Measuring Effectiveness of Antimicrobial Chemical Agents

In this article we will discuss about the two important parameters for determining the effectiveness of antimicrobial chemical agents i.e. Minimal Inhibitory Concentration (MIC) and Minimal Lethal Concentration (MLC).

Minimal inhibitory concentration (MIC) is the lowest concentration of a chemical agent or drug that inhibits growth of a particular microorganism to the extent that there is no visible growth (turbidity) of microorganism.

The MIC is not a constant for a given chemical agent because it is affected by the nature of the target microorganism used, the inoculum size, and the composition of the culture medium, the incubation time, and the conditions of incubation such as temperature, pH and aeration. However, when all conditions are rigorously standardised, different antimicrobial chemical agents can be compared to determine which is most effective against the target microorganism.

The minimal lethal concentration (MLC) is the lowest concentration of a chemical agent or drug that kills the target microorganism. The difference between these two parameters is that the MIC denotes the minimum concentration necessary for complete inhibition of the target microorganism, whereas the MLC is the minimum concentration required to completely kill the target microorganism.

For microbial agents or drugs, the value of MLC is generally higher than MIC because these agents or drugs usually kill target microorganisms at levels only two to four times the MIC. This means that complete inhibition of growth usually does not signify that all target microorganisms have been killed.

Top 5 Methods for Measuring Effectiveness of Antimicrobial Chemical Agents:

1. Tube Dilution Method:

Tube dilution method is the most commonly used method for determination of minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC). In this method, a series of tubes is set up, each containing the same quantity of a standard growth liquid medium (usually Mueller-Hinton broth) and also a gradually increasing concentration of the antimicrobial chemical agent (e.g., antibiotic) to be tested.

The tubes are then inoculated with the same quantity of cell suspension of the test microorganism and incubated till growth has appeared (usually 16 to 20 hours). The first tube in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the chemical agent.

The minimal lethal concentration (MLC) is determined by taking small quantities (usually 0.05 ml) from the tubes showing no growth and sub culturing it into fresh medium lacking the chemical agent.

The lowest concentration of the agent from which the microorganism does not recover and grow when transferred to fresh medium is the minimal lethal concentration (MLC). The tube dilution method for determination of MIC and MLC is diagrammatically represented in Fig. 20.1.

2. Agar Plate Method:

Agar plate method (Fig. 20.2) is very similar to tube dilution method. In this method, Petri plates containing a standard growth agar medium (usually Mueller-Hinton agar) are taken at the place of lubes containing liquid medium. Antimicrobial chemical agents of various concentrations are inoculated on the agar surface of plates already inoculated with the same quantity of the test microorganism.

These plates are incubated and then examined for growth. The first plate in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the chemical agent. Also, the chemical agent to be tested may be applied in the center of the Petri dish and zone of inhibition can be observed. Zone of inhibition develops if the chemical agent is active.

3. Disk Diffusion Tests:

Two disk diffusion tests are used to determine the effectiveness of antimicrobial chemical agents (antibiotics).

These are:

(i) Disk plate method or agar-disk diffusion method and

(ii) Kirby-Bauer method.

(i) Disk Plate Method (or Agar-disk Diffusion Method):

Disk plate method, also called agar-disk diffusion method, is another common method used to evaluate or determine the effectiveness of antimicrobial chemical agent (e.g., antibiotic). In this method, which is very similar to the tube dilution method, a Petri plate containing an agar medium (usually Mueller-Hinton agar) overlayed with a culture of the test microorganism is prepared.

Known amounts of antimicrobial chemical agent are added to filter paper disks, which are then placed on the surface of the agar. During incubation, the agent diffuses from the filter paper into the agar; the further from the filter paper, the lower the concentration of the chemical agent. The effective minimal inhibitory concentration (MIC) is reached at some distance away from the filter paper disc.

Distal to the point of MIC there is visible growth, but growth is absent closer to the disc, A zone of inhibition appears with a diameter proportional to the amount of the antimicrobial agent added to the filter paper disc, the solubility of the agent, the diffusion coefficient, and the overall effectiveness of the agent. Larger the size of the zone of inhibition greater is the effectiveness of antimicrobial chemical agent.

Absence of the zone of inhibition denotes that the test microorganism is resistant to the antimicrobial chemical agent. Agar plate method is normally used to determine the effectiveness of antibiotics against microbial pathogens. The diagrammatic representation of disk plate method or agar-disk diffusion method is shown in Fig. 20.3.

(ii) Kirby-Bauer Method:

Kirby-Bauer method was developed in the early 1960s by W. Kirby, A.W. Bauer, and their colleagues and is most often used to test antimicrobial drug susceptibility. In this method, agar media are inoculated by evenly spreading a defined density of a suspension of the microbial pathogen’s pure culture on the agar surface.

After the agar surface has dried for about 5 minutes, filter paper disks containing a defined quantity (µg/disk) of the antimicrobial drug are then placed on the inoculated agar surface with the help of either with sterilized forceps or with a multiple applicator (Fig. 20.4). The Petri plate is immediately placed in an incubator at 35°C. After 16 to 18 hours of incubation, the diameters of the zones of inhibition around each disk are measured.

Inhibition zone diameters are then interpreted into the degree of microbial resistance with the help of Table 20.1 that relates zone diameter to the degree of microbial resistance.

The values in this table were derived by finding the MIC values and diameter of zones for many different strains of microbial pathogen. A plot of MIC (on a logarithmic scale) versus zone inhibition diameter (arithmetic scale) is prepared for each antimicrobial drug (Fig. 20.5).

These plots are then used to find the zone diameters corresponding to the drug concentrations actually reached in the body of the host. If the zone diameter for the lowest level reached in the body of the host is smaller than that observed with the test microbial pathogen, the latter should possess a MIC value low enough to be destroyed by the drug. A microbial pathogen possessing too high and MIC value (too small a zone diameter) is resistant to the drug at normal body concentrations.

4. Well Method:

The minimal inhibitory concentration (MIC) procedure for evaluation of antimicrobial chemical agent, usually an antibiotic, is also done in wells of a microtiter plate (Fig. 20.6). Wells filled with serial dilutions of chemical agent are inoculated with a standard amount of the test microorganism. Growth in the presence of various concentrations of the agent is observed by measuring turbidity.

Effectiveness of the chemical agent is usually expressed as the highest dilution (lowest concentration) of the agent that completely inhibits growth of the test microorganism.

This defines the value of the minimal inhibitory concentration (MIC). For example, the Fig. 20.6 shows that the end point of the growth of the test microorganism is the third well in rows 1 and 2. In row 3, the antimicrobial chemical agent is ineffective at the concentration tested, since there is microbial growth in all the wells. In row 4, the end point of the microbial growth is in the first well.

5. Phenol-Coefficient Method:

Phenol-coefficient method is considered to be the best-known disinfect screening test and is recognised by Association of Official Analytical Chemist’s (AOAC) and Food Drug Administration (FDA) as standard method of evaluation of disinfectant. This method in which the potency of a disinfectant is compared with that of phenol is sometimes called FAD method.

In phenol-coefficient method, the test organism used is a specific strain of either Staphylococcus aureus or Salmonella typhi. The temperature at which the test is performed, the manner of preparing subcultures, the composition of the subculture medium, the size of the test tubes, and other details of the test are spelled out in the official procedure.

1. Procedure and method of determination:

To determine the phenol coefficient, different dilutions of phenol and test disinfectants (5 ml per tube) are added separately to tubes containing 0.5 ml of 24 hour old broth culture (test culture) of Staphylococcus aureus or Salmonella typhi.

All these tubes are then placed in a 20°C water both. At intervals of 5, 10 and 15 minutes, an aliquot from each tube is transferred to a nutrient broth medium with a loop transfer needle for sub-culturing.

The inoculated subculture tubes are incubated for 2 days and subsequently examined for visible growth. The highest dilution of the disinfectant and the highest dilution of phenol killing the test organism (Staph, aureus or Sal. typhi) in 10 minutes but hot in 5 minutes are recorded.

The calculation of the phenol coefficient of the test disinfectant (Lysol) is performed in the following manner with the help of following equation:

Phenol-coefficient of the test disinfectant = Reciprocal of the test disinfectant dilution recorded /Reciprocal of the phenol dilution recorded

Table 20.2, for convenience, shows the records of the highest dilution of the disinfectant Lysol and the highest dilution of the phenol. Lysol dilution of 1:450 showed no growth at 10 minutes, but growth at 5 minutes; and phenol dilution of 1: 90 showed no growth at 10 minutes, but growth at 5 minutes.

Then the phenol-coefficient of Lysol would be:

Phenol-coefficient of Lysol = (1/450)/(1/90)

= 450/90 = 5

A phenol-coefficient greater than 1 suggests that the test disinfectant is more effective than phenol.

2. Modifications:

Several workers have modified phenol-coefficient method from time to time; the widely accepted on is called Rideal- Walker test, which is the following:

This test was designed by Rideal and Walker to determine the phenol coefficient of test disinfectants using Salmonella species. In this test strain of Salmonella typhi NTCC 786 is used, Serial dilutions of phenol and the disinfectant under test are inoculated with 0.2 ml of the test organism. After 2.5, 5, 7.4 and 10 mm a loopful of culture is transferred to 5 ml broth tubes that are maintained at 37°C on water bath (Table 20.3).

After inoculation the tubes are incubated at 37°C for 48 hr. The phenol coefficient is obtained by dividing the lowest concentration of disinfectant showing growth after 5 min and before 7.5 min by the lowest concentration of phenol giving the same result.

It is, however, notable that no single evaluation test method is suitable for all animicrobial chemical agents. Therefore, care should be taken in selecting a test method for a specific chemical agent, so that the results obtained are meaningful and reproducible and lend themselves to some degree of practical interpretation.

The ultimate criterion for the effectiveness of a chemical agent is its performance under practical conditions. However, the laboratory test should provide a reliable index of its practical value.