Microbiological assay-Principles and methods of different microbiological assay.

Principles and methods of different microbiological assay. Methods for standardization of
antibiotics, vitamins and amino acids. Assessment of a new antibiotic.

1. Microbiological Assay By S.D.Mankar Assistant Professor, Department of Pharmaceutics Pravara Rural College Of Pharmacy,Loni

2. Definition microbiological assay may be defined as qualitative or quantitative determination of any chemical compound from simple or even complex material with the use of microorganism.

3. Need It is necessary to assay antimicrobiological agents for determination of any potency, for determining the pharmacokinetics of a drug in animals or man and for monitoring and controlling antimicrobial chemotherapy. It is simple, specific, inexpensive and convenient mtd . Very useful for determining changes in potency of antibiotics and their preparation.

4. Microbiological assay of Antibiotics: Principal:- The inhibition of microbial growth under standardised condition may be used for demonstrating the therapeutic efficacy of antibiotics. The microbiological assay is based upon a comparison of the inhibition of growth of microorganism by measured conc. Of antibiotics to be examined with that produced by known conc. Of std. preparation of the antibiotic having a known activity.

5. Requirement 1.Test Solution 2. standard solution 3. preparation of inoculum.

6. Media Used for antibiotic solution media required for the preparation of test M.O. inoculum are made from different nutrients. Dissolve the ing . As shown in table in sufficient water to produce 1000 ml and add sufficient 1 M NaoH or 1 M HCL, as required. After sterilization the pH is adjusted.

7. Media Used for antibiotic solution media required for the preparation of test M.O. inoculum are made from different nutrients. Dissolve the ing . As shown in table in sufficient water to produce 1000 ml and add sufficient 1 M NaoH or 1 M HCL, as required. After sterilization the pH is adjusted.

8. Preparation of standard 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. Ex: Dissolve a quantity of the standard preparation of a given antibiotics in the solvents(table3). Dilute the preparation to get the required concentration as stated and stored in a refrigerator. Usually prepared in the ratio of 1:1.5

9. Preparation of the test sample 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 Table4 but with the same final diluents 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.

10. Preparation of Test organism The t e s t o r g a nism f or eac h ant i b i otic i s l i st e d i n T ab l e, t o ge t h e r w i th i t s identification number in the American Type Culture Collection (ATCC).

11. Preparation of inoculums Inoculums is the mixture of microbes along with the culture media in which it is growing. Steps involved: Maintain the test microbes on slant of medium A and transfer to a fresh slant once a week. Incubate the slant at the specified temperature for 1day Using 3ml of slant solution, wash the microbes from agar slant on to a large surface of medium A such as a Roux bottle containing 250ml of agar media Incubate for 1day at the required temperature Wash the growth from the nutrient surface using 50ml of saline solution. Store the test microbes under refrigerator

12. Methods of Microbiological Assay A. Cylinder plate or Cup plate method B. Turbidimetric or Tube Assay method

13. A. Cylinder plate or Cup plate method Principle: This method depends on the diffusion of an antibiotic from a vertical cavity or cylinder , through the solidified agar layer in a petri plate. The growth of test microorganism is inhibited entirely in a circular area or zone around the cavity or cylinder containing a solution of antibiotic .

14. Procedure:- A liquefied assay medium ( 43 -45 ˚ C) is inoculated by suspension of test M.O. and the inoculated medium is poured immediately into sterile petri plate or pre-prepared agar plates by using assay medium and then sprayed the test culture or M.O. on the surface of plates. Solution of known conc. Of the std. preparation and test antibiotic are prepared in appropriate solution. Preparation of the std. solution and potency of antibiotics for assay of penicillin and assay of streptomycin is given as previous table.

15. These solutions are added in sterile cavities or cylinders prepared in a solid medium. The volume of solution added to each cavity or cylinder must be uniform and sufficient to fill the holes. The plates are left standing for 1 to 2 hrs at room temp. or at 4 ˚ C. All plates are then incubated for about 18 to 24 hrs at the given temp. Accurately measure the diameter or areas of the circular inhibition zone produced by std. and test antibiotic solution. Plot the graph which relates zone diameter to the log of conc.of antibiotics and is plotted and the unkown conc. Of test antibiotics is calculated.

16. B. Turbidimetric or Tube Assay method This method depends upon the 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. Five different concentration of the standard solution are prepared for preparing the standard curve . 1ml of each concentration of the standard solution & of the sample solution are placed in each of the tubes in duplicate at 9 ml of nutrients medium previously seeded with the appropriate test organism at to each other

17. At the same time 03 control tubes, one containing the inoculated culture medium ( culture control) another identical with it but treated immediately with 0.5 ml of dil. Formaldehyde solution (blank) and a third containing uninnoculated culture medium are prepared. All the tubes are placed in an incubator at the specified temp. for 4 to 5 hrs. after incubation add 0.5 ml of dil. Formaldehyde solution to each tube. The growth of test M.O. is measured by determining the absorbance at about 530 nm of each of the solutions in the tubes against the blank.

18. Microbiological Assay of Vitamins Principal:- Vitamins are imp. Growth factors needed for growth and multiplication of M.O. they are very sensitive to small ampounts of the growth factors. It is the ability of the test M.O. to synthesize the factor being assayed that forms the basis of microbiological assay of vitamins and amino acids. Methods:- 1. titrimetric method 2.turbidimetric method.

19. Procedure Clean 10 test tubes and add 0.0ml, 0.5ml, 1.0ml, 1.5ml, 2ml, 2.5ml, 3ml,4ml,4.5ml and 5ml respectively of standard cyanocobalamin solution. To each test tube add 5ml basal medium stock solution. Adjust final volume 10ml by using water. To other four test tubes, add 1ml, 2ml,3ml,4ml respectively of the test solution to be assayed. To each test tube add 5ml basal medium stock solution. Adjust final volume 10ml by using water.

20. Sterilize all test tubes in autoclave at 121 C for 5 minutes. After sterilization, cool all test tubes upto room temperature and inoculate with one drop of inoculum (bacterial culture). Incubate the test tubes for 64 to 72 hours at any chosen temperature with in the range of 30- 37 O C . Titrate the contents of each tube with 0.05N NaOH using 0.1%w/v bromothymol blue as an indicator (converts to green colour). Determine the average of the titration values for each level of standard and test sample used . Plot the graph of titration value versus Std. Cyanocobalamin solution concentration.

21. Turbidimetric method Apparatus, reagents and procedure are same as per titrimetric method but in these test includes two more test tubes to which neither std. cynocobalamin solution nor inoculum is added. Incubate all test tubes at 30-37 ˚C for 16 – 24 hrs. By using uninoculated blank test tube adjust the transmittance value against corresponding level of std cynocobalamin solution. Draw a smooth curve and calculate the con. Of test solution of cynocobalamin .

22. Assessment of new antibiotic Minimum inhibitory concentration(MIC ) Minimum inhibitory conc. Is the lowest conc. Of antimicrobial compound found to inhibit the growth of a particular test M.O. It may be applied to disinfectant, antiseptic, preservative & antibiotic. MIC values usually expressed in terms of μ g/ml or units/ml.

23. Methods 1.Liquid dilution method or test tube method: Use series of test tube which contains double strength medium and labelled as 0,0’,and 1 to 10. In first test tube( un-inoculated), inoculum is not added which is used for checking sterility of medium. All other eleven test tubes, inoculum is added to reach the final conc. Of M.O. is 10 5 to 10 6 cells/ml. In all test tubes test chemical is added ranging from 0.5 to 5 ml except uninoculated & control test tube.

24. The second tube (control) is used to check the susceptibility of the medium for growth of test M.O. and the viability of the inoculum. Adjust the final volume (10 ml) in all test tube by using sterile water. All contents of test tubes are properly mixed and incubate at 37 C for 2- 3 days. after incubation all test tubes are examined for the growth in form of turbidity and record the result. Calculate the MIC.

25. Solid dilution method In this method, first test chemical is mixed into molten agar and then poured into petri plates. After solidification, inoculum is spread on surface of agar medium. All plates are incubated at 37 C for 2- 3 days . After incubation, all plates are observed for growth of inoculum and calculate the MIC.

26. Advantages 1. several M.O. can be tested at the same time by use of multipoint inoculator. Contaminations are easily detected, bcz colony features on solid media are more distinctive than turbidity diff. in fluid media.