Microbiological assays – General Method

 Microbiological assays – General Method

Contents

       Significance of microbiological
assay

       Microbiological assay methods

       Agar diffusion method – General
principle

Learning objectives

At the
end of this lecture, student will be able to:

       Compare microbiological assays with
other methods of assay

       List the methods of microbiological
assays

       Describe the general method of agar
diffusion method of assay

       Describe the method for
turbidometric method of assay

Introduction

       Microbiological assay determines the
potency or concentration of a chemical substance by its effect on the growth of
microorganism

       Growth promotional effect – vitamins
and amino acids

       Growth inhibitory effect –
antibiotics

       The microbiological assay is based
upon a comparison of the inhibition/promotion of growth of micro-organisms by
measured concentration of the test compound with that produced by known
concentrations of a standard preparation having a known activity.

Need for microbiological assay

       The inhibition of growth under
standardized conditions may be utilized for demonstrating the therapeutic
efficacy of antibiotics.

       Any subtle change in the antibiotic
molecule which may not be detected by chemical methods will be revealed by a
change in the antimicrobial activity 

       Microbiological assays are very
useful for resolving doubts regarding possible change in potency of antibiotics
and their preparations.

Types of Microbiological
Assays

Method
A:

                “Cylinder plate” or
“plate” assay

Method
B:

                “Turbidometric” or
“tube” assay

 

Cylinder Plate or Plate Assay

      
The cylinder-plate method (Method A) depends upon
diffusion of the antibiotic from a vertical cylinder through a solidified agar
layer in a Petri dish or plate to an extent such that growth of the added
micro-organism is prevented entirely in a zone around the cylinder containing a
solution of the antibiotic.

Turbidometric” or “Tube” assay

       The turbidimetric method (Method B)
depends upon the inhibition of growth of a microbial culture in a uniform
solution of the antibiotic in a fluid medium that is favourable to its rapid
growth in the absence of the antibiotic.

Preparation of media

       The media required for the
preparation of test organism are made from the ingredients.

       Minor modifications of the
individual ingredients may be made, or reconstituted dehydrated media may be
used provided the resulting media have equal or better growth-promoting
properties and give a similar standard curve response.

       Dissolve the ingredients in
sufficient water to produce 1000 ml and add sufficient 1M Sodium hydroxide or 1M
Hydrochloride acid, as required so that after sterilization the pH is b/w 6.5
to 7.5.

Preparation of standard
solutions

To prepare
stock solution,

       Dissolve a quantity of the Standard
Preparation of a given antibiotic, accurately weighed and previously dried

       Use specified solvent

       Dilute to the required concentration
as indicated.

       Store in a refrigerator and use
within the period indicated.

The cylinder-plate (or
cup-plate) method

Also
known as Agar Plate Diffusion Assay (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’.

Plate method – types

Cylinder
plate method

       This method was first devised by
Abraham et al  and later modified by
Schmidt and Moyer

       Depends upon diffusion of the
antibiotic from vertical steel cylinders placed on the surface of inoculated
agar medium.

       This produces zones of inhibition
around the cylinder containing antibiotic solution depending upon the
concentration of the antibiotic

Punched-hole
method

       Holes are punched out of the
inoculated culture medium and the antibiotic solutions are then loaded into
them

Paper-disc
method

       Paper discs with a diameter of 9 mm
are impregnated with the antibiotic solution and placed on the culture medium.
Antibiotic can also be applied to the disc after it has been placed on the
medium. Plates containing a single layer of medium with 2 mm thickness may be
used for these tests

The cylinder-plate (or cup-plate) method

The zone diameter observed depends on

      Initial population density

      Rate of diffusion of ‘antibiotic’

      Rate of growth of ‘organism’

      Thickness of agar layer

Designing
an assay method:

      Proper choice of ‘indicator
organism’

      Suitable culture medium

      Appropriate sample size

      Exact incubation temperature.

       Inoculate
a previously liquified medium appropriate to the assay with the requisite
quantity of suspension of the micro organism

       Add
the suspension to the medium at a temperature between 40° and 50° and
immediately pour the inoculated medium into the petridishes or large
rectangular plates to give a depth of 3 to 4 mm

       Ensure
that the layers of medium are uniform in thickness, by placing the dishes or
plates on a level surface.

       The
prepared dishes or plates must be stored in a manner so as to ensure that no
significant growth or death of the test organism occurs before the dishes or
plates are used and that the surface of the agar layer is dry at the time of
use.

 

       Using
the appropriate buffer solutions, prepare solutions of known concentrations of
the antibiotic to be examined.

       Where
directions have been given in the individual monograph for preparing the
solutions, these should be followed and further dilutions made with buffer
solution.

       Apply
the solutions to the surface of the solid medium in sterile cylinders or in
cavities prepared in the agar.  

       The
volume of solution added to each cylinder or cavity must be uniform and
sufficient almost to fill the holes when these are used.

       When
paper discs are used these should be sterilized by exposure of both sides under
a sterilizing lamp and then impregnated with the standard solutions or the test
solutions and placed on the surface of the medium.

       When
Petri dishes are used, arrange the solutions of the Standard Preparation and
the antibiotic to be examined on each dish so that, they alternate around the
dish and so that the highest concentrations of standard and test preparations
are not adjacent.

       When
plates are used, place the solutions in a Latin square design, if the plate is
a square, or if it is not, in a randomized block design.

       Leave
the dishes or plates standing for 1 to 4 hours at room temperature or at 4°, as
appropriate, as a period of pre-incubation diffusion to minimise the effects of
variation in time between the applications of the different solutions.

       Incubate
them for about 18 hours at the temperature. Accurately measure the diameters or
areas of the circular inhibition zones and calculate the results. 

Summary

       Microbiological assay determines the
potency or concentration of a chemical substance by its effect on the growth of
microorganism

       Any substances having either growth
promoting or growth inhibiting effect can be evaluated

       Two types – Cup plate and
Turbidometric method

       Cup plate method based on
measurement of zone of inhibition

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