Evaluation Of The Colistin Broth Disk Elution Method For Determine The MIC Of Colistin Against Gram Negative Bacilli

Scope: 

To determine the minimum inhibitory concentration (MIC) in mcg/ml of colistin against microorganisms.

Principle: 

The disk elution method is a quantitative technique performed in vitro, for determining the MIC of colistin against microorganisms.

Material Required:

a)   Cation Adjusted Muller Hinton Broth

b)   Colistin sulfate disk  (10µl)

c)    Saline

d)   Screw cap Tubes

 Equipment Required:

a) Biological Safety Cabinet.

b) Incubator

Procedure:

Standardized Inoculum Preparation

Using a loop pick 3–5 colonies from a fresh (18–24 hours) nonselective agar plate and transfer to sterile saline (4–5 mL).

Adjust turbidity to equivalent of a 0.5 McFarland turbidity standard.

Test procedure:

1. Let the CAMHB tubes (10 mL) and colistin disks warm to room temperature. Label 4 tubes of CAMHB for each isolate to be tested with 1, 2, and 4 µg/mL and control.

2. Using aseptic technique, carefully add colistin disk:

a)      1 colistin disk to the tube labeled “1 µg/mL”

b)     2 colistin disks to tube labeled “2 µg/mL”

c)      4 colistin disks to the tube labeled “4 µg/mL”

3. Gently vortex the tubes with the added disk and let the colistin elute from the disks for   at least 30 minutes but no longer than 60 minutes at room temperature.

4. Add 50 µL standardized inoculum to the control and 1-, 2-, and 4-µg/mL tubes to attain a final inoculum concentration of approximately 7.5 × 105 CFU/mL.

5. Using a 10-µL loop, subculture from the original inoculum tube to a blood agar plate as a purity check.

6. Cap the tubes tightly and vortex each inoculated tube on slow speed to mix. Slow speed is suggested to prevent colistin from sticking to the cap and glass surface above the meniscus of liquid and loosen the caps slightly before incubation.

 7. Incubate the tubes and purity plate at 33 to 35°C; ambient air for 16–20 hours.

Reading tubes and Interpreting Results:

1.    Examine the purity plate to ensure inoculum was pure.

2.    Examine the growth control tube, which must demonstrate obvious turbidity for the test to be valid.

3.     Read the MIC as the lowest concentration that completely inhibits growth of the test isolate.

For Enterobacterales and P. aeruginosa:

 ≤ 2 μg/mL = intermediate

 ≥ 4 μg/mL = resistant

 Figure: 1:- Colistin Broth Disk Elution.

 

Fig: 1 - Results for routine QC strain P. aeruginosa ATCC 27853 with an MIC ≤ 1 μg/mL

Additional testing and reporting

If there is an inconsistent growth pattern (Eg, no growth in 2 μg/mL but growth at 1 μg/mL and 4 μg/mL), repeat the test.

An inconsistent growth pattern may occur as a result of:

• Contamination at higher dilutions
• Hetero-resistant isolates.
• Inadequate colistin concentration in tubes.
• Error inoculating the tubes

Quality control:  

It is suggested to test a positive and negative control each time a batch of ten determinations is prepared:

Escherichia coli ATCC 25922 (MIC range <=1 – 2 µg/ml). 

P. aeruginosa ATCC 27853 (MIC range <=1 – 4 µg/ml)

Reference:

1.    Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests M100, Vol.42 No: 2, Page no: 146.