Microbiological (Microbial) Assays: Antibiotics-Vitamins- Amino Acids

MICROBIOLOGICAL (MICROBIAL) ASSAYS : ANTIBIOTICS–VITAMINS– AMINO ACIDS

INTRODUCTION

There are, in fact, three most critical and highly explicite situations, wherein the absolute neces-sity to assay the ‘antimicrobial agents’ arise, namely :

(a) Production i.e., in the course of commercial large-scale production for estimating the ‘po-tency’ 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), gas-tric 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.

1. Importance and Usefulness

The actual inhibition of the observed microbial growth under stringent standardized experimen-tal parameters may be judiciously utilized and adequately exploited for demonstrating as well as estab-lishing 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 resolv-ing the least possible doubt(s) with respect to the change in potency of antibiotics and their respective formulations i.e., secondary pharmaceutical products.

2. Principle

The underlying principle of microbiological assay is an elaborated comparison of the ‘inhibi-tion 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 known activity.

3. Methodologies

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 :

(a) 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’.

(b) 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 log-dose-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.

4. 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 :

·        Enzymatic assays,

·        Immunological assays,

·        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.