of inhibition are measured. The results are compared with established
values to determine the organism’s susceptibility or resistance to each antibiotic.
 Materials: Broth suspension of bacteria, Mueller Hinton agar, antibiotic discs, cotton swab, forceps, Vernier caliper
 Procedure: Inoculation of the Mueller-Hinton Agar
1. Dip a sterile swab into the inoculum tube. Rotate the swab against the side of the tube (above the fluid level) using firm pressure,
to remove excess fluid. The swab should not be dripping wet.
2. Inoculate the dried surface of a MH agar plate by streaking the swab three times over the entire agar surface; rotate the plate
approximately 60 degrees each time to ensure an even distribution of the inoculum.
3. Rim the plate with the swab to pick up any excess liquid. Discard the swab into an appropriate container. Allow the plate to sit at
room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to
the next step.
4. Using an alcohol-flamed, fine pointed forceps (cool before using) place the antibiotic discs on the previously inoculated plate.
Press the discs firmly into the agar so that it is in full contact with the agar surface. The disc should be placed equidistant from
each other.
5. Incubate plates at 37C. Measure and interpret the zone of inhibition after incubation.