Types of Microbiological (Microbial) Assays

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Chapter: Pharmaceutical Microbiology : Microbiological (Microbial) Assays: Antibiotics-Vitamins- Amino Acids

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.


TYPES OF MICROBIOLOGICAL (MICROBIAL) ASSAYS

 

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 :

 

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 :

 

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.

 

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.**

 

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 :


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 :

 

(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 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.

 

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 :

·        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 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).



 

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.

 

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 :

 

(a) 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 log10 concentration, or

·        [zone size]2  Vs log10 concentration,

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


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.

 

(b) 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.


 

2. Rapid-Reliable-Reproducible Microbial Assay Methods

 

It is worthwhile to mention here that the usual ‘conventional agar-plate assays’ not only re-quire stipulated incubation for several hours but also are rather quite slow. Furthermore, reasonably judicious constant, rigorous, and honest attempts do prevail for the development of ‘rapid-reliable-reproducible microbial assay methods’ based on the exploitation of techniques that essentially meas-ure definite cognizable variations in the pattern of growth-rate invariably after a short incubation.

 

Nevertheless, these so called ‘rapid methods’ generally suffer from the similar critical problems usually encountered in the ‘slow methods’ namely :

·        inadequate specificity, and

·        lack of precision.

 

In actual practice there are two well-known techniques that provide rapid-reliable-reproduc-ible microbial assay methods, namely :

(a) Urease Activity, and

(b) Luciferase Assay.

 

These two aforesaid techniques shall now be discussed briefly in the sections that follows :

 

(a) Urease Activity

 

Urease refers to an enzyme that specifically catalyzes the hydrolysis of urea to ammonia (NH3) and carbon dioxide (CO2) ; it is a nickel protein of microbes and plants which is critically employed in carrying out the clinical assays of plasma-urea concentration.

 

Importanlty, the microorganism Proteius mirabilis grows significanlty in a urea-containing culture medium, whereupon it particularly causes the hydrolysis of urea to ammonia, and thereby helps to raise the pH of the medium. However, the actual production of urease is reasonably inhibited by the so called ‘aminoglycoside antibiotics’,* such as : amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, doxorubicin, cephalosporins, cephamycius, thienamycin, lincomycin, clindamycin, erythromycin, clarithromycin, azithromycin, oleandomycin, spramycins, and the like.

 

Methodology : The various steps involved are as follows :

 

(1) Assay is performed with two series of tubes of urea-containing culture medium that have been duly incorporated with a range of calibrator solutions.

 

(2) First series of tubes in duly added a certain volume of the sample which is essentially equivalent to the volume of the calibrator.

 

(3) Second series of tubes is duly added exactly half the volume of the sample.

 

(4) Both ‘set of tubes’ are subsequently inoculated with P. mirabilis, and duly incubated for a duration of 60–70 minutes.

 

(5) pH of the resulting solution is measured accurately upto 0.01 pH units.

 

(6) In fact, it is possible to obtain two distinct ‘calibration curves’ by plotting pH Vs log10 i.e., the ensuing calibrator concentration for each of the two series.

 

(7) The ‘vertical distance’ existing between the two curves is found to be almost equal to the legarithm of 1/2 the concentration of ‘drug substance’ present in the sample.

 

Note : (1) In usual practice, it is rather difficult to obtain ‘reliable’ results by adopting the ‘Urease Activity’ method.

(2) A standardized, senstitive, and reliable pH Meter is an absolute must for this particular assay.

 

(b) Luciferase Assay

 

In the specific ‘Luciferase Assay’, the firefly luciferase** is made use of for the actual meas-urement of small quantum of ATP*** duly present in a microbial culture, whereby the levels of ATP get proportionately reduced by the ensuing action of the aminoglycoside antibiotics (see Section 10.3.2.1).

 

Methodology : The various steps involved in the ‘Luciferase Assay’ are as enumerated under sequentially :

 

(1) Both test solutions (i.e., after preliminary heating provided the matrix is serum) along with calibrators are carefully added into the various tubes of the culture medium specifically containing a growing microbial culture (i.e., organism).

 

(2) After adequate incubation for a 90 minute duration the cultures are duly treated with ‘apyrase’ so as to ensure the complete destruction of the extracellular ATP.

 

(3) The resulting solution is duly extracted with EDTA/sulphuric acid, and thus the intracellular ATP critically assayed with the firefly enzyme using a ‘Luminometer’.

 

(4) Finally, a ‘calibration curve’ is constructed meticulously by plotting the two vital compo-nents, namely : (a) intracellular ATP content, and (b) log10 i.e., the calibrator concentration.

 

Note : As to date, the ‘Luciferase Assay’ has not yet accomplished a wide application ; however, it may find its enormous usage in the near future with the advent of such ‘luciferase formula-tions’ that would turn out to be even much more active, reliable, and dependable.

 

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