Microbiological assays of Antibiotic and Vitamins

In detailed overview of Microbiological assays of Antibiotic(Streptomycin, Penicillin) and Vitamins(B12) and their principles

1. UNIT III 4.2 Microbiological assays: General Methods and Assays of Antibiotics and Vitamins Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. 3 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.

4. 4 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.

5. 5 Types of Microbiological Assays Method A: “Cylinder plate” or “plate” assay diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish growth of the added micro-organism is prevented entirely in a zone around the cylinder containing a solution of the antibiotic. Method B: “ Turbidometric ” or “tube” assay the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium

6. 6 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.

7. 7 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.

8. 8 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 Punched-hole method Paper-disc method

9. 9 Plate method – types Cylinder plate method F irst 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

10. 10 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.

11. 11 Assay of antibiotics Method Both Method A (cup plate) and Method B (Turbidimetric) can be used Method A – Depends on measurement of zone of inhibition Method B – Depends on measurement of turbidity Method Method A (Cup plate method) Method B (Turbidimetric method) Standard Stock solution Conc Streptomycin sulphate - 1mg/ml Solvent used for stock solution Water Median dose 1µg/ml (for cup plate method) 30 µg/ml (for turbidimetric method Test Organism Bacillus subtilis Test Media Peptone Yeast extract Beef extract Agar pH after sterilization 7.8 to 8.0 Incubation temperature 32 - 35⁰C

12. 12 Method Method A (Cup plate method) Method B (Turbidimetric method) Standard Stock solution Conc Crystalline salt of Benzyl penicillin -1000 Units/ml Solvent used for stock solution Potassium phosphate buffer Median dose 1 Unit/ml Test Organism Staphylococcus aureus Test Media Peptone Yeast extract Beef extract Agar pH after sterilization 6.6 ± 0.1 Incubation temperature 32 - 35⁰C Assay of Penicillin

13. Microbiological assay of Vitamins Principle Vitamins and amino acids are essential for the growth of microorganisms. The basis of this assay is to measure the ability of test organism to utilize the substance being assayed under a proper nutritional condition. The organisms require these growth factors (vitamins & amino acids) in micro or nanograms. The response (growth of test organism) is proportional to the dose (amount of factor) added to medium. Nutrient medium will contain an abundance of all nutrients essential for growth of the organism except the test substance On adding small amounts of the test substance to tubes, growth takes place Growth depends on the quantity of test substance added 13

14. Microbiological assay of Cyanocobalamine About Vit B12: Also known as cyanocobalamin It is a water soluble vitamin Structure is similar to that of heme where the iron is replaced with cobalt as a centre of molecule Its main sources are liver, eggs, milk, meat & fish Vit B12 deficiency causes Macrolyticanemia and Pernicious anemia. National Research Council,USA recommends a daily in take of about 5mg of vitB12 14

15. Principle of assay: The test organism selected must be capable of utilizing free cyanocobalamin Lactobacillus Liechmanni is found to satisfy the requirements Gram negative bacilli, non-pathogenic, easy to culture & easily available Isolated from milk, cheese, & other dairy products Assay is performed by using either titrimetric or turbidimetric method. 15

16. 16 Method Method B (Turbidimetric method) Or Titrimetric method Standard Stock solution Conc Standard cobalamine stock solution 0.01ng to 0.04ng per ml Solvent used for stock solution Water Median dose 1 Unit/ml Test Organism Lactobacillus leichamannii Test Media Contains complex nutrients except vit B2 Incubation temperature 37⁰C A ssay of Cyanocobalamine

17. 17 Method Method B (Turbidimetric method) Or Titrimetric method Standard Stock solution Conc Standard cobalamine stock solution 0.01ng to 0.04ng per ml Solvent used for stock solution Water Median dose 1 Unit/ml Test Organism Lactobacillus casei Test Media Contains complex nutrients except vit B12 Incubation temperature 37⁰C Microbiological assay of Riboflavin

18. Titrimetric Method   Principle Estimation of amounts of acid produced by Lactobacillus by titrimetric method It is a much slower method compared to turbidimetric It takes almost 3 days for the acids to build up Titrimetric procedures have been largely replaced by turbidimetric procedure 18

19. Methods of standardization of amino acids Standard conditions are required for a microbiological assay to determine the degree of activity of a compound based on the amount required to develop the predicted effect. Microbiological analyses take a lot of time and are not applicable to all amino acids. Some microorganisms need amino acids to grow and reproduce. These microbes rely on specific amino acids in many strains. The growth of such microorganisms will be limited if a small amount of an amino acid is supplied, as measured by turbidimetry, or if only a small amount of pH levels are increased by microtitration. 19

20. Guithrie and Susi developed a modified version of the microbiological test using diffusion in gels to screen blood samples for the presence of elevated phenylalanine levels in clinical biochemistry laboratories. It is named after its inventors as the Guthrie test, and it is one of the most commonly used microbiological methods to evaluate amino acids. In the assay, phenylalanine is evaluated by comparing its ability to inhibit Bacillus subtilis growth against the chemical competitor, β-2- theienylalanine . This test makes use of an agar plate, onto which suspensions of B.subtilis spores are added, along with growth nutrients and an additional amount of β-2 thienylalanine for  supplementation. 20

21. In addition to the filter paper soaked in blood, phenylalanine standards are also soaked in blood and placed on the agar surface. They are then incubated overnight at 37°C. When phenylalanine concentrations in blood discs are high enough, β-2 thienylalanine can't prevent bacterial growth. The discs themselves show growth zones around them. We measure the circumference of each zone of growth the next day, and correlate this with phenylalanine levels. 21

22. 22 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 • Antibiotic assay – determines growth inhibitory property • Antibiotic assay can be carried out by cup plate or turbidometric method • Vitamin assay determines growth promoting ability of vitamins • Vitamin assay is carried out by turbidometric method

23. 23 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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