Assay methods of antibiotics a laboratory manual

Preparation of the standard solution To prepare a stock solution, dissolve a quantity of the Standard Preparation of a given antibiotic, accurately weighed and previously dried where so indicated in Table 3, in the solvent specified in the table, and then dilute to the required concentration as indicated. Store in a refrigerator and use within the period indicated. On the day of assay, prepare from the stock solution five or more test dilutions, the successive solutions increasing stepwise in concentration, usually in the ratio Use the final diluent specified and a sequence such that the middle or median has the concentration specified in Table 3.

The assay with 5 levels of the Standard requires only one level of the unknown at a concentration assumed equal to the median level of the standard. Maintain a culture on slants of the medium and under the incubation conditions specified in Table 5, and transfer weekly to fresh slants. Test Organism. Staphylococcus aureus. Saccharomyces cerevisiae. Micrococcus luteus. Mycobacterium smegmatis.

Pseudomonas aeruginosa. Bacillus pumilus. Bacillus subtilis. Staphylococcus epidermidis. Bacillus cereus var, mycoides. Bordetella bronchiseptica. Klebsiella pnumoniae. Bacillus cereus. Subscribe to Pharmatutor Alerts by Email.

CULTURE MEDIA Media for the tests may be prepared as described below, or equivalent commercially available dehydrated mixtures yielding similar formulations may be used provided that when reconstituted as directed by the manufacturer, they comply with the growth promotion test. Other media may be used provided that they have been shown to sustain the growth of a wide range of micro-organisms.

The following culture media have been found to be suitable for the test. Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for the culture of both fungi and aerobic bacteria. Fluid Thioglycollate Medium — For use with clear fluid products. L-Cystine 0. Sodium chloride 2. Granular agar moisture content. Yeast extract water-soluble 5.

Pancreatic digest of casein Sodium thioglycollate or 0. Thioglycollic acid 0. Resazurin sodium solution. Distilled water to ml. Mix the ingredients other than the thioglycollate or thioglycollic acid and the resazurin sodium solution, in the order given above, in a mortar, with thorough grinding. Stir in some heated distilled water , transfer to a suitable container, add the remainder of the distilled water , and complete the solution by heating in a boiling water-bath.

Dissolve the sodium thioglycollate or thioglycollic acid in the solution and, if necessary, add 1M sodium hydroxide so that, after sterilisation, the solution will have a pH of 7.

If filtration is necessary, heat the solution again without boiling and filter while hot through moistened filter paper.

Add the resazurin sodium solution, mix and distribute the medium into suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a colour change indicative of oxygen uptake at the end of the incubation period. If more than the upper one-third of the medium has acquired a pink colour, the medium may be restored once by reheating in a water-bath or in free-flowing steam until the pink colour disappears, and cooling rapidly, taking care to prevent the introduction of non-sterile air into the container.

When ready for use, not more than the upper one-tenth of the medium should have a pink colour. Medium more than 4 weeks old should not be used. Alternative Thioglycollate Medium — For use with turbid and viscid products and for devices having tubes with small lumina.

Heat the ingredients in a suitable container until solution is effected. Mix, add 1M sodium hydroxide , if necessary, so that, after sterilisation, the medium will have a pH of 7. The medium is freshly prepared or heated in a water-bath and allowed to cool just prior to use. It should not be reheated. Soyabean-casein Digest Medium. Papaic digest of soyabean meal 3.

Sodium chloride 5. Dipotassium hydrogen phosphate 2. Dissolve the solids in distilled water , warming slightly to effect solution.

Cool to room temperature and add, if necessary, sufficient 1M sodium hydroxide so that after sterilisation the medium will have a pH of 7. If the suspensions are prepared by these methods, growth characteristics are sufficiently uniform so that the inoculum can be adequately determined by the trials given below. Use Medium A containing mg of manganese sulphate per litre.

For Pseudomonas aeruginosa in the assay of Carbenicillin, use the dilution yielding 25 per cent light transmission, rather than the stock suspension, for preparing the inoculum suspension. Maintain the test organism on slants of Medium A and transfer to a fresh slant once a week. Incubate the slants at the temperature indicated above for 24 hours.

Using 3 ml of saline solution , wash the organism from the agar slant onto a large agar surface of Medium A such as a Roux bottle containing ml of agar. Incubate for 24 hours at the appropriate temperature. Wash the growth from the nutrient surface using 50 ml of saline solution. Store the test organism under refrigeration. Determine the dilution factor which will give 25 per cent light transmission at about nm.

Determine the amount of suspensions to be added to each ml of agar of nutrient broth by use of test plates or test broth. Store the suspension uder refrigeration. Proceed as described in Method 1 but incubate the Roux bottle for 5 days. Centrifuge and decant the supernatant liquid. Wash the spore suspension three times with 50 to 70 ml of saline solution. Resuspend in 50 to 70 ml of saline solution and heat- shock again for 30 minutes.

Use test plates to determine the amount of the suspension required for ml of agar. Store the suspension under refrigeration. Maintain the test organism on 10 ml agar slants of Medium G. Inoculate ml of nutrient broth. Proceed as described in Method 1 but wash the growth from the nutrient surface using 50 ml of Medium 1 prepared without agar in place of saline solution.

For Method A. After the suspension is prepared as given under Table 5, add different volumes of it to each of several different flasks containing ml of the medium specified in Table 3 the volume of suspension suggested in Table 3 may be used as a guide.

Using these inocula, prepare inoculated plates as described for the specific antibiotic assay. While conducting cylinder-plate assays, double-layer plates may be prepared by pouring a seed layer inoculated with the desired micro organism over a solidified uninoculated base layer.

For each Petri dish, 21 ml of base layer and 4 ml of the seed layer may be generally suitable. Fill each cylinder with the median concentration of the antibiotic Table 3 and then incubate the plates. After incubation, examine and measure the zones of inhibition. The volume of suspension that produces the optimum zones of inhibition with respect to both clarity and diameter determines the inoculum to be used for the assay.

For Method B. Proceed as described for Method A and, using the several inocula, carry out the procedure as described for the specific antibiotic assay running only the high and low concentrations of the standard response curve. After incubation, read the absorbances of the appropriate tubes.

Determine which inoculum produces the best response between the low and high antibiotic concentrations and use this inoculum for the assay. Glassware for holding and transferring test organisms is sterilised by dry heat or by steam. Thermostatic control is required at several stages of a microbial assay, when culturing a microorganism and preparing its inoculum and during incubation in a plate assay.

Closer control of the temperature is imperative during incubation in a tube assay which may be achieved by either circulated air or water, the greater heat capacity of water lending it some advantage over circulating air.

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