MINIMUM INHIBITORY CONCENTRATION

San Pedro College – Pharmacy Department

2nd Semester, A.Y. 2019-2020

Minimum Inhibitory Concentration (MIC) is lowest concentration of an antimicrobial that will inhibit the visible growth of a
microorganism following overnight incubation, usually reported as mg/L.

A related concept is the minimum bactericidal concentrations (MBCs), which is the lowest concentration of antimicrobial
that will prevent the growth of an organism after subculture on to antibiotic-free media.

Infections in the ICU are often caused by pathogens with higher MICs compared with other clinical settings. Regular
surveillance of MICs is required due to a continuing decrease in susceptibility to the commonly used antibiotics in critically ill
patients.

Lowest concentration: Realize that it is the least amount of antibiotic that represents the MIC. So in our above
example, both 1 µg/mL (test tube one) and 0.5 µg/mL (test tube two) prevented bacterial growth, but 0.5 µg/mL is the
lowest concentration and therefore the MIC.

of a certain antibiotic: We just discovered the MIC of amoxicillin. Now does this MIC apply to a different antibiotic?
No, it doesn't. The MIC is specific to the antibiotic being used in the test tube.

visible growth: The MIC is determined by observation with the naked eye not a more sensitive instrument, like a
microscope. The technician is looking for which test tubes remain turbid or cloudy after incubation with the antibiotic. A
turbid tube, such as test tube three, represents a tube in which bacterial growth is observable. A clear tube, such as test tubes
one and two, represent a tube in which bacterial growth has been hindered.

specific bacteria: Just like with the antibiotic, the inhibition of growth is only of a specific bacteria. Therefore, if you
have the MIC of amoxicillin inhibiting the growth of bacteria A, then you cannot apply this same MIC when using amoxicillin to
inhibit the growth of bacteria B.

period of incubation: The standard period of incubation is overnight (24hrs).

The common uses of MICs include:

• evaluating the efficiency of certain antibiotics against certain strains of bacteria, including newly developed
antibiotics
• confirming drug resistance

More strains of bacteria are becoming drug resistant or less susceptible to the curing power of an antibiotic.
MICs help to show which types of bacteria are resistant to which antibiotics.

DETERMINATION
• by agar or broth dilution methods
• strips with graded changes in antibiotic concentrations provided
o when placed on plates the point at which the bacterial inhibition intersects with strip is the MIC

Page 1 of 3
CLINICAL APPLICATION
• generally drug concentrations need to be 4-5x the MIC to ensure that an antibiotic is effective
• most laboratories routinely report susceptibility with the classification ‘S, I and R’ (Susceptible, Intermediate-
Susceptible and Resistant) based on MIC breakpoints
o The breakpoint is the chosen concentration of an antibiotic which defines whether a species of bacteria is
susceptible or resistant to the antibiotic
o If the MIC is less than or equal to the susceptibility breakpoint the bacteria is considered susceptible to the
antibiotic

PROS AND CONS OF USING THE MIC

Advantages:
• Easy to perform
• Familiar and widely used
• usually automated
• Highly reproducible (due to simplicity and automation)
• Rapid turnaround of results

Disadvantages:
• MIC can vary greatly with minor variations in methodology can result in large variations of the MIC.
o e.g. prolonged incubation may increase the apparent MIC
o e.g. smaller inoculum concentrations may decrease the apparent MIC
o e.g. MIC may change with freezer storage of samples
• Comparisons between laboratories are hindered by differences in techniques used inhibition of visible growthdoes
not mean the organisms were killed (cf. minimum bactericidal concentration)
• MIC is not truly a single number, but a range depending on the dilution series used during its determination
• MIC does not necessarily equate with in vivo efficacy, for example:
o an antibiotic will be ineffective if it does not penetrate the affected tissues
o antibiotics with high MICs may still be effective if concentrated at the site of infection (e.g. treatment of UTI
with gentamicin, which concentrates in the urine)
o antibiotic kill characteristics are important (e.g. concentration versus time dependent killing)
o critically ill patients may have altered pharmacodynamics and pharmacokinetics
o ‘breakpoints’ are often subjective and may not correlate with clinical outcomes

Müller-Hinton Agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. It is also
used to isolate and maintain Neisseria and Moraxella species. It was co-developed by microbiologist John Howard Mueller and
veterinary scientist Jane Hinton at Harvard university as a culture for gonococcus and meningococcus, who published the
method in 1941.

It typically contains:
• 2.0g beef extract
• 17.5g casein hydrolysate
• 1.5g starch
• 17.0g agar dissolved in 1 liter of distilled water.
• pH adjusted to neutral at 25 °C.[1]

Principle of MHA
Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar. Beef Extract and Acid
Hydrolysate of Casein provide nitrogen, vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch is added
to absorb any toxic metabolites produced. Starch hydrolysis yields dextrose, which serves as a source of energy. Agar is the
solidifying agent.
The use of a suitable medium for testing the susceptibility of microorganisms to sulfonamides and trimethoprim is
essential. Antagonism to sulfonamide activity is demonstrated by para-aminobenzoic acid (PABA) and its analogs. Reduced
activity of trimethoprim, resulting in smaller inhibition zones and innerzonal colonies, is demonstrated on unsuitable Mueller
Hinton medium possessing high levels of thymidine. Both the PABA and thymine/thymidine content in Mueller Hinton Agar
are reduced to a minimum, thus markedly reducing the inactivation of sulfonamides and trimethoprim when the media is
used for testing the susceptibility of bacterial isolates to these antimicrobics.

Page 2 of 3
Uses of MHA
1. The major use of Mueller Hinton Agar is for antimicrobial susceptibility testing. It has become the standard medium
for the Bauer Kirby method and its performance is specified by the NCCLS.
2. It can be used to cultivate Neisseria
3. It is specified in FDA Bacteriological Analytical Manual for food testing, and procedures commonly performed on
aerobic and facultative anaerobic bacteria.

Why MHA is used for antibiotic susceptibility testing?

1. It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow.
2. It contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with
the antibiotics. It also mediates the rate of diffusion of the antibiotics through the agar.
3. It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a
truer zone of inhibition.
4. MHA shows acceptable batch-to-batch reproducibility for susceptibility testing.
5. MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e. concentration of inhibitors thymidine and
thymine is low in MHA).
6. Both the para-aminobenzoic acid (PABA) and thymine/thymidine content in Mueller Hinton Agar are reduced to a
minimum, thus markedly reducing the inactivation of sulfonamides and trimethoprim when the media is used for
testing the susceptibility of bacterial isolates to these antimicrobics.

Preparation of MHA
1. Suspend 38 gm of the medium in one liter of distilled water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. Autoclave at 121°C for 15 minutes. Cool to room temperature.
4. Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface to give uniform depth.
5. Allow to cool to room temperature.
6. Check for the final pH 7.3 ± 0.1 at 25ºC.
7. Store the plates at 2-8 ºC.

Limitations of MHA
1. Numerous factors can affect results: inoculum size, rate of growth, medium formulation and pH. Strict adherence to
protocol is required to ensure reliable results.
2. Drug inactivation may result from the prolonged incubation times required by slow growers.
3. Variation in the concentration of divalent cations, primarily calcium and magnesium affects result of aminoglycoside,
tetracycline, and colistin test with P. aeruginosa isolates.

Page 3 of 3