Assessment of a New Antibiotic

It is the process of discovery, evaluation and the Establishment of efficacy of identified synthetic or natural chemicals.

Identification means, finding of any chemical synthesized or obtained from natural sources like microbial cells or herbal / plant extracts.

Establishment of efficacy related with the findings that the new antimicrobial agent is effective against which organism, i.e. against bacteria (which strain), or fungi.

Establishment of effective concentration, means determination of concentration at which the chemical effectively kills the bacterial strains and also determination of MInimum Inhibitory Concntration (MIC).

So in the assesment of any new Antibiotic some properties will be established like

The antibiotic is effetive against which microbial strain.
What is the efficacy of new chemical (Antibiotic), and
what is the minimum inhibitory concentration, which can show bacteriostatic or bacteriocidal activity.

Agar Diffusion Method:

This method is most applied method for assessment of antimicrobial activity.

In this method agar plates are inoculated with any microbial cells (against which the antimicrobial activity is to be tested).

For this, the microbial cells are spread evenly (with the help of sterile cotton swab) on the surface of solidified nutrient agar media.

Then this seeded culture media is allowed to incubate in optimized conditions to allow the growth of microbial cells. After appropriate incubation, microbial cells formed a uniform layer over the surface of nutrient agar media (This is called Lawn Culture).

Then a filter paper discs are taken and wetted or impregnated with the fixed quantity and known concentration of the chemical to be tested.

Then these wet paper disks are placed over the surface of culture media. Due to this the test chemical is diffused from paper disk and also diffuse into the agar media.

After sometime there will be a clear area is formed around the paper discs. This area is called zone of inhibition.

Interpretation of Results

Zone of inhibition formation shows that the microbial cells are killed or destroyed by the test chemical, this indicated that the test chemical is effective against selected microbial cells.

The diameter of the zone of inhibition shows the effectiveness of test chemical, as much as the diameter of zone of inhibition, as much as the test chemical is effective against selected microbial cells.

Antimicrobial Gradient Test (E-test)

This method provides two types of information about any test chemical:

Sensitivity of selected microbial cells towards any test chemical
Information about Minimum Inhibitory Concentration (MIC) of test chemical. MIC of any chemical is that concentration below which there is no antimicrobial activity.

The first step of this method is preparation of bacterial cell lawn culture.

Then a paper strip is taken and over that paper strip, test chemical is applied in increasing concentration at fixed interval or gap. Suppose first spot is of concentration 1 µg/ml, then next is of 2 µg/ml, than 3 µg/ml, 4 µg/ml …… 8 µg/ml like that.

In this way we get a wet strip having test chemical of different concentrations, on the strip at one end the test chemical is at lowest concentration and at the another end, the test chemical is at highest concentration.

Then this strip is place over bacterial cell culture and allow to stand for some time.

After that, we can see that there is formation of clear area (zone of inhibition), because the test chemical is diffused from the stripe to the culture medium and kills bacterial cells as per its efficacy.

At which point of concentration, the distance of clear zone is maximum, that concentration of test chemical is maximum effective concentration, and at which concentration there is minimum distance of zone, it is called Minimum Inhibitory Concentration (MIC).

Below concentration of MIC, any chemical not show any antimicrobial activity.

Dilution Method:

This is most basic method for assessment of any newer or established antimicrobial compounds.

In this method several dilutions of test chemical are prepared in two fold concentrations i.e. 1 µg/ml, 2 µg/ml, 4 µg/ml, 8 µg/ml, 16 µg/ml, 32 µg/ml……and so on, in nutrient growth broth medium (2 mL). (Broth is liquid medium, not solid culture medium).

These dilutions are then filled in small tubes or 96 well tubes.

Then a fix quantity of bacterial cell suspension is added in these tubes and allowed to grow in suitable conditions.

After some time the dilutions are checked for survival of bacterial cells. For this viability indicators are used. Viability indicators are the compounds metabolized by the microbial cells and produce coloured products.

So after addition of viability indicators, colour production in each tube was checked.

Viability indicators are: Tetrazolium salts, Alamar blue dye (Resazurine)

The tube which showed no colour production, indicate that there is no viable cell. By using this principle the minimum concentration is determined which kills the bacterial cells.