MICROBIAL ASSAY OF ANTIBIOTICS

About Authors:
Nilesh Sovasia*, Arshad Hala
Seth G.L.Bihani S.D.College Of Technical Education,
Institute Of Pharmaceutical Science & Drug Research,
Sri Ganganagar, Rajasthan, India
*nilesh.sovasia@yahoo.com

ABSTRACT
The microbiological assay of an antibiotic is based upon a comparison of the inhibition of growth of micro-organisms by measured concentrations of the antibiotics under examination with that produced by known concentrations of a standard preparation of the antibiotic having a known activity.

Reference Id: PHARMATUTOR-ART-1560

INTRODUCTION:
Two general methods are usually employed, the cylinder-plate (or cup-plate) method and the turbidimetric (or tube assay) method.
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. 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.

The assay is designed in such a way that the mathematical model on which the potency equation is based can be proved to be valid. If a parallel-line model is chosen, the two log doseresponse lines of the preparation under examination and the standard preparation should be parallel; they should be rectilinear over the range of doses used in the calculation. These conditions should be verified by validity tests for a given probability. Other mathematical models, such as the slope ratio method, may be used provided that proof of validity is demonstrated.

MEDIA:
Prepare the media required for the preparation of test organism inocula from the ingredients listed in Table 1. 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 waterto produce 1000 ml and add sufficient 1 M sodium hydroxideor 1 M hydrochloric acid, as required so that after sterilization the pH is as given in Table 1.

Table 1– Media: Quantities in g of ingredients per 1000 ml

* Quantity in ml, to be added after boiling the media to dissolve the agar.

STANDARD PREPARATION AND UNITS OF ACTIVITY
A Standard Preparation is an authentic sample of the appropriate antibiotic for which the potency has been precisely determined by reference to the appropriate international standard. The Potency of the standard preparation may be expressed in International Units or in μg per mg of the pure antibiotic.

The Standard Preparations for India are certified by the laboratory of the Indian Pharmacopoeia Commission or by any other notified laboratory(ies) and are maintained and distributed by the agency(ies) notified for the purpose.

A Standard Preparation may be replaced by a working standard prepared by any laboratory which should be compared at definite intervals under varying conditions with the standard.

Buffer solution
Prepare by dissolving the following quantities given in Table 2 of dipotassiumhydrogen phosphate and potassium dihydrogen phosphatein sufficient waterto produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acidor 10 M potassium hydroxide.

Table 2 – Buffer Solutions

Buffer No.

      Dipotassium        Hydrogen Phosphate,

        K2HPO4(g)

Potassium      Dihydrogen phosphate,

KH2PO4(g)

pH adjusted after sterilization to

1

2

3

4

5

6

2.0

16.73

-

20.0

35.0

13.6

8.0

0.523

13.61

80.00

-

4.0

6.0±0.1

8.0±0.1

4.5±0.1

6.0±0.1

10.5±0.1*

7.0±0.2

* After addition of 2 ml of 10M potassium hydroxide

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 1:1.25 for Method A or smaller for Method B. Use the final diluent specified and a sequence such that the middle or median has the concentration specified in Table 3

Preparation of the sample solution
From the information available for the substance under examination (the “unknown”), assign to it an assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for each antibiotic in Table 3 but with the same final diluent as used for the Standard Preparation. 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.

Test organisms
The test organism for each antibiotic is listed in Table 4, together with its identification number in the American Type Culture Collection (ATCC). Maintain a culture on slants of the medium and under the incubation conditions specified in Table 5, and transfer weekly to fresh slants.

Table 4 - Test Organisms for Microbiological Assay of Antibiotics

Antibiotic

Test Organism

ATCC1 No.

Amikacin

Amphotericin B

Bacitracin

Bleomycin

Carbenicillin

Chlortetracycline

Erythromycin

Framycetin

 

Gentamicin

Kanamycin sulphate

 

Neomycin

Novobiocin

Nystatin

Oxytetracycline

 

Polymyxin B

Spiramycin

Streptomycin

 

Tetracycline

 

Tobramycin

Tylosin

Staphylococcus aureus

Saccharomyces cerevisiae

Micrococcus luteus

Mycobacterium smegmatis

Pseudomonas aeruginosa

Bacillus pumilus

Micrococcus luteus

Bacillus pumilus

Bacillus subtilis

Staphylococcus epidermidis

Bacillus pumilus

Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus epidermidis

Saccharomyces cerevisiae

Bacillus cereus var, mycoides

Staphylococcus aureus

Bordetella bronchiseptica

Bacillus pumilus

Bacillus subtilis

Klebsiella pnumoniae

Bacillus cereus

Staphylococcus aureus

Staphylococcus aureus

Staphylococcus aureus

29737

9763

10240

607

25619

14884

9341

14884

6633

12228

14884

29737

12228

12228

2601

11778

29737

4617

6633

6633

10031

11778

29737

29737

9144

1. American Type Culture Collection, 21301 Park Lawn Drive, Rockville, MD20852, USA

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