MICROBIAL ASSAY OF ANTIBIOTICS

 

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                                                 0.5 g

Sodium chloride                                         2.5 g

Dextrose monohydrate/anhydrous                 5.5 g/5.0 g

Granular agar (moisture content

less than 15 per cent, w/w)                        0.75 g

Yeast extract (water-soluble)                      5.0 g

Pancreatic digest of casein                         15.0 g

Sodium thioglycollate or                              0.5 g

Thioglycollic acid                                       0.3 ml

Resazurin sodium solution

(0.1 per cent), freshly prepared                   1.0ml

Distilled water to                                       1000 ml

pH of the medium after sterilization              7.1 ± 0.2

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 7.1 ± 0.2. 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. Sterilise in an autoclave at 121º for 20 minutes. If the medium is to be stored, cool promptly to 25º and store at 2º to 30º, avoiding excess of light. 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.

Use fluid thioglycollate medium by incubating it at 30º to 35º.

Alternative Thioglycollate Medium — For use with turbid and viscid products and for devices having tubes with small lumina.

L-Cystine                                           0.5 g

Sodium chloride                                   2.5 g

Dextrose monohydrate/anhydrous           5.5 g/5.0 g

Yeast extract (water-soluble)                5.0 g

Pancreatic digest of casein                    15.0 g

Sodium thioglycollate or                         0.5 g

Thioglycollic acid                                  0.3 ml

Distilled water to                                  1000 ml

pH of the medium after sterilization         7.1 ± 0.2

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 7.1 ± 0.2. Filter, if necessary, place in suitable vessels and sterilise at 121º for 20 minutes. Store at a temperature between 2º and 25º in a sterile sealed container, unless it is intended for immediate use.

The medium is freshly prepared or heated in a water-bath and allowed to cool just prior to use. It should not be reheated.

Use alternative thioglycollate medium in a manner that will assure anaerobic conditions for the duration of the incubation at 30º to 35º.

Soyabean-casein Digest Medium

Pancreatic digest of casein                                  17.0 g

Papaic digest of soyabean meal                            3.0 g

Sodium chloride                                                 5.0 g

Dipotassium hydrogen phosphate                          2.5 g

(K2HPO4)

Dextrose monohydrate/anhydrous                        2.5 g/2.3 g

Distilled water to                                               1000 ml

pH of the medium after sterilisation                       7.3 ± 0.2

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 7.3 ± 0.2. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121º for 20 minutes.

Use soyabean-casein digest medium by incubating it at 20º to 25º under aerobic conditions.

PREPARATION OF THE INOCULUM
Prepare the microbial suspensions for the inoculum for the assay as given in Table 5. 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.

1. Use Medium A containing 300 mg of manganese sulphate per litre.
2. 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.

METHODS OF PREPARATION OF TEST ORGANISM SUSPENSION:
1. 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 250 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 530 nm. Determine the amount of suspensions to be added to each 100 ml of agar of nutrient broth by use of test plates or test broth. Store the suspension uder refrigeration.

2. Proceed as described in Method 1 but incubate the Roux bottle for 5 days. Centrifuge and decant the supernatant liquid. Resuspend the sediment with 50 to 70 ml of saline solution and heat the suspension for 30 minutes at 70º. 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 100 ml of agar. Store the suspension under refrigeration.

3. Maintain the test organism on 10 ml agar slants of Medium G. Incubate at 32º to 35º for 24 hours. Inoculate 100 ml of nutrient broth. Incubate or 16 to 18 hours at 37º and proceed as described in Method I.

4. 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.

DETERMINATION OF INOCULUM

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 100 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.

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