Module content -AST by agar dilution and E test method

Minimum Inhibitory concentration:

Minimum inhibitory concentration (MIC) is defined as lowest concentration of an antimicrobial agent which inhibits the visible growth of microorganisms, MIC is considered a gold standard for determining the susceptibility of an organism against an antimicrobial agent

Advantages of MIC:

  • It is used to confirm unusual resistance pattern
  • When borderline results are obtained by disk diffusion method
  • When disk diffusion method testing a particular antibiotic against a microrganism may give unreliable results.
  • Widely used in antimicrobial resistance surveillance
  • Used in comparative testing of new antimicrobial drugs
  • Epidemiological studies of susceptibility patterns
  • When quantitative results are needed for clinical management of patient

 

MIC can be performed by different methods

  1. Agar dilution
  2. Gradient diffusion method
  3. Broth dilution
  4. Macro broth dilution
  5. Micro broth dilution method

  1. Agar dilution method: Prepare the antibiotic stock and antibiotic dilution as described previously. Now prepare the agar dilution plates as follows

If we need the dilution range e.g. 0.125 -128 ug/ml mark 12 test tubes/universal containers with dilutions 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.125, 0 mg/l. From the 10,000 mg/L stock add 256, 128, 64, 32 into the tubes no antibiotic is added in the tube marked as 0 which serves as antibiotic free growth medium

 

Preparation of agar dilution plates: Pour 20 ml of molten agar into the each container including the antibiotic free control tube. Mix well and pour each dilution into 90 mm plates, let the agar solidify, use the plates preferably in the same day or store at 4-8 0C

 

Preparation of inoculum:  Prepare the suspension of test isolate by picking isolated colonies from non-selective agar plate and emulsify in sterile distilled water till the turbidity is equivalent to 0.5 McFarland standard.

For streptococci, pseudomonas, acinetobacter, enterobacteriacae, staphylococcus, bacteroides species inoculum is further diluted 1:10 using sterile distilled water (104 cfu/spot)          

Inoculation:    Inoculate 1-2 uL spot onto the agar surface, may be inoculated manually or using a multipoint inoculator, allow the spots to dry before incubation.

Incubation: Incubate the plates at 35-37 0C in ambient air for most of the organisms if testing Haemophilus or pneumococci plates should be incubated with 4-6% CO2

Reading and interpretation:  First observe the controls, all organisms should grow on antibiotic free plates, MIC is the lowest concentration of antibiotic that inhibits the visible growth, single colony at the site of inoculation should be disregarded. MIC of reference strain should be within the acceptable ranges

Advantages of agar dilution method:

  • A number of isolates can be tested at the same time
  • Exact MIC value can be determined
  • Any mutatnts or contaminants can be easily recognized in agar dilution method
  • Range of antibiotic concentration can easily be extended

 

Disadvantages:

  • only one antibiotic can be tested on a single set of plates
  • This method is time consuming and laborious, requiring extensive technical resources
  • Swarming of proteus species may hinder result interpretation
  • Plates prepared with antibiotics have limited expiry dates, delay in using these plates may result in antibiotic deterioration

 

For Example we tested ceftazidime against P.aeruginosa isolate and MIC turns out to be 2 ug/mL, refer to CLSI guidelines for MIC values and this falls into category labelled as “Sensitive”

 

 

  1. E test method:

E test strip is a non-porous plastic strip coated with predefined stable gradient of antibiotic concentrations, upper side of the strip is marked with MIC scale. This method is useful to determine the MIC of fastidious, slow growing or nutritionally deficient organisms, it is helpful in detection of low level resistance and specific antimicrobial resistance phenotypes such as ESBL, AMPC etc.

Principle: E test is a quantitative technique that is based on concept of both dilution and diffusion principle for susceptibility testing.

Procedure: Take out the strips from the freezer -20 0C and bring it to room temperature.

Pick several well isolated colonies from a non-selective agar medium and emulsify in sterile saline to prepare a suspension with turbidity equivalent to 0.5 McFarland standard

With a sterile cotton swab prepare a uniform lawn of inoculum onto the agar surface

Allow the surface to dry

Using a sterile forcep gently drop the E strip on to the agar surface with MIC scale facing upwards, taking care not to move the strip once it touches the agar surface

Incubate the plates according to the organism’s specific growth requirements

Observe and read the zone of inhibition, read MIC value as the point where ellipse intersects the strip

 

Test is not valid if the lawn is too light or too heavy or there is mixed growth of test organisms

Interpret the MIC readings following the CLSI/EUCAST guidelines, to go to CLSI 2016 guidelines click the following link

http://ljzx.cqrmhospital.com/upfiles/201601/20160112155335884.pdf