There are mainly two different types of microbiological assays usually encountered bearing in
mind the response of an ever-growing population of microbes vis-a-vis ascertaining the profile of
antimicrobial agent measurements, such as :
(a) Agar Plate diffusion assays, and
(b) Rapid-reliable-reproducible microbial assay methods.
Each of the two aforesaid types of microbiological assays will now be discussed individually in
the sections that follows :
Agar Plate Diffusion Assays (Method-A)
In the agar-plate diffusion assays the ‘drug substance’ gets slowly diffused into agar seeded
duly with a susceptible microbial population. Subsequently, it gives rise to a ‘specific zone of growth
inhibition’. However, the agar-plate diffusion assay may be one-, two- or three-dimensional (i.e.,
1D, 2D or 3D).
All these three different types shall now be discussed briefly in the sections that follows :
In this particular assay the capillary tubes consisting of agar adequately seeded with ‘indicator
organism’ are carefully overlaid with the ‘drug substance’. The drug substance e.g., an antibiotic
normally gets diffused downwards into the agar thereby giving rise to the formation of a ‘zone of inhibition’.
However, this specific technique is more or less obsolete now-a-days.
Merits : There are three points of merits, such as :
perfectly applicable for the assay of antibiotics anaerobically,
may efficiently take care of very small samples, and
exhibits an appreciable precision,
Demerit : It essentially has a critical demerit with regard to the difficulty in setting up and
As to date, the 2D- or 3D-assay methods represent the commonest and widely accepted form of
the microbiological assay. Nevertheless, in this particular instance the samples need to be assayed are
adequately applied in a certain specific type of reservoir viz., cup, filter-paper disc, or well, to a thinlayer
of agar previously seeded with an indicator microorganism aseptically in a Laminar Air Flow
Bench. In this way, the ‘drug substance’ gets gradually diffused into the medium, and after suitable
incubation at 37°C for 48–72 hrs. in an ‘incubation chamber’, a clear cut distinctly visible zone of
growth inhibition comes into being*. However, the diameter of the zone of inhibition very much
remains within limits, provided that all other factors being constant, and the same is associated with the
concentration of the antibiotic present in the reservoir.**
Dynamics of Zone Formation
It has been duly observed that during the process of incubation the antibiotic gets diffused from
the reservoir. Besides, a proportion of the bacterial population is moved away emphatically from the
influence of the antibiotic due to cell-division.
Important Observations : Following are some of the important observations, namely :
(1) Edge of a zone is usually obtained in a situation when the minimum concentration of the
antibiotic that will effectively cause the inhibition in the actual growth of the organism on the agar-plate
(i.e., critical concentration accomplished) attains, for the very first time, a specific population density
which happens to be excessively too big in dimension and quantum for it to inhibit effectively.
(2) The precise and exact strategic position of the zone-edge is subsequently determined by
means of the following three vital factors, such as :
initial population density,
rate of diffusion of ‘antibiotic’, and
rate of growth of ‘organism’.
(3) Critical Concentration (C′) : The critical concentration (C′) strategically located at the
edge of a ‘zone of inhibition’ and formed duly may be calculated by the following expression :
Graphical Representation : It is feasible and possible to have a ‘graphical representation’ to
obtain a zone of inhibition in different ways, for instance :
(1) An assay wherein the value of To and D happen to be constant, an usual plot of In C Vs d2 for
a definite range of concentrations shall, within certain limits, produce a ‘straight line’ that may be
conveniently extrapolated to estimate C′ i.e., critical concentration.
(2) In fact, C′ duly designates the obvious minimum value of C that would yield a specific zone
of inhibition. Evidently, it is absolutely independent of D and To.
(3) However, the resulting values of D and To may be manipulated judiciously to lower or enhance
the dimensions of zone based on the fact that the concentrations of C is always greater than C′.
i.e., the concentration of ‘drug’ in reservoir > critical concentration of the ‘drug’.
(4) Pre-incubation would certainly enhance the prevailing number and quantum of microbes
present actually on the agar-plate ; and, therefore, the critical population density shall be duly accomplished
rather more rapidly (i.e., To gets reduced accordingly) thereby reducing the observed zones of
(5) Minimizing the particular microbial growth rate suitably shall ultimately give rise to relatively
‘larger zones of inhibition’.
(6) Carefully enhancing either the sample size or lowering the thickness of agar-layer will
critically increase the zone size and vice-versa.
(7) Pre-requistes of an Assay—While designing an assay, the following experimental parameters
may be strictly adhered to in order to obtain an optimized appropriately significant fairly large
range of zone dimensions spread over duly the desired range of four antibiotic concentrations, such
proper choice of ‘indicator organism’,
suitable culture medium,
appropriate sample size, and
exact incubation temperature.
10.3.1.4. Management and Control of Reproducibility
As the observed dimensions of the zone of inhibition depend exclusively upon a plethora of
variables*, as discussed above, one should meticulously take great and adequate precautionary measures
not only to standardise time, but also to accomplish reasonably desired good precision.
Methodologies : The various steps involved in the management and control of reproducibility
are as stated under :
(1) A large-size flat-bottomed plate [either 30 × 30 cm or 25 × 25 cm] must be employed, and
should be meticulously levelled before the agar is actually poured.
(2) Explicite effects of variations in the ‘composition of agar’ are adequately reduced by preparing,
and making use of aliquots of large batches.
(3) Inoculum dimension variants with respect to the ‘indicator organisms’ may be minimized
proportionately by duly growing a reasonably large volume of the organism by the following two ways
and means, such as :
dispensing it accordingly into the aliquots just enough for a single agar plate, and
storing them under liquid N2 so as to preserve its viability effectively.
(4) In the specific instance when one makes use of the ‘spore inocula’, the same may be adequately
stored for even longer durations under the following two experimental parameters, for
absolute inhibition of germination, and
effective preservation of viability.
(5) It is a common practice to ensure the ‘simultaneous dosing’ of both calibrators and samples
onto a single-agar plate. In this manner, it is possible and feasible to achieve the following three
cardinal objectives :
thickness of the agar-plate variants,
critical edge-effects, and
incubation temperature variants caused on account of irregular warming inside the ‘incubator’
must be reduced to bare minimum by employing some sort of ‘predetermined random layout’.
(6) ‘Random Patterns’ for Application in Microbiological Plate Assay : In usual practice, we
frequently come across two prevalent types ‘random patterns’ for application in the microbiological
plate assay, namely :
(a) Latin-Square Arrangement – in this particular case the number of replicates almost equals
the number of specimens (samples) ; and the ultimate result ensures the maximum precision,
as shown in Fig. 10.1(a).
(b) Less Acceptable (Demanding) Methods – employing rather fewer replicates are invariably
acceptable for two vital and important purposes, such as :
clinical assays, and
Measurement of Zone of Inhibition
To measure the zone of inhibition with an utmost precision and accuracy, the use of a Magnifying
Zone Reader must be employed carefully. Besides, to avoid and eliminate completely the subjective
bias, the microbiologist taking the reading of the incubated agar-plate must be totally unaware of the
ground realities whether he is recording the final reading of either a ‘treat zone’ or a ‘calibrator’.
Therefore, the judicious and skilful application of the ‘random’ arrangements as depicted in Fig. 10.2
may go a long way to help to ensure critically the aforesaid zone of inhibition. However, the ‘random
pattern’ duly installed could be duly decephered after having taken the reading of the agar-plate.
Calibration may be accomplished by means of two universally recognized and accepted
methods, namely :
(a) Standard Curves, and
Each of these two methods will now be discussed briefly in the sections that follows :
. Standard Curves
While plotting the standard curves one may make use of at least two and even up to seven
‘calibrators’ covering entirely the required range of operational concentrations. Besides, these selected
concentrations must be spaced equally on a ‘Logarithmic Scale’ viz.,starting from 0.5, 1, 2, 4, 8, 16 and
up to 32 mg. L– 1.
However, the exact number of the ensuing replicates of each calibrator must be the bare minimum
absolutely necessary to produce the desired precision ultimately. It has been duly observed that a
‘manual plot’ of either :
zone size Vs log10 concentration, or
[zone size]2 Vs log10 concentration,
A microcomputer may by readily installed and programmed to derandomise the realistic and
actual zone pattern by adopting three steps in a sequetial manner viz., (a) consider the mean of
the ‘zone sizes’ ; (b) compute the standard curve ; and (c) calcuate the ultimate results for the
tests ; and thereby enabling the ‘zone sizes’ to be read almost directly from the incubated
agar-plate right into the computer.
The 2-by-2-assay is particularly suitable for estimating the exact and precise potency of a plethora
of ‘Pharmaceutical Formulations’. In this method a relatively high degree of precision is very much
required, followed by another two critical aspects may be duly taken into consideration, such as :
Latin square design with tests, and
Calibrators at 2/3 levels of concentration.
Example : An 8 × 8 Latin square may be employed gainfully in two different ways :
First— to assay 3 samples + 1 calibrator, and
Second— to assay 2 samples + 2 calibrators,
invariably at two distinct levels of concentrations* each, and having a ‘coefficient of variation’ at
Evidently, based on this technique, one may obtain easily and conveniently the ‘parallel dose–
response lines’ strategically required for the calibrators vis-a-vis the tests performed at two distinct
dilutions, as depicted in Fig. 10.3. Importantly, it is quite feasible and possible to establish the exact and
precise potency of samples may be computed effectively or estimated from meticulously derived