The ‘radioenzymatic assays’ have gained their abundant acceptance and recognition for the
assay of aminoglycoside antibiotics e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin,
tobramycin, doxorubicin, cephalosporins, cephamycins, thienamycin, lincomycin, clindamycin, erythromycin,
clarithromycin, azithromycin, oleandomycin, spramycins etc ; and chloramphenicol (or
Chloromycetine). Importantly, the radioenzymatic assays are exclusively based upon the fact that the
prevailing inherent microbial resistance to the said aminoglycoside antibiotics and chloramphenicol
is predominantly associated with the specific as well as the critical presence of certain highly specialized
enzymes* that particularly render the ‘antibiotics’ absolutely inactive via such biochemical means as :
acetylation, adenylation, and phosphorylation.
It has been duly proved and established that :
􀂳 aminoglycoside antibiotics—are susceptible to prominent attack by these critical and
specific enzymes as :
Aminoglycoside acetyltransferases (AAC) ;
Aminoglycoside adenylyltransferases (AAD) ;
Aminoglycoside phosphotransferases (APH).
􀂳 Chloramphenicol—is prone to predominant attack by the enzyme :
Chloramphenicol acetyl transferases (CAT).
Mechanism of Action : The mechanism of action of these enzymes viz, AAC, AAD, and APH
are not the same :
Acetyltransferases [i.e., AAC]—invariably attack the most susceptible amino moieties (–NH2),
and to accomplish this critical function may require acetyl coenzyme A (AcCoA).
Adenylyltransferases [i.e., AAD] and Phosphotransferases [i.e., APH]—these enzymes usually
attack the most susceptible hydroxyl moieties (–OH), and specifically requires adenosine
triphosphate [ATP] i.e., another nucleotide triphosphate.
Applications : As to date quite a few AAC and AAD enzymes have been judiciously employed
for the radioenzymatic assays.
Example : Both the enzyme and the suitable radiolabelled cofactor [1 – 14C]** acetyl coenzyme
A, or [2 – 3H]*** ATP are used frequently in order to specifically radiolabel the ‘drug substance’ under

Method— The various steps involved in the assay are as follows :
(1) Enzymes are normally prepared by anyone of the following two techniques,
(a) Osmotic Shock i.e., by breaking the cells of an appropriate microbial culture by exposing
than to a change of strength of solution therby affording a definite perceptible alteration in
the ‘osmotic pressure’, and
(b) Ultrasonic Sound-waves i.e., by breaking the cells of a suitable bacterial culture by means
of the high-frequency ultrasonic sound waves.
Thus, the said two methods do break open the cells to a considerable extent, and no purification
is required at all.
(2) Radiolabelled drug substance is subsequently separated from the ensuing reaction mixture
soonafter the said reaction has attained completion duly. Thus, the exact quantum of the extracted
radioactivity is observed to be directly proportional to the exact quantum of the drug substance present
in the given sample.
Note : Separation of two types of antibiotics are accomplished duly as stated under :
(a) Aminoglycoside Antibiotics—by binding them suitably to phophocellulose paper, and
(b) Chloramphenicol—by making use of an organic solvent.

In a particular situation when the reactants are adequately present in enough quantum, and the
prevailing reaction attains completion in due course, one may conveniently plot a graph of the counts
per minute (min–1) Vs concentration of calibrator, which is found to be linear, as illustrated

Non-Isotopic Modification
The calibration accomplished by using the radiolabelled drug essentially needs either a Geiger
Müller Counter or a Scintillation Counter, for
measuring the ensuing radio activity (in mC) of the radioactive
chemicals, which being an enormously expensive
equipment, and a skilled technician. Therefore,
in order to circumvent these glaring untoward serious
problems one may adopt a photometric variation
of the aminoglycoside acetyltransferases [AAC] assay meticulously. For this the sulphydry reagent
viz., 5, 5′-dithiobis (2-nitrobenzoic acid) is incorporated carefully into the on-going assay-system.
Thus, the said reagent specifically interacts with the corresponding coenzyme A (reduced form)
duly generated thereby producing a distinct yellow-coloured product that may be quantitatively assayed
by using a previously standardized UV-Visible Spectrophotometer.
(a) Reactions : The two reactions are as follows :
(b) Aminoglycoside + Acetyl CoA —→ Acetyl – Aminoglycoside + CoASH
CoASM + DTNB —→ Yellow Product

Rapid-Reliable-Reproducible Microbial Assay Methods

Rapid-Reliable-Reproducible Microbial Assay Methods

It is worthwhile to mention here that the usual ‘conventional agar-plate assays’ not only require
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-reliablereproducible
microbial assay methods’ based on the exploitation of techniques that essentially measure
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-reproducible
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 :
 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
 Luciferase Assay
In the specific ‘Luciferase Assay’, the firefly luciferase** is made use of for the actual measurement
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
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 components,
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 formulations’
that would turn out to be even much more active, reliable, and dependable.



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 :
 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 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
subsequent standardization.

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
as :
􀂳 proper choice of ‘indicator organism’,
􀂳 suitable culture medium,
􀂳 appropriate sample size, and
􀂳 exact incubation temperature. 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
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 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
􀁏 pharmacokinetic studies,

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
(b) 2-By-2-Assay.
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. 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



There are several well-recognized variants in assay profile for antibiotics, vitamins, and amino
acids, namely :
(a) Calibration of assay,
(b) Precision of assay,
(c) Accuracy of assay, and
(d) Evaluation of assay performance.
The various aspects of assay profile stated above shall now be treated briefly in the sections that
follows :
 Calibration of Assay
Irrespective of the method adopted for the microbial assay it is absolutely necessary to work out
a proper calibration in case the ultimate result is necessarily expected in terms of the absolute units viz.,
mg.L– 1.
Calibrator Solutions — The calibrator solutions are essentially prepared either from a pure
sample of the drug to be assayed or a sample of known potency.
Importantly, there are certain drug substances that are hygroscopic in nature ; and, therefore,
their inherent potency may be expressed as :

(a) ‘as-is’ potency — which refers to — ‘the potency of the powder without drying’,* and
(b) ‘dried potency’— which refers to — ‘the potency after drying to constant weight under
specified/defined experimental parameters’.
Importantly, in as-is potency, the drug should be stored in such a manner that it may not lose or
absorb water ; whereas, in dried potency the drug should always be dried first before weighing.
Thus, once an appropriate ‘standard materials’ is actually accomplished, the calibrator solutions**
usually covering a suitable range of concentrations should be prepared accordingly. However,
the actual number and concentration range of the collaborators shall solely depend on the specific
type of assay being carried out. Likewise, the matrix*** wherein the calibrators are dissolved duly is
also quite vital and important, unless it may be shown otherwise, must be very much akin to the respective
matrix of the samples.
Note : (1) It should be absolutely important when carying out the assay of drugs present in ‘serum’, due
to the fact that protein-binding may invariably influence the ultimate results of microbiological
assay predominantly.
(2) No assay can give rise to fairly accurate results unless and until the suitable ‘calibrator solutions’
(i.e., calibrators) precisely prepared in an appropriate matrix.
 Precision of Assay
Precision refers to – ‘agreement amongst the repeated measurements’.
Alternatively, precision is an exact measure of reproducibility, and is duly estimated by replicating
a single sample a number of times thereby determining :
􀁏 mean result (X) ,
􀁏 standard deviation (SD), and
􀁏 coefficient of variation (SD/ X × 100).
Intra-Assay Precision—usually refers to the precision within a single-run exclusively.
Inter-Assay Precision—normally refers to the precision between two or more runs.
Degree of Precision—required in a specific instance essentially will determine two cardinal factors,
namely :
􀁑 number replicates actually needed for each calibrator, and
􀁑 number plus concentration range of calibrators.

Accuracy of Assay
Accuracy may be defined as — ‘a measure of the correctness of data as these correspond to
the true value’.
Considering that the calibrator solutions were prepared correctly from the suitable ‘drug’, the
resulting accuracy of a specific result shall exclusively depend upon two important aspects, namely :
􀁏 precision of assay, and
􀁏 specificity of assay.
Poor Specificity is encountered usually in the following three instances, such as :
􀁏 samples comprising of endogenous interfering materials,
􀁏 presence of other antibacterial agents, and
􀁏 active metabolites of the ‘drug’ being assayed.
Positive Bias i.e., if the other drugs or drug metabolites are present simultaneously, accuracy
of assay shall be expressed predominantly as a positive bias.*
Negative Bias i.e., if there are antagonists present in an appreciable quantum, accuracy of assay
will be expressed mostly as a negative bias.
Note : In fact, inaccuracy caused due to apparent poor precision will invariably exhibit absolutely
‘no bias’, and that caused on account of either under–or over-potent calibrators will exhibit
positive and negative bias respectively.
Evaluation of Assay Performance
It has been duly proved and established that while assessing the performance characteristics of an
altogether newly developed assay, both intra–and inter–assay precision duly spread over the entire
range of expected concentrations must be estimated precisely.
Important Points : These are as stated under :
(1) It is extremely important to check the accuracy with the help of the ‘spiked samples’** very
much spread over the entire range of concentrations used in the assay.
(2) Assaying ‘drug substances’ in biological fluids e.g., urine, blood, serum, sputum, cerebrospinal
fluid (CSF) etc.
(3) Samples withdrawn from individual subjects who have been duly administered with the drug
either enterally*** or parenterally**** by virtue of the fact that in vitro metabolites may only be
apparent in these instances.

(4) Such substances that might have an inherent tendency to interfere in the assay should be
thoroughly checked for there possible interference either alone or in the presence of the ‘drug substance’
being assayed.
(5) In an ideal situation, preferentially a relatively large number of samples must be assayed
both by the ‘new method’ and the ‘reference method’ individually, and the subsequent results obtained
may be meticulously by linear regression ; and thus the ensuing correlation coefficient of the
said two methods determined.
(6) Routinely employed methods may be tackled with ‘internal controls’* almost in every run ;
and, therefore, the laboratories that are actively engaged in the assay of clinical specimens must take
part in an external quality control programme religiously.



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
known activity.
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 :
􀁏 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.

Natural Killer Cells [NK Cells]

Natural Killer Cells [NK Cells]

It has been amply proved and widely accepted that the body’s cell-mediated defense system
usually makes use of such cells that are not essentially the T cells***. Further, certain lymphocytes that
are known as natural killer (NK) cells, are quite capable of causing destruction to other cells, particularly
(a) tumour cells, and (b) virus-infected cells. However, the NK cells fail to be immunologically

specific i.e., they need not be stimulated by an antigen. Nevertheless, the NK cells are not found to be
phagocytic in nature, but should definitely get in touch (contact) with the target cell to afford a lysing
 Interferons [IFNs]
Issacs and Lindenmann (1957)* at the National Institute of Medical Research, London (UK)
discovered pioneerly the interferons (IFNs) while doing an intensive study on the various mechanisms
associated with the ‘viral interference’**.
It is, however, an established analogy that viruses exclusively depend on their respective host
cells to actually cater for several functions related to viral multiplication ; and, therefore, it is almost
difficult to inhibit completely viral multiplication without affecting the host cell itself simultaneously.
Importantly, interferons [IFNs] do handle squarely the ensuing infested host viral infections.
Interferons [IFNs] designate — ‘a particular class of alike antiviral proteins duly generated
by some animal cells after viral stimulation’.
It is, therefore, pertinent to state here that the critical interference caused specifically with viral
multiplication is the prime and most predominant role played by the interferons.
 Salient Features : The salient features of interferons may be summarized as stated
under :
(1) Interferons are found to be exclusively host-cell-specific but not virus-specific

(2) Interferon of a particular species is active against a plethora of different viruses.
(3) Not only do various animal species generate interferon variants, but also altogether various
kinds of cells in an animal give rise to interferon variants.
(4) All interferons [IFNs] are invariably small proteins having their molecular weights ranging
between 15,000 to 30,000. They are observed to be fairly stable at low pH range (acidic),
and are quite resistant to heat (thermostable).
(5) Interferons are usually produced by virus-infected host cells exclusively in very small
(6) Interferon gets diffused into the uninfected neighbouring cells as illustrated in Fig. 9.7.
Explanation : The various steps involved are as follows :
(1) Interferon happens to interact with plasma or nuclear membrane receptors, including the
uninfected cells to produce largely mRNA essentially required for the critical synthesis of
antiviral proteins (AVPs).
(2) In fact, AVPs are enzymes which causes specific disruption in the different stages of viral
Examples : These are as given under :
(a) One particular AVP inducts the inhibition of ‘translation’ of viral mRNA by affording
complete blockade in the initiation of the ensuing protein synthesis,
(b) Another AVP causes the inhibition of the phenomenon of ‘polypeptide elongation’,
(c) Still another AVP takes care of the process of destruction with regard to mRNA before
 Interferon : An Ideal Antiviral Substance : Various cardinal points are as stated
below :
• Prevailing ‘low concentrations’ at which interferon affords inhibition of viral multiplication
are found to be absolutely nontoxic to the uninfected cells.
• Interferon possesses essentially a good number of beneficial characteristic properties.
• Interferon is distinguishably effective for only short span.
• Interferon plays a pivotal and vital role in such critical infections which happen to be quite
acute and transient in nature, for instance : influenza and common colds.
Drawback : Interferon has a serious drawback, as it has practically little effect upon the viral
multiplication in cells that are already infected.
Interferon Based on Recombinant DNA Technology : In the recent past ‘interferon’
has acquired an enormous recognition and importance by virtue of its potential as an antineoplastic
agent, and, therefore, enabled its production in a commercial scale globally on a top public-health
priority. Obviously, the interferons specifically produced by means of the recombinant DNA technology
are usually termed as recombinant interferons [rINFs]. The rINFs have gained an overwhelming
global acceptability, popularity, and utility due to two extremely important reasons, namely : (a) high
purity, and (b) abundant availability.

Usefulness of rINFs : Since 1981, several usefulness of rINFs have been duly demonstrated
and observed, such as :
Antineoplastic activity – Large dosage regimens of rINFs may exhibit not so appreciable overall
effects against certain typical neoplasms (tumours), whereas absolute negative effect on others.
However, the scanty results based on the exhaustive clinical trials with regard to the usage of
rINFs towards anticancer profile may be justifiably attributed to the following factual observations,
such as :
􀁑 several variants of interferons vis-a-vis definitive antineoplastic properties,
􀁑 rINFs in cojunction with other known chemotherapeutic agents might possibly enhance
the overall antineoplastic activity,
􀁑 quite significant and encouraging results are duly achievable by making use of a combination
of :
rINFs + doxorubicin*
or rINFs + cimetidine**
􀁑 subjects who actually failed to respond reasonably well earlier to either particular chemotherapy
or follow up treatment with interferon distinctly showed remarkable improvement
when again resorted to the ‘original chemotherapy’.
 Classical Recombinant Interferons [rIFNs] : There are quite a few classical
recombinant interferons [rIFNs] have been meticulously designed and screened pharmacologically to
establish their enormous usefulness in the therapeutic armamentarium. A few such rIFNs shall now be
treated briefly in the sections that follows :
[A] Interferon-α [Syn : Alfa-interferon ; Leukocyte interferon ; Lymphoblastoid interferon ;]
Interferon-α is a glycopeptide produced by a genetic engineering techniques based on the human
sequence. It does affect several stages of viral infections, but primarily inhibits the viral-protein translation.
It is invariably employed to prevent and combat the hepatitis B and C infections. In usual
practice the drug is administered either via subcutaneous (SC) route or intramuscular (IM) route.
However, it gets rapidly inactivated but generally the overall effects outlast the ensuing plasma
Toxicities – include neurotoxicity, flu-like syndrome, and bone-marrow suppression.
Drug interactions – may ultimately result from its ability to minimize the specific hepatic syndrome
P450-mediated metabolism.
[B] Interferon Alfa-2A, Recombinant [Syn : IFA-α A ; R0-22-8181 ; Canferon ; Laroferon ; Roferon-
A ;]
Interferon alfa-2A refers to the recombinant HuIFN-α produced in E. coli, and made up of
165 amino acids

Characteristic Pharmacologic Activities : These are as follows :
(1) Enhances class I histocompatibility molecules strategically located on lymphocytes.
(2) Increases the production of ILs-1 and -2 that critically mediates most of the therapeutic and
toxic effects.
(3) Regulates precisely the antibody responses.
(4) Increases NK cell activities.
(5) Particularly inhibits the neoplasm-cell growth via its distinct ability to inhibit appreciably
the protein synthesis.
(6) Being antiproliferative in nature it may exert its immunosuppressive activity.
(7) Action on the NK cells happens to be the most vital for its antineoplastic action.
(8) Approved for use in hairy-cell leukemia and AIDS-related Kaposi’s sarcoma.
(9) Drug of first choice for the treatment of renal-cell carcinoma.
(10) Preliminary clinical trials ascertained virtually its promising efficacy against quite a few
typical disease conditions as : ovarian carcinoma, non-Hodgkin’s lymphoma, and metastatic
carcinoid tumour.
(11) Besides, it exhibits marked and pronouned antiviral activity against the RNA viruses.
(12) Effective in the treatment of varicella in immunocompromised children, non-A and non-
B hepatitis, genital warts, rhinoviral colds, possible opportunistic bacterial infections
in renal and transplant recipients.
(13) Increases the targetting process associated with monoclonal antibody (MAB)-tethered
cytotoxic drugs to the neoplasm cells.
[C] Interferon Alfa-2B, Recombinant [Syn : IFNα2 ; Introna; Intron A ; Viraferon ; Seh-30500 ;
YM-14090 ;] ;
The recombinant HuIFNα is produced in E. coli.
Therapeutic Applications : are as stated under :
(1) Approved for use in several disease conditions as : hairy-cell leukemia, AIDS-related
Kaposi’s sarcoma, myclogenous leukemia, melanoma, chronic hepatitis, and
condylomata acuminata.
(2) Most of its actions are very much similar to those of rIFN-αA.



Phygocytosis may be defined as — ‘the engulfing of microorganisms or other cells and foreign
particles by phagocytes’.
Alternatively, phagocytosis (from the Greek words for eat and cell) referts to — ‘the phenomenon
of ingestion of a microorganism or any particulate matter by a cell’.
Interestingly, the human cells which critically carry out this ardent function are collectively
known as phagocytes, such as : all types of WBCs, and derivatives of WBCs.
Actions of Phagocytic Cells : In this event of a contracted infection, both monocytes* and
granulocytes** usually get migrated to the infected area. Interestingly, during this process of migration,
the monocytes do get enlarged to such a dimension and size that they finally develop into the actively
phagocytic macrophages.
Types of Macrophages : There are, in fact, two major categories of the macrophages, such as :
(a) Wandering Macrophages : Based on the glaring fact that these cells (monocytes) do have
a tendency to leave the blood and subsequently migrate via the tissue cells to the desired
infected areas, they are commonly known as wandering macrophages.
(b) Fixed Macrophages (or Histocytes) : A monocyte that has eventually become a resident in
tissue. Fixed macrophages or histocytes are invariably located in certain specific tissues
and organs of the body. In fact, they are found abundantly in various parts of a human body,
for instance :
• Bronchial tubes ; • Lungs (alveolar macrophages) ;
• Bone marrow ; • Nervous system (microglial cells ) ;
• Lymph nodes ; • Peritoneal cavity (surrounding abdominal organs) ;
• Liver (Kupffer’s cells ) ; • Spleen ;

Importantly, the macrophage variants critically present in the body strategically constitute the
mononuclear phagocytic (reticuloendothelial) system.
 Functions of Phagocytes (or Phagocytic Cells) : It has been duly observed that when
an infection gets contracted one may apparently observe a distinct shift taking place predominantly in
the particular types of WBC which runs across the blood stream. Thus, the following cardinal points
may be noted, carefully :
􀁑 Granulocytes – particularly the ‘neutrophils’ occur overwhelmingly in the initial phase of
infection, at this point in time they are found to be extremely phagocytic in nature.
􀁑 Distinct aforesaid dominance is evidently shown by the presence of their actual number in a
differential WBC count.
􀁑 With the progress of contracted infection, the macrophages also predominate – scavenge –
phagocytize remaining live/dead/dying microorganisms.
􀁑 Enhanced number of monocytes, that eventually develop into the corresponding macrophages,
is adequately reflected in the WBC-differential count explicitely.
􀁑 Blood and lymph containing bacteria when made to pass via various organs in the body
having fixed macrophages, cells of the mononuclear phagocytic system ultimately get rid
of the bacteria by phagocytosis.
􀁑 Mononuclear phagocytic system also helps in the critical disposal of the worn-out blood

Mechanism of Phagocytosis : In order to understand the exact and precise mechanism
of phagocytosis, we may have to divide the phenomenon of phagocytosis, as illustrated in Fig. 9.6,
into four cardinal phases, such as : chemotaxis, adherence, ingestion, and digestion. These four distinct
phases shall now be treated briefly in the sections that follows from [A] through [D] :
[A] Chemotaxis [Syn : Chemotropism] :
Chemotaxis may be defined as — ‘the movement of additional white blood cells to an area of
inflammation in response to the release of chemical mediators by neutrophils, monocytes, and
injured tissue’.
In other words, chemotaxis refers to the chemical attraction of the phagocytes to microbes.
Importantly, the various ‘chemotactic chemical susbtances’ which specifically attract the
phagocytes happen to be such microbial products as components of :

• white blood cells (WBCs),
• damaged tissue cells, and
• peptides derived from complement.
[B] Adherence :
Adherence refers to the act or condition of sticking to something. In fact, it represents the ensuing
adherence of antigen-antibody complexes or cells coated with antibody or complement to cells
bearing complement receptors or Fe receptors. It is indeed a sensitive detector of complement-fixing
Because, adherence is intimately related to phagocytosis, it represents the attachment of the
later’s plasma membrane onto the critical surface of the bacterium or such other foreign material.
Nevertheless, adherence may be hampered by the specific presence of relatively larger capsules or
M protein*. Besides, in certain instances adherence takes place quite easily and conveniently, and the
microbe gets phagocytized rapidly.

[C] Ingestion :
In usual practice adherence is followed by ingestion. One may vividly notice that during the
phenomenon of ingestion, the plasma membrane belonging to the phagocyte gets extended in the form
of distinct projections usually termed as pseudopods which eventually engulf the bacterium. Thus,

once the bacterium gets duly surrounded, the pseudopods meet and fuse ultimately, thereby surrounding
the bacterium with a particular ‘Sac’ known as phagocytic vesicle or phagosome.
[D] Digestion :
Digestion refers to the particular phase of phagocytosis, wherein the respective phagosome*
gets detached from the plasma membrane and duly enters the cytoplasm. Later on, within the cytoplasm
the phagosome meticulously gets in touch with the lysosomes** which essentially comprise of two
important components, namely :
• digestive enzymes, and
• bactericidal substances.
Modus Operandi [or Mode of Action] : The various steps involved are as given below :
(1) Both phagosome and lysosome membranes upon contacting each other invariably gets fused
to result into the formation of a ‘single larger structure’ termed as ‘phagolysosome’.
(2) Interestingly, the integral contents of the phagolysosome usually ‘kills’ most types of
microorganisms within a span of 10–30 minutes. The most plausible and possible reason for
such a marked and pronounced bactericidal effect is perhaps due to the specific contents of
the lysosomes.
(3) Residual body : After completion of the process of digestion the actual contents of the
phagolysosome are duly brought into the cell by ‘ingestion’ ; and, therefore the
phagolysosome essentially and exclusively comprises of the indigestible material, which is
usually known as the ‘residual body’.
(4) Residual body subsequently takes a step forward toward the cell boundary and critically
discharges its ‘waste products’ very much outside the cell.
A Few Exceptions : These exceptions are as stated below :
(a) Toxins of certain microorganisms viz., toxin-producing Staphylococci plus the bacterium
Actinobacillus (present in dental plaque, may actually exert a cidal effect upon the phagocytes.
(b) Some other microbes, for instance : Chlamydia, Leishmania, Mycobacterium, and Shigella
together with the ‘malarial parasites’ may possibly dodge and evade the various components
of the immune system by gaining an access into the phagocytes.
(c) Besides, the said microorganisms may virtually block the ultimate fusion between phagosome
and lysosome, as well as the adequate process of acidification (with HCl) of the digestive

Internal Defense Mechanisms

Internal Defense Mechanisms

Internal defense mechanisms emphatically constitute the ‘second-line of defense’ comprising
of the body’s internal mechanisms that may be critically mobilized against the highly specific invading
Mechanisms : The internal defense mechanisms are of two different types, such as :
(a) Non specific in action – e.g., phagocytosis, and
(b) Specifically aimed at the pathogens – e.g., sensitized cells, and antibodies.
Importantly, the above two different types are usually designated as nonspecific defense mechanisms
and specific acquired immunity*.
However, it is pertinent to state here that while the infection is active the two aforesaid mechanisms
virtually exert their action simultaneously in order to rid the body of the so called ‘invading
microbes’. In fact, this very interrelationship, and the interrelationships prevailing between the defense
mechanisms may be explicitely depicted

Nonspecific Defense Mechanisms
Mother nature has enabled the ‘human body’ so splendidly as to critically mobilize several
factors that act nonspecifically against the possible wide spread invasion by the ‘foreign organisms’.
Interestingly, such cardinal and vital factors essentially consist of the following four typical examples,
namely :
􀁑 complement system,
􀁑 phagocytosis,

􀁑 naturally occurring cytotoxic lymphocytes, and
􀁑 in terferon.
Each of the aforesaid factors shall now be treated individually in the sections that follows :
 Complement System
Higher animal’s serum usually made up of a particular group of ‘eleven proteins’, which are
highly specific in nature, and are widely referred to collectively as the so called complement system by
virtue of the fact that its action complements predominantly to that of some prominent antibody-mediated
reactions. In other words, the complement system critically enacts a pivotal role with respect to
the overall generalized resistance against the infection caused by the ‘pathogens’ ; and, therefore,
accounts for as the ‘principal mediator’ of the ensuing specific inflammatory response.
Mode of Action (Modus Operandi) : The various steps involved are as follows :
(1) When the very ‘First Protein’, belonging to cluster of elevan proteins, gets duly activated
there exist distinctly a prominent ‘sequential cascade’ whereby the ‘active molecules’ duly
come into being via the inactive precursors*.
(2) Some of the protein variants do get activated very much along the ‘sequential cascade’ that
may function as mediators of a specific response, and eventually serves as activators of the
next step.

Complement Fixation (or Attachment) : In a broader perspective, the complement system is
quite capable of attacking and killing the invading cells exclusively after the antibody gets bound to the

in cell attack. Summararily, it represents as the classical or antibody-dependent pathway that prevalently
need to be activated by specific antibody : C1, C4, C2 and C3.



In a broader sense the ensuing interaction existing between a host (human body) and a
microorganism designates an excellent unique dynamic phenomenon whereby each and every protagonist
critically serves to maximize its overall survival. It has been duly observed that in certain typical instances,
after a specific microbe gains its entry or comes in contact with a host, a distinct positive mutually
beneficial relationship takes place which ultimately becomes integral to the final health of the host. In
this manner, the microorganisms turn out to be the normal microbiota*. However, in other such cases,
the particular microorganism causes, induces or produces apparent devastating and deleterious overall
effects upon the host ; and, therefore, may finally even cause death of the host via a dreadful ailment.
Interestingly, the prevailing environment of a ‘host’ is heavily surrounded with microorganisms,
and there lies an ample scope and opportunity to come in their contact every moment of the day.
Nevertheless, quite a few of these microbes are pathogenic in nature (i.e., cause disease). Surprisingly,
these pathogens are at times duly guarded and prevented from producing a disease due to the inherent
competition offered by the normal microbiota. In reality, the invading pathogens are squarely kept
away from the host by the ‘normal microbiota’ by using nutrients, resources, space, and may even yield
such chemical substances which would repel them ultimately.
In addition to the above stated glaring scientific fact and evidences these ‘normal microbiota’
grossly prevent colonization of pathogens to a great extent ; and, thereby, most probably checking the
disease (to the host) via ‘bacterial interference’.
Example : An excellent typical example is stated as under :
Lactobacilli – present strategically in the female genital tract (FGT) usually maintain a low pH
(acidic), and thereby exclusively afford the colonization by the pathogenic microbes. Besides, the
corynebacteria located critically upon the skin surface give rise to the formation of ‘fatty acids’ which
ultimately inhibit the phenomenon of colonization by the pathogenic organisms.
Note : It is an excellent example of ‘amensalism’. (i.e., symbiosis wherein one population (or individual)
gets affected adversely and the other is unaffected).
Interestingly, the ‘normal microbiota’ usually give rise to protection confined to a certain degree
from the invading pathogens ; however, they may themselves turn into pathogenic in character and
cause disease under certain particular circumstances. Thus, these ‘converted pathogens’ are invariably
known as ‘opportunistic microorganisms’** or pathogens.
Based on the above statement of facts and critical observations one may conclude that on one
hand pathogen makes use of all the opportune moments available at its disposal to cause and induct

infection, the host’s body possesses a plethora of ‘defense mechanisms’ to encounter the infection. In
fact, the observed intricacies prevailed upon by the host-pathogen relationship are not only numerous
but also quite divergent in nature, which may be classified under the following three heads, such as :
(a) Natural Resistance,
(b) Internal Defense Mechanisms, and
(c) Nonspecific Defense Mechanisms.
The aforesaid three categories shall now be discussed separately in the sections that follows :

Natural Resistance
It has been observed that the two cardinal aspects, namely : (a) physiological needs, and (b) metabolic
requirements, of a pathogen are an absolute necessity in establishing precisely the extent vis-a-vis
the range of potentially susceptible hosts. However, the naturally resistant hosts exert their action in
two variant modes, such as :
􀁑 miserably fail to cater for certain urgently required environmental factors by the microbes
for their usual growth, and
􀁑 essentially possess defense mechanisms to resist infection considerably.
Besides, there are some other factors pertaining to the host’s general health, socioeconomic status,
level of nutrition potentiality, and certain intangible conditions viz., stress, mental agony, depression
Natural resistance essentially comprises of the following four vital and important aspects :
 Species Resistance
In general, the fundamental physiologic characteristics of humans, namely : normal body temperature
may give a positive clue whether or not a specific bacterium can be pathogenic in nature.
Likewise, in host-specific e.g., human and bovine species, the tubercle bacillus is found to crossinfect
both humans and cattle having almost an identifical body temperature.
Salient Features : The salient features of species resistance are as given under :
(1) inability of a bacterium to induct disease in the resistant species under the natural environments,
(2) critical production in the specific resistant species of either a localized or a short-period
infection caused solely due to an experimental inoculation vis-a-vis a progressive or generalized
ailment in naturally susceptible species, and
(3) introduction of experimental disease particularly in the resistant species exclusively caused
by massive doses of the microbes, usually in two different ways :
(a) under unnatural parameters, and
(b) by an unnatural route.

Racial Resistance
Exhaustive and intensive studies have amply proved that the very presence of a pathogen in the
isolated races give rise to a gradual selection for resistant members, because the susceptible members
die of progressive infection ultimately. It may be further expatiated by the following three glaring
examples :

Examples :
(i) Incorporation of altogether ‘new pathogens’ e.g., tubercle bacillus, by the relatively resistant
Europeans into an isolated American Indians population*, finally caused epidemics
that almost destroyed a major proportion of the ensuing population.
(ii) African Blacks (Negros) invariably demonstrate a relatively high resistance to the tropical
diseases, namely : malaria, yellow fever, and
(iii) Orientals do exhibit a much reduced susceptibility to syphilis.
 Individual Resistance
It may be critically observed that there are certain individuals who apparently experience fewer
or less severe infections in comparison to other subjects, irrespective of the fact that :
􀁏 both of them essentially possess the same racial background, and
􀁏 do have the same opportunity for ultimate exposure.
Causation : Individual resistance of this nature and kind is perhaps on account of :
􀁏 natural in-built resistance factor, and
􀁏 adaptive resistance factor.
Age Factor – is equally important, for instance :
􀁏 aged people are more prone to such ailments as : Pneumonia – most probably due to a
possible decline of the ‘immune functions’ with advancement in growing age.
􀁏 children i.e., very young individuals are apparently more susceptible to such ‘children’s
disease’ as : Chicken-pox, measles–just prior to their having acquired enough in-built
resistance/immunity that essentially follows both inapparent and overt contracted infections.
Genetic Factor – Immunodeficiencies** found in some, individuals are caused solely due to
‘genetic defects’, that largely enhance the probability and susceptibility to disease.
Other Factors – include malnutrition, personal hygiene, and an individual’s attitude to sex profile
; hazards and nature of work-environment ; incidence of contacts with infected individuals, and an
individual’s hormonal vis-a-vis endocrine balance – they all do affect the overall frequency as well as
selectivity of some critical ailments.
External Defense Mechanisms
In fact, the external defense mechanisms do represent another cardinal and prominent factor in
natural resistance ; however, they essentially involve the chemical barriers as well. Besides, two other
predominant factors viz., (a) mechanical barriers, and (b) host secretions, essentially make up the
body’s First-Line of Defense Mechanism against the invading microorganisms.
Mechanical Barriers – actually comprise of such materials as : intact (unbroken) skin and
mucous membranes that are practically incapable of getting across to the infectious agents. However,
the said two mechanical barriers viz., intact skin and mucous membranes do afford a substantial ‘effective
barrier’, whereas hair follicles, dilatation of sweat glands, or abrasions do allow the gainful
entry for the microbes into the human body.

Examples : Various typical examples are as given under :
(1) Large segment of microbes are duly inhibited by such agents as :
􀁏 low pH (acidity),
􀁏 lactic acid present in sweat, and
􀁏 fatty acids present in sweat.
(2) Mucous secretions caused by respiratory tract (RT), digestive tract (DT), urogenital
tract (UT) plus other such tissues do form an integral protective covering of the respective
mucous membranes thereby withholding and collecting several microorganisms until they
may be either disposed of effectively or lose their infectivity adequately.
(3) Chemical Substances – Besides, the ensuing mechanical action caused by mucous, saliva,
and tears in the critical removal of microorganisms, quite a few of these secretions do contain
a number of chemical substances which critically cause inhibition or destruction of
Examples : A few typical examples are as stated under :
(a) Lysozyme – an enzyme invariably observed in several body fluids and secretions viz.,
blood, plasma, urine, saliva, cerebrospinal fluid, sweat, tears etc., that predominantly do
exert an effective antimicrobial action on account of its inherent ability to lyse some
particular Gram positive microbs by specifically affording the hydrolysis of
(b) Several other hormones and enzymes are capable of producing distinct chemical, physiological,
and mechanical effects that may ultimately cause minimization of susceptibility
to reduction, and
(c) The prevailing inherent acidity or alkalinity of certain ‘body fluids’ possess an apparent
deleterious effect upon several microbes, and helps to check and prevent the potential
pathogens for gaining an easy access to the deeper tissues present in the body.
(d) Lactoferrin-Lactoferrin is an iron-containing red-coloured protein found in milk (viz.,
human and bovine) that essentially possesses known antibacterial characteristic features.
It is also found in a plethora of body-secretions that specifically and profusely bathe the
human mucosal surfaces, namely :
• bronchial mucous ; • seminal fluids ;
• hepatic bile ; • saliva ;
• nasal discharges ; • tears ; and
• pancreatic juice ; • urine.
Lactoferrin forms a vital and important constituent of the highly particular granules of the
‘polymorphonuclear leukocytes’*.
(5) Transferrin : It represents the serum counterpart of lactoferrin. In fact, both these typical
proteins essentially possess high molecular weights ~ 78,000 daltons, besides having several
metal-binding critical sites.



gets adequately reflected by the ‘antibody titer’, that accounts for the total quantum
antibody** present in the serum. Soonafter the very first initial contact with an antigen, the serum of
the exposed person emphatically comprises of absolutely no detectable antibodies upto even several
days at a stretch. However, one may distinctly notice a gradual rise in the
‘antibody titer’ i.e., first andforemost
IgM*** antibodies are produced and subsequently IgG****
Ultimately, a slow decline in antibody titer takes place. Importantly, the ensuing pattern of decline
duly designates the characteristic feature of a
primary response to an antigen. However, the immune
of the host gets adequately intensified immediately after a second exposure to an antigen.Nevertheless, this secondary response is usually termed as memory or anamnestic response
It has been observed that there exists certain
activated B lymphocytes that fail to turn into the so
antibody-producing plasma cells, but do persist and sustain as the long-lived memory cells.
After a long span even stretching over to several decades, when such
‘cells’ are duly stimulated by the
‘same antigen’
, they invariably tend to differentiate rapidly into the much desired antibody-producing
plasma cells.
Actually, this ultimately affords the fundamental basis of the secondary immune responseas depicted


Evidences from an exhaustive survey of literature has revealed that during the early stages
pertaining to the historical development of immunological experimentation, the, biologists duly learned
‘certain specific types of immunity’
may be meticulously transferred between animals (belonging
to the same species) by actually
transferring serum from immunized to nonimmunized animals.
Importantly, other kinds of immunity could not be transferred effectively
via blood serum. Obviously, at a
much later stage it was duly understood that these special types of immunity may be easily and conveniently
transferable only when certain specific lymphocytes were transferred actually.
Based on the further extensive and intensive researches carried out by the
‘immunologists’ across
the globe ultimately accumulated copious volumes of valuable informations and results that are now wellknown
to reflect the
two major segments of the so called vertebrate immune system, namely :
a) immunity associated with serum-transfer reflecting the activities of the humoral (antibody-mediated) immune system*,
) immunity associated with transfer of lymphocytes reflecting the activities of the cellmediated
immune system*.
Nevertheless, these
two aforesaid major segments exert their actions both individually, and togetherin order to safeguard the humans from ailment irrespective of their age, race, and gender.


may be defined as — ‘the state of being immune to or protected from a disease
especially an infectious disease’.
Importantly, this particular state is invariably induced by having been exposed to the
on an microorganism that critically invades the body or by having been duly immunized with a
capable of stimulating the production of specific antibodies.
the generation of an immune response solely depends upon the prevailing
interaction of
three cardinal components of the immune mechanism, such as :
immunogen stimulation,
humoral immune system, and
cellular immune system.
Since 1901 and as to date the epoch making discovery and spectacular evolution of
i.e., conglomeration of immune system variants, across the world has
revolutionized not only the safer quality of life of human beings but also provided a broad spectrum of
newer avenues in combating the complicated dreadful not-so-easy diseases of the present day.
Immune Response :
In reality, the immune responses do refer to such processes whereby animals
(including humans) give rise to certain
specifically reactive proteins (known as ‘antibodies’) and
adequate cells in response to a great number of
foreign organic molecule and macromolecule variants.
Based on the scientifically demonstrated proofs and evidences the generalized
immune response
essentially possesses
four major primary characteristic features, such as :
a) discrimination,
b) specificity,
c) anamnesis, and(
d) transferability by living cells.
It usually designates the
‘ability of the immune system’ to have a clear-cut discrimination
‘self’ and ‘nonself’ ; and, therefore, it invariably responds exclusively to such materials that
happen to be
foreign to the host.
It refers to such a response that is extremely specific either solely for the
inducing material or
to which the immune cells or antibodies would interact in a much prominent and greater
It most commonly refers to the critical ability to elicit a larger specific response much more
rapidly on being induced by a
‘second exposure’ to the same very foreign antigen. It is also termed as
anamnestic response or the immunologic memory, as illustrated in Fig. 9.1.
Transferability by Living Cells
Interestingly, the
active immunity is observed to be exclusively transferable from one particular
inbred animal specimen to another by the respective
‘immune cells’ or ‘lymphocytes’, and definitelynot by
immune serum*
Adjuvants :
It has been duly observed that there exist quite a few nonspecific substances,
namely :
alum, mineral oil, that essentially do possess the abiliy to prolong as well as intensify the
immune response to a particular antigen on being injected simultaneously with the antigen.
In fact, such materials are termed as
adjuvants by virtue of the fact that they profusely aid the immuneresponse.
In other words,
acquired immunity invariably refers to the ‘protection’ an animal inherently
develops against certain types of microorganisms or foreign substances. In reality, the
acquired immunity
gets developed in the course of an
individual’s lifespan. Fig. 9.2 depicts the different types of
acquired immunity in a summarized form.
 Active Immunity
Active immunity
refers to the specific immunity obtained from the development within the body
antibodies or sensitized T lymphocytes (T Cells) which critically neutralize or destroy the infective
agent. It may eventually result from the
immune response to an invading organism or from inoculation
with a vaccine essentially containing a
foreign antigen.
Cell-Mediated Immunity [or T-cell Mediated Immunity]
It has been duly observed that the
regulatory and cytotoxic actions of T cells during the specific
immune response
is known as the cell-mediated immunity. However, the entire process essentially
needs almost 36 hr to accomplish its full effect. It is also called as
T cell mediated immunity.
Physiological Actions :
Interestingly, unlike B cells, T cells invariably fail to recognize the so
foreign antigens on their own. A foreign antigen is duly recognized by a macrophage which
engulfs it and displays part of the antigen on its surface next to a
histocompatibility or ‘self’ antigen
(macrophage processing). Finally, the presence of these two markers together with the secretion of a
cytokine, interleukin-1 (IL-1)
by macrophages and other antigen-presenting cells duly activates CD4+/
CD8 T cells (
i.e., helper T cells), that categorically modulate the activities of other cells adequately
involved in the
immune response.
Thus, the
CD4+T cells secrete interleukin-2 (IL-2), that stimulates the activity of natural killer
cells (NK cells), cytotoxic T cells,
and B cells ; and ultimately promotes the proliferation of CD+T cells
in order that the invading pathogen may be destroyed or neutralized effectively. Besides,
secreted by CD+T cells increases distinctly the macrophage cytotoxicity and antigen
However, the T-cell mediated immunity plays a significant and pivotal role in the rejection
of transplanted tissues
and in ‘tests for allergens’ i.e., the delayed hypersensitivity reaction.
 Congenital Immunity
congenital immunity refers to the immunity critically present at birth. It may be either
natural or acquired, the latter predominantly depends upon the antibodies solely received from the mother’s
Herd Immunity
herd immunity represents the immune protection duly accomplished via vaccination of a
portion of a population, that may eventually minimise the spread of a disease by restricting the number
of potential hosts for the respective pathogen.
 Humoral Immunity [or B-cell Mediated Immunity]
Humoral immunity
respresents the immunity duly mediated by antibodies in body fluids e.g.,plasma or lymph. As these antibodies are adequately synthesized and subsequently secreted by B cells,
that protect the body against the
infection or the reinfection by common organisms, such as : streptococci
staphylococci, it is also known as B-cell mediated immunity. In reality, the B cells are stimulated
by direct contact with a foreign antigen and differentiate into the
plasma cells that yield antibodies
against the antigen ; and the corresponding
memory cells which enable the body to rapidly produce
these antibodies if the same antigen appears at a later time.
It is, however, pertinent to state here that
B cell differentiation is also stimulated duly by interleukin-2
secreted by the T4 cells, and by foreign antigens processed by macrophages.
 Local Immunity
Local immunity
is usually limited to a given area or tissue of the body.
 Natural Immunity
Natural immunity
refers to the immunity programmed in the DNA, and is also known as the
genetic immunity.
It has been observed that there are certain pathogens that fail to infect some species
due to the fact that the cells are not exposed to appropriate environments, for instance : the
cannot reproduce in the canine cells ; and, therefore, dogs do have natural immunity to measles.
 Passive Immunity
Passive immunity
specifically refers to the immunity acquired by the introduction of preformed
into an unprotected individual. It may take place either through injection or in utero from
antibodies that usually pass from the mother to the foetus
via the placenta. It can also be acquired by thenewborn by ingesting the mother’s milk.