MICROBIOLOGIC

AL ASSAY
 Biological Assay: -
Biological Assay refers to
measurement of the relative potency
or activity of compound by
determining the amount required to
produce a stipulated effect on
suitable test animal or organ under
standard condition.
 Microbiological Assay:

The term microbial assay designates a type of

biological assay, specifically, a biological assay
performed with micro organism e.g. bacteria years
and molds.
One notable difference involves the relative size of
the experimental population. In a typical bioassay
procedure the response of each individual test
animal is noted and the results obtained when a
series of calculate mean activity standard error. In
typical microbial assay each evaluation is
performed with a culture of microorganism and
measurements represents the average response of
an extremely large population.
 Microbiological assays are used to
evaluate the in vitro activity of drug. The
specific strains of test organism are used
to evaluate the activity of the specific
anti -microbial agent, and specific
method of maintenance or preparation of
micro organism medium in which the
micro organism hold for testing the
agents. Specific mediums are used for
specific microbial testing, adopting the
specific condition of the test.
 Microbiological assay may be used to
evaluate the agent quantitatively or
qualitatively. In qualitative evaluation,
we consider test organism as sensitive,
intermediate or resistant to the agent
and in quantitative evaluation in terms of
concentration of the agent which inhibit
the growth of micro organism the
minimum inhibitory concentration (MIC)
or that which kill microbes, the minimum
bactericidal concentration (MBC).
 We should be aware of the meaning of
the categories of susceptibility of
micro organism.
SENSITIVE: Indicates the dose of
agent that should be appropriate for
killing the strain of micro organism.
INTERMEDIATE: - Referred to as
moderately resistant, moderately
susceptible to the agent. This indicates
that the strain may be inhibited by
larger doses of agent or may be
inhibited in sites where the agent is in
excess. Larger doses of agents are
required to produce results.

 RESISTANT: -This indicates that

microorganism is unlikely to respond
to the anti microbial agent. It is failure
of response of antimicrobial caused by
specific strain of micro organism.
Break point concentration of anti
microbial may be used to separate
susceptible form resistant or
intermediate Isolates on the basis of
growth or no growth at the selected
concentration. These may be used to
interpret MIC results
 on the basis of growth or no growth
at the selected concentration. These
may be used to interpret MIC results
in establishing zone size and
relationship of zone size to MIC by
closely defined methods have been
established for agent tested.
 MEDIA: -
A substance use to provide nutrients for
the growth and multiplication of micro
Organism.
TYPE OF MEDIA
1. Selective media
2. Differential media
3. Assay media
4. Media for enumeration of bacteria
5. Solid & semi solid media
6. Maintenance media
7. Media for characterization of bacteria.
 PREPARATION OF MEDIA:-
All microbiological media must be
prepared by accurate measurement of
weight and volumes through mixing
and dissolution of the dehydrate
medium using heating as appropriate,
neither under heating nor overheating,
and finally sterilizing by an appropriate
heat process or other means.
 METHOD OF
MICROBIOLOGICAL ASSAY: -
Following are the four micro
biological methods: -
Macro dilution broth susceptibility
test.
Agar dilution susceptibility test.
Disk diffusion susceptibility test.
Micro dilution susceptibility test.
 They have several common features.
a. The compound being assayed must
influence the growth of the test
organism.
b. A varying response in growth must
be produced by addition of varying
quantities of the test material.
c. The growth medium must contain an
excess of the compound required by the
test organism for growth.
d. The assumption is made that the
compound being assayed is the only
growth promoting or inhibiting
compound present.
 1. MACRODILUTION BROTH
SUSCEPTIBILITY TEST: -
The macro dilution broth
susceptibility test was among the
first to be developed and still serves
as a reference method. Serial
dilutions of anti- -microbial agent are
made in broth after which a
standardized bacterial suspension is
added.
 Figure shows 10 test tubes that contain
nutrient broth. Quantities of antibiotic
are serially diluted from 100ug/ml to 0.4
ug/ml. Tube number 10 is free of
antibiotic and serves as a growth
control. Each of the 10 tubes is
inoculated with a calibrated suspension
of the microorganism to be tested and
incubated at 35 C for 18 hours. At the
end of the incubation period, the tubes
are visually examined for turbidity. Note
in figure that the five tubes to the left
are clear, the five to the right appear
cloudy.
 Cloudiness indicates that the bacterial growth
has not been inhibited by the concentration of
antibiotic contained in the medium. Figure
illustrates that the breakpoint of growth
inhibition in figure is between 6.25ug/ml and
3.12ug/ml of antibiotic. This breakpoint
represent the MIC, defined as the lowest
concentration of antibiotic in microorganism / ml
that prevents the in vitro growth of bacteria.
Thus, in the example shown in figure MIC lies
some where b/w 6.25ug/ ml and 3.12 ug/ ml.
However , by convention , the MIC is interpreted
as the concentration of the antibiotic ,contained
in the first tube in the series ,that inhibit visible
growth . Therefore ,in this example , the MIC is
6.25ug/ml.
 2.AGAR DILUTION SUSCEPTIBILITY TEST: -
The agar dilution procedure, which is the
second reference method has been successfully
adopted for routine use in large laboratories by
testing only selected concentration of antibiotic. A
standardized suspension of bacteria is inoculated
onto a series of agar plates each containing a
different concentration of antibiotic,
encompassing the therapeutic range of the drug.
For example, if the therapeutic range for a given
antibiotic is 2 to 12 ug/ml. A series of agar plates
containing I, 4,8,16 and 32ug/ml of antibiotic
might be used to determine the susceptibility of
the organism being tested. If the organism grows
on the first three plates but not in the plate
containing 16ug/ml. Of antibiotic, an MIC value of
16ug/ml can be established, similar to the
interpretation of the end point in the broth
dilution technique.
 Agar dilution plates ready for
interpretation are shown in figure
note that those microorganism that are
sensitive to the concentration of
antibiotic contained in any given agar
plate do not produce a circle of growth
at the inoculum site , whereas those
that are resistant appear as circular
colonies. The agar plates are marked
with grid so that each micro-organism
can be identified by a number and the
results entered on the worksheet.
 3. DISK DIFFUSION SUSCEPTIBILITY TEST.
Figure illustrate the basic principle of the disk
diffusion method of antimicrobial susceptibility
testing.
As soon as the antibiotic-impregnated disk comes in
contact with the moist agar surface, water is
absorbed into filter paper and the antibiotic into the
surrounding medium. The rate of extraction of the
antibiotic out of the disk is grater then its outward
diffusion into the medium, so that the concentration
immediately adjacent to the disk may exceed that in
the disk itself. As the distance from the disk
increases, however, there is a logarithmic reduction
in the antibiotic concentration. If the plate has been
previously inoculated with a bacterial suspension,
simultaneous growth of bacteria occurs on the
surface of the agar. When critical cell mass of
bacteria is reached, the inhibitory activity of the
medium and temperature of incubation. The lateral
extent of antimicrobial
 diffusion before the critical time is reached will be
affected by the depth of agar because diffusion
occurs in three dimensions. The points at which
the critical cell mass is reached appear as a
sharply middle of the disk forming the center of
the circle if the test bas been preformed properly.
The concentration of diffused antibiotic at this
interface of growing and inhibited bacteria in
known as the critical concentration and
approximates the MIC obtained in dilution tests.
Although directed calculation of inhibitory
concentration is not done in practice the MIC an
actually be calculated with reasonable accuracy if
the characteristics of antimicrobial diffusion and
bacterial growth are known.
 Development of a
standardized disk diffusion
procedure.: -
 Initially it was believed the zone sizes
around disks impregnated with various
antibiotics could be correlated with the
relative susceptibility of the organism to
those antibiotics could be correlated with the
relative susceptibility of the organism to
those antibiotics; that is, the larger the zone,
the more effective the antibiotic should be
therapeutically. As more was learned of the
mechanisms by which diffusion occurred and
inhibitory zones were formed, it became
apparent that factors influencing the
diffusion rate in vitro did not necessarily co-
relate with anti microbial in vitro.
 INTERPRETATION OF THE RESULTS: -
The zone size that is observed in a disk
diffusion test has no meaning in and of itself.
The interpretative standards provided by the
NCCLS are derived from a correlation between
zone sizes and MICs of those species that can be
tested by the disk diffusion method. A prototype
of regression curve providing such a correlation
is illustrated in figure. A large number of strains
have been tested against a single antibiotic both
by the disk diffusion technique and by a dilution
method. Each triangle represents the results of
both tests for a single strain. A regression line
has been drawn through the many individual
points. Once the regression line is established,
an approximate MIC result can be inferred from
any zone diameter.
 In this example, a zone size of 18mm
corresponds to an MIC less than 6.25
ug/ml the break point of the broth
dilution test illustrated in fig. Thus anti
microbial agent that produces a zone
diameter greater then 18mm would
theoretically has an MIC less then 6.25
ug/ml, and the organism would be
considered susceptible, one producing a
zone size less than 18 mm would
conversely, be considered resistant.
 4. MICRO DILUTION BROTH
SUSCEPTIBILITY TEST
Concurrent with the development of
a usable disk diffusion test, other
investigators have at tempted to
simplify both dilution test. The
microtube dilution procedure is similar
in principle to the macrotube method,
except that the susceptibility of
microorganisms to antibiotics is
determined in a series of microtube
wells that are molded into a plastic
plate, each plate may contain 80,
 96, or more wells, depending on the
number and concentration of antibiotics
that are to be included in the
susceptibility test panel.
The advantages of the microtube method
are that small volumes of bacteria can be
tested simply and inexpensively against
a panel of antibiotics.
The intensive labor involved in
preparing the multi-well plates has been
a major impediment to their routine use.
 TESTS FOR DETERMINATION OF
BACTERICIDAL ACTIVITY : -
Indications.
The indications for determination of
bactericidal activity are few. The infection
must either be a serious one in which anti
microbial activity that is lethal to the
microbe is desired, because other host
defense mechanisms are compromised, or
be located in a site that is difficult to reach
with antibiotics. Historically, streptococcal
endocarditic has been the primary
indication for determining bactericidal
activity.
 Situations in which bacterial testing,
has been suggested include
osteomyelitis, meningitis, and
septicemia in patients who are
neutropenic.
A satisfactory method for
performing bactericidal tests has not
been found.