AIM :

To determine the sensitivity of a given culture to the given antibiotics using the agar cup method.

THEORETICAL BACKGROUND :

Microbiological assay may be defined as qualitative and/or quantitative determination of any chemical compound from a simple or even complex material with the use of microbes. Thus, microbes can be and are exploited as analytical tools in an analytical laboratory. Today, a new branch of microbiology, analytical microbiology, has been developed wherein various analytical techniques involving microbes are dealt with. Microbes which are important in analytical work include bacteria (e.g. lactic acid bacteria), fungi (e.g. mutant of Neurospora crassa), and protozoa (e.g. Tetrahymena pyriformis). The principles of microbiological assay were discovered and used by many early workers, including Fleming, in the 1920s for the assay of the enzyme, lysozyme, and penicillin. But it was not until the development of penicillin manufacture in 1940 that the method had any widespread application. Large-scale manufacture of antibiotics made it imperative that analytical procedures capable of detecting the amount of antibiotic produced during fermentation were further developed and factors affecting their sensitivity and reproducibility elucidated.

Microbiological assay of growth factors (such as vitamins andamino acids) evolved and developed from purely academic studies on the nutritional requirements of bacteria and fungi.

The principle of the microbiological assay of antibiotics is different from that of the microbiological assay of other substances such as growth factors The growth of a sensitive test microorganism does not occur in the presence of an antibiotic, provided a sufficient quantity of the antibiotic is present. There are several methods for quantitative determination of antibiotics in test samples.

In the agar diffusion method, the diameter of the inhibition zone is directly proportional to the quantity of an antibiotic. The antibiotic present in the solution diffuses radially through the agar medium layers and exerts its toxic effect on the sensitive test microorganism present in the seed agar medium. Subsequently, a zone of inhibition develops around the cup or paper disc.

Considering the sequence of events which may occur when an antibiotic diffuses through agar in which bacterial cells capable of multiplying in the medium are present, it is evident that a number of laws play a role in the process of the assay. These include the laws of diffusion (that describe the changes in concentration which take place in time at each point on the agar; such as Fick's law), the laws of growth (that describe the rate of production of cells and of cell constituents), the laws of absorption, permeability and partition (which govern the way in which the antibiotic-outside the .cell reaches the affected cell receptors controlling metabolism), the laws of specific antibiotic action (concerned with the chemistry of the effects of the antibiotic on metabolism) and the laws of statistics (enable an estimate to be made of the probability that differences in experimental results are due to chance variations).

Hence, there are many parameters affecting results of a bioassay. They may be understood by exemplifying the penicillin assay and can be summarized as :

1. Test microorganisms :

Generally, the following microorganisms are usedin the determination of the potency of penicillin content in samples : Staphylococcus aureus (ATCC 6538P or ATCC 9144) and Sarcina lutea (ATCC 9341). Recently, Bacillus subtilus 6633 has also found a place in the bioassay of penicillin. The features that make these organisms ideal for the assay of penicillin include :

• Great sensitivity to penicillin.
• Give well-defined zone edges.
• Thermoduric and hence hot agar does not necessarily kill the organisms.

The range of the plate assay is from 0.05 to 2 u./ml. when Sarcina lutea is the test organism. The range of the plate assay is from 0.5 to 4 u./ml. when Staphylococcus aureus is the test organism. The stock cultures of these microorganisms are maintained by weekly transfers to fresh sterile slants of Grove and Randall agar medium No. 1. In the preparation of the inoculum suspension, the freshly prepared slants are incubated at 32 to 35°C. for 16 to 24 hours in the specific medium i.e. Groove and Randall medium number 1. The culture is sedimented and washed free of media components before being used for the test.

2. Standard solutions :

A stock of solution of penicillin working standard is prepared by careful weighing of the dried standard and then diluted to a suitable concentration (usually 100 u/rnl.). The dilution is made with a sterile pH 6 phosphate buffer. In some penicillins (not G), it is necessary to solubilize them in a smali quantity of solvent (e.g. formamide) before diluting in a buffer. Such stock solution is stored in the refrigerator. The stock solution can be used for a maximum of 2 days.

It is to be kept in mind that all penicillins or penicillin derivatives do not exhibit identical dose-response curves even under identical assay conditions. Therefore, it is extremely important that one makes use of a standard identical to the compound being assayed. Two important precautions to be taken are :

• A stock solution of penicillin workingstandard shouldbe prepared from the solid penicillin that has been preserved in tightly stoppered vials over a desiccant (e.g. silica gel) in a desiccator.
• Weighing of the solid penicillin should be done in an area with controlled humidity (preferably a relative humidity of 50 per cent or less).

3. Sample preparation :

There are many different materials to be handled by the assayist for testing purposes. Therefore, it is impossible to narrate here all the methods of preparing samples for estimation. A description of the method of preparing the samples for assay in an industry may be given as under:

Penicillin solutions and bulk powders to be tested may be diluted directly with pH 6 phosphate buffer before assay.

In the case of some penicillins, it is essential to bring about the solubilisation of the sample using a small quantity of solvent (e.g. methanol or formamide) before dilution. Certain conditions (e.g. pH) of the sample solution are kept identical with that of standard solution.

4. Mechanics of the assay :

In the routine work, a 100-mm. flat bottom petri plate is used. Such a plate can accommodate four wells or discs per plate. Ideally, for an unknown sample, it is a good idea to inciude dilutions in one of the wells on the plate containing standards to decrease plate-to-plate variations. With samples likely to contain contaminants with microbial activity (either stimulatory or inhibitory), at least two dilutions of the sample should be assayed. If this is done, and parallelism demonstrated, the assayist may have considerably more faith in the validity of the results. Generally, such assay methods make use of the two-layer agar system. In this system, the first layer called 'base' layer is prepared by pouring melted agar into the plate and then allowed to harden on a levelled surface. The next layer called 'seed' agar is prepared by pouring melted agar that has been cooled to approximately 48°C, and inoculated with the appropriate test microorganism. There are two reasons for the use of the two-layer system. They are :

• to improve the definition of the resulting zones of inhibition.
• to provide the optical contrast necessary for' the precise measurement of the zones.

The selection and application of agars in this phase of the assay is of utmost importance to the success of the method. Inoculum to be added in the seed agar is an important factor so far as validity of the assay results is concerned. The cellular mass is produced in a Roux bottle containing appropriate medium (e.g. Groove and Randall agar medium No. 1 for Staphylococcus aureus). Then the resultant growth is harvested by washing the agar surface with a suitable quantity of sterile 0-85 per cent sodium chloride solution. Finally, the inoculum suspension is standardized by a suitable method. In the case of S. aureus, a quantity of 0.2-0.4 ml. of standardized inoculum suspension (108 viable cells/ml.) is added to 20 ml. of liquefied and cooled(48°C.) agar.

The standard stock solution is diluted according to the requirements to obtain a linear response. This dilution operation is carried out immediately before use on the day of the assay. The number and spacing of doses in the standard response curve is a matter which must be largely decidedupon by the assayist. Like standard penicillin, the sample to be assayed is diluted in the range of the standard curve, giving the importance of the diluent that one chooses. There are many ways to apply the standards and samples to the seeded agar surface. In any event, it is probably advisable to hold all assay plates under refrigeration until immediately before use and then to apply all solutions as rapidiy as is consistent with good technique. The plates are incubated at 28 to 30°C for 16 to 18 hours for development of satisfactory inhibition zones.

5. Measuring the response :

After incubation under optimum conditions, circular zones surrounding the wells containing the antibiotic develop. The size of the circular zone depends upon the potency of the antibiotic present in the well. In other words, the diameter of the inhibition zone is proportional to the potency of the antibiotic. Therefore, response in such assays is measured in terms of the diameter of the inhibition zone. This task of measuringthe diameter may be accomplished by one of the following listed devices: Callipers, Millimetre rules, Scale projectors, and Automatic and semi-automatic measuring devices.

The selection of any of the above measuringdevices is governed by the number of assays being run, the degree of accuracy required, and the allowable cost. A valid assay is possible only when standard and sample give the same type of zones.

6. Computation of answers :

The first requirement in the computation of the answer is of the standard dose-response curve. The standard curve is plotted by plotting the average response value (millimetres) for each of the standard levels against the logarithm of the respective doses, andby joining these plotted points. By the use of such a standard curve, the potency of the unknown antibiotic (e.g. penicillin G) may be determined. The advantages and disadvantages of microbiological assay procedure as compared to other methods (e.g. chemical, physico-chemical, chromatographic, enzymatic and biological) are as follows.

• Advantages of the microbiological assay method:it is relatively as accurate as chemical methods; simple, can be specific, inexpensive, convenient, only small amounts of material required; sometimes can be made specific for one enantiomorphic form; instrumentation is not elaborate; excellent for estimation of one or more compounds in an unknown.
• Disadvantages of the microbiological assay method : somewhat larger amounts of material are required than for chromatography when a number of amino acids are to be determined; continuous checks are necessary for consistency, specificity, and interferences.

7. Precautions :

• The lowest concentration of the antibiotic taken should give atleast 12mm diameter zone for better results.

• Petri-dishes (whether glass or plastic) are poor conductors of heat.Hence, there will be considerable plate-to-plate variation if plates are incubated in stacks. Therefore, the plates must be incubated as a single layer in a large tray.

• Spillage from cups must be avoidedas it results in irregular zones. Hence, considerable' care sirouin"be taken in'transporting the plates to the refrigerator for prediffusion and later to the incubator.
• Penicillinmoleculesarethermolabile.Hence,pipettesshouldnotbeheated as it will inactivate penicillin while pipetting.
• Plates must be prediffused at 4°C for 15-20 minutes before incubation at 37°C for 24 hours to allow maximum diffusion of the antibiotics resulting in sharper zones of inhibition.
• Phosphate buffer (pH=6.0)shouldbeusedfor dilutionof both;thestandard as well as the unknown.