There are, in fact, three most critical and highly explicite situations, wherein the absolute necessity
to assay the ‘antimicrobial agents’ arise, namely :
(a) Production i.e., in the course of commercial large-scale production for estimating the ‘potency’
and stringent ‘quality control’,
(b) Pharmacokinetics i.e., in determining the pharmacokinetics* of a ‘drug substance’ in
humans or animals, and
(c) Antimicrobial chemotherapy i.e., for strictly managing, controlling, and monitoring the
ensuing antimicrobial chemotherapy**.
Summararily, the very ‘first’ situation i.e., (a) above, essentially involves the assay of relatively
high concentration of ‘pure drug substance’ in a more or less an uncomplicated solution, for
instance : buffer solution and water. In addition to the ‘second’ and ‘third’ i.e., (b) and (c) above,
critically involve the precise and accurate measurement at relatively low concentration of the ‘drug
substance’ present in biological fluids, namely : serum, sputum, urine, cerebrospinal fluid (CSF), gastric
juice, nasal secretions, vaginal discharges etc. Nevertheless, these biological fluids by virtue of their
inherent nature invariably comprise of a plethora of ‘extranaceous materials’ which may overtly and
covertly interfere with the assay of antibiotics
Importance and Usefulness
The actual inhibition of the observed microbial growth under stringent standardized experimental
parameters may be judiciously utilized and adequately exploited for demonstrating as well as establishing
the therapeutic efficacy of antibiotics.
It is, however, pertinent to state here that even the slightest and subtle change duly incorporated
in the design of the antibiotic molecule may not be explicitely detected by the host of usual ‘chemical
methods’, but will be revealed by a vivid and clear-cut change in the observed ‘antimicrobial activity’.
Therefore, the so called microbiological assays do play a great useful role for ascertaining and resolving
the least possible doubt(s) with respect to the change in potency of antibiotics and their respective
formulations i.e., secondary pharmaceutical products.
The underlying principle of microbiological assay is an elaborated comparison of the ‘inhibition
of growth’ of the microbes by a measured concentration of the antibiotics under investigation
against that produced by the known concentrations of a ‘standard preparation of antibiotic’ with a
In usual practice, two ‘general methods’ are employed extensively, such as :
(a) Cylinder-plate (or Cup-plate) Method, and
(b) Turbidimetric (or Tube-assay) Method.
Each of the two aforesaid methods shall now be discussed briefly in the sections that follows :
Cylinder-Plate Method (Method-A)
The cylinder-plate method solely depends upon the diffusion of the antibiotic from a vertical
cylinder via a solidified agar layer in a Petri-dish or plate to an extent such that the observed growth of
the incorporated microorganism is prevented totally in a zone just around the cylinder containing a
solution of the ‘antibiotic’.
Turbidimetric (or Tube-Assay) Method (Method-B)
The turbidimetric method exclusively depends upon the inhibition of growth of a ‘microbial
culture’ in a particular uniform solution of the antibiotic in a fluid medium which is quite favourable
and congenial to its rather rapid growth in the absence of the ‘antibiotic’.
Conditionalities : The various conditionalities required for the genuine assay may be designed
in such a manner that the ‘mathematical model’ upon which the potency equation is entirely based
can be established to be valid in all respects.
Examples : The various typical examples are as stated under :
(a) Parallel-Line Model — If one happens to choose the parallel-line model, the two logdose-
response lines of the preparation under investigation and the standard preparation
must be parallel, i.e., they should be rectilinear over the range of doses employed in the
calculation. However, these experimental parameters need to be critically verified by the
validity tests referred to a given probability.
(b) Slope-Ratio Method : It is also feasible to make use of other mathematical models, for
instance : the ‘slope-ratio method’ provided that proof of validity is adequately demonstrated.
Present Status of Assay Methods
Based on the copious volume of evidences cited in the literatures it may be observed that the
‘traditional antimicrobial agents’ have been duly determined by microbiological assay procedures.
Importantly, in the recent past significant greater awareness of the various problems of poor assay
results specificity associated with such typical examples as :
partially metabolized drugs,
presence of other antibiotics, and
urgent need for more rapid/reproducible/reliable analytical techniques ;
has appreciably gained ground and equally encouraged the judicious investigation of a host of other
fairly accurate and precise methodologies, namely :
Chromatographic assays, including :
—High Performance Liquid Chromatography (HPLC)
—Reverse-Phase Chromatography (RPC)
—Ion-Pair Chromatography (IPC)
This chapter will cover briefly the underlying principles of these aforesaid techniques.