Agar Plate Diffusion Assays (Method-A)

10.3.1. 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 :

10.3.1.1. One-Dimensional Assay

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 inhi- bition’. 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

subsequent standardization.

* Internal Controls i.e., samples having known value.

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10.3.1.2. 2D- or 3D-Assay

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 thin- layer 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.**

10.3.1.3. 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 :

In Cd 2 In C ′= 4D To

where,

C = Concentration of drug in Reservoir,

d = Distance between Reservoir and zone-edge,

D = Diffusion coefficient***, and To = Critical time at which the position of zone-edge was determined critically. Graphical Representation : It is feasible and possible to have a ‘graphical representation’ to

obtain a zone of inhibition in different ways, for instance : * In this instance – a circle around the reservoir can be observed.

** Reservoir : Cup, filter-paper disc (soaked with ‘antibiotic’ soln., or well. *** Constant for a given ‘antibiotic’ in a given ‘matrix’ being diffused into a given medium at a given tem-

perature.

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2 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 en- hance 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 accom- plished rather more rapidly (i.e., To gets reduced accordingly) thereby reducing the observed zones of

inhibition.

(5) Minimizing the particular microbial growth rate suitably shall ultimately give rise to rela- tively ‘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 param- eters 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 as :

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 meas- ures not only to standardise time, but also to accomplish reasonably desired good precision.

Methodologies : The various steps involved in the management and control of reproducibil-

ity 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 pre-

paring, 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 :

* Variables e.g., sample size, agar-thickness, indicator organism, population density, organism growth rate, and drug-diffusion rate.

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dispensing it accordingly into the aliquots just enough for a single agar plate, and

storing them under liquid N 2 so as to preserve its viability effectively. (4) In the specific instance when one makes use of the ‘spore inocula’, the same may be ad-

equately stored for even longer durations under the following two experimental parameters, for instance :

absolute inhibition of germination, and effective preservation of viability.

(5) It is a common practice to ensure the ‘simultaneous dosing’ of both calibrators and sam- ples 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 ‘incuba- tor’ 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 preci-

sion, 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

pharmacokinetic studies,

as illustrated in Figs. 10.1(b) and (c). 34817652

(a) Latin Square (b) 16 Doses [Calibrators/Samples in Quadruplicate]

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C1 S2 C2 C4 C3 S1 C5 S3 S4 S5 S3 C3 C1 S2 C4 S4 S1 S5 C5 C2 C2 C4 C3 S4 C1 S2 C5 S1 S5 S3

(c) 5 Samples and 5 Calibrators in triplicate

Fig. 10.1. (a) (b) and (c) : Specific Examples of ‘Random Patterns’ for use in Microbiological Plate Assay.

10.3.1.5. Measurement of Zone of Inhibition

To measure the zone of inhibition with an utmost precision and accuracy, the use of a Magnify- ing 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.

10.3.1.6. Calibration

Calibration may be accomplished by means of two universally recognized and accepted methods, namely :

(a) Standard Curves, and (b) 2-By-2-Assay. Each of these two methods will now be discussed briefly in the sections that follows :

10.3.1.6.1. 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 mini- mum absolutely necessary to produce the desired precision ultimately. It has been duly observed that a ‘manual plot’ of either :

zone size Vs log 10 concentration, or

[zone size] 2 Vs log 10 concentration,

will give rise to the formation of ‘near straight line’, as depicted in Fig. 10.2.

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Log Antibiotic Concentration 10

Fig. 10.2. Standard Curve for an Agar-Plate Diffusion Assay

Note : 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.

10.3.1.6.2. 2-By-2-Assay

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 about 3%.

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

nomograms.**

* Usually spread over a two- or four-fold range. ** Nomogram : Graphic representation comprising of several lines marked off to a scale, arranged in such a

way that by using a straight edge to connect known values on two lines an unknown value may be read at the point of intersection with a third line ; used to determine drug doses for specific persons.

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Standard Sample

Test Sample

X = Horizontal distance

mm

between two lines.

( er

Antilog of X = Relative

et

potency of ‘Test’ and

Log Dose 10

Fig. 10.3. A Graphical Representation of a 2-By-2-Assay Response Between a Standard and a Test Sample.