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MIC of polymyxin B and Colistin sulphate

E-strips of  polymyxin B and colistin sulphate  use to determine MIC value  as shown above picture.

Principle of E- test

It is a recent modification of the agar diffusion sensitivity test. It uses  an absorbent strip with a known gradient of antibiotic concentration along its length. When the strip is placed on the agar plate inoculated with the  test , organism , the test antibiotic diffuses into the medium. The MIC is recorded as the lowest concentration of the gradient which inhibits the growth of the  organism.

Requirements for Epsilometer test

• Muller-Hinton agar
• E-test strips (5X50 mm antibiotic carrier)
• McFarland Densitometer/ O.5 McFarland Std
• Forceps
• Sterile cotton swabs
• Sterile normal saline
• Bunsen burner
• Inoculating wire

Procedure of Epsilometer test

Opening an E-test package

1. Remove the package stored at -20 °C or -70°C.
2. Equilibrate at room temperature. This time taken approximately 30 minutes if stored at –  20 ° and approximately one hour if stored at-70°C. Ensure all the moisture has evaporated before opening.
3.  Inspect the package for holes or crack. Do not use if damaged.
4. Cut along with broken line  at the top of a blister. Do not cut between the blisters.
5. Tip the strip out of the opening slightly and take them out with forceps.
6. If strips stick together, twist them apart with your fingers.
7. Only the handle i.e. the area labeled E.
8.  Place the strips to be used into a dry clean Petri dish.

Application of strips

• Apply E -test strips with forceps. Ensure the MIC scale is facing upward i.e. towards the opening of the plate.
• Emulsify 3 or 4 individual test strain colonies and transfer to a tube of saline.
• Compare turbidity to that in the 0.5 McFarland standards. Adjust turbidity of inoculum to match that standard or read with McFarland Std. densitometer.
• Ensure that agar surface is dry before swabbing it. Dip a swab in  the inoculum, remove excess fluid and swab the entire agar surface evenly in 3 directions (60°) .
• Allow the agar surface to dry for 10-15 minutes.
• Open the E-test package  and place the strips in a dry Petri dish
• Apply the strips to the  agar surface with a forceps. Always apply the strip with  the MIC scale facing the opening of the plate. Do not apply it upside down.
• Use the templates to position 4-6 strips on a 150 mm plate or 1-2 strips on a 90 mm plate.
• Place the handle of the strip closest  to the rim of the plate.
• Incubate at 37°C for 18-24 hours.

Observation of Epsilometer test

After incubation an elliptical zone of growth inhibition is seen around the strip. The MIC is read from the the scale at the intersection of the zone with the strip( as shown above figure).

Result and Interpretation of E-test

1. Read MIC at the point where ellipse intersects the scale. If a MIC value between two twofold dilutions is seen, always round up to the highest value.
2. Read the MIC value at complete inhibition of all growth.
3. If the intersect differs on either side of the strip, read the MIC as the greater value. Ignore any growth at the edge of the strip.
4. MIC values of the bacteria should be interpreted as S (Susceptible), I (Intermediate) or R (Resistant) by comparing the breakpoints values of each antibiotic with the criteria recommended by CLSI.

MIC readings of

• Polymyxin B: 1.0 µg/ml
• Colistin: 1.5 µg/ml

Precautions

1. Be firm when applying the strip. Once applied, do not move the strip.
2. Always store unused strips in airtight containers at -20°C or -70°C.

Related Video

# How to test MIC of antibiotics for bacteria as shown below-#

References

1. Koneman’s color atlas and textbook of diagnostic microbiology ,sixth edition,Washington winn,jr.,Stephen allen,Willam janda,Elmer koeman,Paul schreckenberger,Gail woods
2. Collier Leslie, Balows Albert & Sussman Max ; Topley and Wilson’s Microbiology and Microbial Infections, 9th edition Vol-2;Arnold.
3. Mackie & McCartney pratical medical microbiology , 14th edition, churchill livingstone.
4. Textbook of DIAGNOSTIC MICROBIOLOGY,Third edition ,CONNIE R. MAHON,DONALD C.LEHMAN,GEORGE MANUSELIS.