To Determine The Response of Test Organisms To Antibiotics

To determine the response of test organisms to antibiotics
The response of microorganisms to Antibiotics are majorly measure by two methods .

namely
1. Disc diffusion test
2. Minimum inhibitory concentration
A true antibiotic is an antimicrobial chemical produced by microorganisms against other

microorganisms. Mankind has made very good use of these antimicrobials in its fight against
infectious disease. Many drugs are now completely synthetic or the natural drug is manipulated to
change its structure somewhat, the latter called semisynthetics. Bacteria respond in different ways
to antibiotics and chemosynthetic drugs, even within the same species. For example,
Staphylococcus aureus is a common normal flora bacterium found in the body. If one isolated this
bacterium from 5 different people, the 5 isolates would likely be different strains, that is, slight
genetically different. It is also likely that if antibiotic sensitivity tests were run on these isolates,
the results would vary against the different antibiotics used.
1. The Kirby-Bauer test for antibiotic susceptibility (also called the disc diffusion test) is a
standard that has been used for years. First developed in the 1950s, it was refined and by
W. Kirby and A. Bauer, then standardized by the World Health Organization in 1961. It
has been superseded in clinical labs by automated tests. However, the K-B is still used in
some labs, or used with certain bacteria that automation does not work well with. This test
is used to determine the resistance or sensitivity of aerobes or facultative anaerobes to
specific chemicals, which can then be used by the clinician for treatment of patients with
bacterial infections. The presence or absence of an inhibitory area around the disc identifies
the bacterial sensitivity to the drug.
The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs
are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts
the further it is away from the disc. If the organism is killed or inhibited by the concentration
of the antibiotic, there will be NO growth in the immediate area around the disc: This is
called the zone of inhibition. The zone sizes are looked up on a standardized chart to give
a result of sensitive, resistant, or intermediate. Many charts have a corresponding column
that also gives the MIC (minimal inhibitory concentration) for that drug. The MIC is
currently the standard test run for antibiotic sensitivity testing because it produces more
pertinent information on minimal dosages.
The Mueller-Hinton medium being used for the Kirby-Bauer test is very high in protein.
2. Minimum Inhibitory Concentration (MIC) is a one of criteria that establishes efficiency of
an antibiotic.
Minimum Inhibitory Concentration is the minimum concentration at which an antibiotic
inhibits the growth of microorganism but not necessarily kills it.
MIC the term is applicable to antibiotics, disinfectants, antiseptics and preservatives.
MIC of an antibiotic is tested either by one of the following ways,
• Liquid Dilution Method.
• Solid Dilution Method
• Liquid Dilution Method (Test Tube Method)
•
• Double strength medium is added in all test tubes.
• First test tube (Uninoculated) inoculum is not added and is used to check sterility of the
medium used.
• In all other remaining eleven test tubes 3-4 drops of inoculum is added to reach 10 -
10 cells/ ml concentration of the test microorganism.
• In all test tubes except “Uninoculated” and “Control” chemical agent under test are added
from 0.5 ml to 5.0 ml.
• The final volume of 10 ml is adjusted using sterile water in all test tubes.
• Contents of all test tubes are mixed well and incubated @ 37℃ for 2-3 days.
• After incubation all tests tubes are examined for growth of microorganism and the
minimum inhibitory concentration is calculated.
• It is necessary to carry out a preliminary experiment to determine the range of MIC of the
chemical agent under test.
Solid Dilution Method:
• In this method the chemical agent under test is mixed in the molten agar and then is poured
in the petri dish.
• After solidification, the inoculum is spread on the surface of the agar medium.
• All the plates are incubated @ 37℃ for 2-3 days.
After incubation all petri dishes are examined for growth of microorganism and the minimum
inhibitory concentration is calculated
Advantages
1. Several microorganisms can be studied once by using a multipoint inoculator.
2. Contaminations can easily be detected as colony features are distinctive here rather than
turbidity difference.
Amino acid response determination
Microbiological assays were used for quantitation of amino acids since many years as they were
the most reliable, sensitive, and specific available tests. They can be used for any type of biological
material, however sometimes they are affected by the activators or inhibitors present. in the
material being standardised. These inexpensive methods are used for analysing large batches of
samples, but are time-consuming and also not suitable for all amino acids. Amino acids are
essential for the growth and replication of some microorganisms. Many strains of such microbes
depend on a particular amino acid. Thus, if such microorganisms are cultured using a small amount
of that particular amino acid, a limited degree of growth will be observed, which is measured by
turbidimetry or by measuring the increased lactic acid production by either microtitration or pH
change.
Vitamin response determination
Vitamins are essentially required for the growth and multiplication of microorganisms (which are
highly sensitive even to small amounts of growth factors). These test microorganisms can
synthesise the factor being assayed which forms the basis of microbiological assay of vitamins and
amino acids. The test microorganisms used for the standardisation of water-soluble vitamins are
given in table 2.
Table 2: Test Microorganisms and Conditions for the Microbial Assay of Vitamins
Principle: Vitamins are essential growth factor for microorganisms. In the standardisation of
vitamins, the ability of test organisms to use the substance being standardised under a proper
nutritional condition is measured. These growth factors (vitamins) are required by the organisms
in micro or nano grams. The growth response produced by the test organism is proportional to the
amount of growth factor added to the medium.
• Microbiological assay of Vitamin B12 is can be performed by using one of the following
methods,
• Tritrimetric Method.
Turbidimetric Method

To Determine The Response of Test Organisms To Antibiotics

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To determine the response of test organisms to antibiotics

The response of microorganisms to Antibiotics are majorly measure by two methods .

A true antibiotic is an antimicrobial chemical produced by microorganisms against other

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