Alexander Fleming’s discovery of Penicillin in 1929 began a new era of antibiotic chemotherapy. In the 1940’s industrial production of penicillin started.
Many antibiotics are currently available in the market and many more will still be discovered. Whether for a synthetic chemical or an antibiotic, the trial and error procedure involves testing for antimicrobial activity mainly in vitro against known pathogens.
Antimicrobial susceptibility testing is done in order to determine whether an organism is susceptible to a series of antibiotics that might be relevant in treatment.
To ascertain the susceptibility of the organism to a single concentration of the antibiotic, the concentration chosen is often related to the concentration found in the infected site. These guidelines are usually based on serum levels attainable by the antibiotic but are sometimes also related to antibiotic levels in urine, bile spectrum, and sinus fluid.
Current prevalence of resistant strains in a community or hospital unit are detected by frequently collecting together individual test results to provide survey data. Antibiotic sensitivity tests are also often used as an aid in the identification of infecting organisms e.g the almost constant resistance by Proteus spp to tetracycline.
Test for susceptibility of pathogens to antimicrobials is now 4 common clinical practice. These methods are now used to standardize and test the antimicrobial effect of herbs.
The tests are used to determine the following:
1. Minimum Inhibitory Concentration (MIC)
The aim of this test is to determine the lowest concentration of the drug required to inhibit the growth of the organism.
2. Minimal lethal concentration (MLC)
This is used to determine the lowest concentration required to kill the organisms. It is often referred to as minimal bactericidal concentration (MBC).
The methods of susceptibility testing that are commonly used are Disk agar diffusion technique or Kirby – Bauer method and the Broth dilution method.
Antibiotic sensitivity testing using kirby – Bauer method
This is an agar diffusion method in which an agar plate is first inoculated with a test microorganism and paper discs impregnated with antibiotics are placed on the agar.
The antibiotics radially diffuse through the agar. Depending on the sensitivity of the test organism to the antibiotic different zones of inhibition are formed around the discs. The inhibition zone diameter (IZD) is measured in millimeters (mm) and is compared with a standard and thereafter the microorganism is rated as resistant or slightly sensitive or sensitive to each antibiotic.
This rating is usually based on standards of zones of inhibition which have been defined through extensive research.
Discs are always commercially available and are readily grouped for gram positive and gram negative organisms. It is important that Mueller-Hinton medium adjusted to a pH of 7.2 to 7.4 with a uniform depth of about 4mm in a petri dish is used.
This is to ensure that an easily reproducible result is obtained. To obtain a uniform depth of about 4mm in a petri dish, pour 25ml of the medium in a 9cm dish.
This test could be easily used to compare the effect of various antibiotics on a particular organism in a single plate.
Evaluation of plant extract for antibacterial activity
The problem of antimicrobial resistance has revitalised the use of plant root extracts, leaf extracts, stem bark extracts etc for the treatment and control of diseases.
Scientific knowledge helps in the consideration of these traditional herbal remedies in conjunction or as an affordable alternative to Orthodox medical treatment. This evaluation involves testing crude aqueous extracts, ethanolic extracts of the known plant on a bacterial isolate.
For example the antibacterial effect of a plant leaf or root extract that is used by a community to treat typhoid fever can be determined in the laboratory using Salmonella isolates.
The antimicrobial effect of an extract used in controlling wound infections can be assayed using organisms that are implicated in wound infections. The pre-treatment of roots, leaves or stem barks depend on the kind of test and interest of the researcher or scientist.
Often phytochemical analysis are carried out to determine the active ingredients in the various extracts as this can be indicative of the particular active ingredient that have antibacterial or antifungal activity.
Testing for Penicillinase Production
The presence of Penicillinase (B-lactamase) results in po the breakdown of the B-lactam ring of penicillin with the production of penicilloic acid.
In this test, the penicilloic acid to, react with iodine and this can be detected by decolourisation of the blue black colour of the starch paper.