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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