Microbiological Assay

Principal and methods of diffrent microbiological assay.
Cup plate method and turbiditymetric method

1. TOPIC : Principle and me t hod’s of different microbiological assay Subject: Pharmaceutical microbiology SUBMITTED TO : Dr. Dharemendra jain ( Asstt. Professor ) Department of Pharmaceutical science’s SUBMITTED BY: Vaibhav Namdeo ( Y181501 67 ) B.PHARM, 3 rd Semester DEPARTMENT OF PHARMACEUTICAL SCIENCES DR. HARISINGH GOUR VISHWAVIDYALAYA , SAGAR,M.P. A CENTRAL UNIVERSITY

2. Microbiological Assay Biological Assay :- Biological assay refers to measurement of the relative potency or activity of compounds. Relative potency :- Relative potency is term used to express the biological activity of sample preparation compared to a standard preparation Microbiological Assay “ It is type of biological assay employed on microorganisms for determination of the relative potency of activity of any microbiological compound” Ex. Antibiotic preparation, vitamins and amino acid.

3. Objective of microbiological assay Assay :- Assay is a process employed for determining the content or quality of any substence. Objective :- many therapeutic agent which either inhibit the growth of microoganism (antibiotic ) or are essential for their growth (vitmins and amino acid can be standardized by microbial assay “ microbiological assay are performed to measure the activity of antibiotics (extent of ability to inhibit the growth of microoganism or vitmin & amino acid (extent of ability to support the growth of microoganism

4. Test organism that are used for test purpose Yeast Moulds becteria

5. Basic Principle “ Determination of relative potency of activity of a compound is based upon a comparison of the predieted effect by known concentration of the standard preparation of the microbiological compound having a known activity” Two general methods are used for microbiological assays Cylinder plate method or cup plate method. Tube assay method or titrimetric method.

6. Reference standard and units of activity The potency (activity) of an antibiotic product is expressed as the ratio of the does that inhibits the growth of a suitble susceptible miceooganism to the dose of the an “ international biological standard An international biological reference preparation of that antibiotic that produces similar inhibition. Potency of antibiotic can be expressed in “unit “ or “microgram” of activity per mg of dried material as stated in pharmacopeia.

7. Reference standard and units of activity Microgram of activity is based on single active ingredients. “unit” is used when there are more then one active ingredients in an antibiotic.

8. Cylinder plate method 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 dish. The growth of microoganism is inhibited entirely in a circular area or zone around the cavity or cylinder containing a solution of antibiotic.

9. Preparation of the Assay Preparation of agar plate :- The double layered plates are prepared for cylinder plate method. For this the first the bottom layer is prepared of agar medium (without) microoganism. Over which a inculated agar With microoganism was added. The thickness of the second layer is 4 mm. Store the prepared dishes or plates in a manner so as the ensure that no significant growth or death of the test organism occurs during storage.

10. Preparation of the assay Preparation of sample sol. :- sample sol. Are prepared by disolving the known amount of compound with suitble medium discribed in pharmacopoeia. Preparation of stock sol. :- to prepare stock sol. Disolve accuratly weighed quantity of the standard antibiotic and by disolving it in the solvent specified in the pharmacopoeia and appropriate amount of buffer sol. Also added to archived specific ph for growth.

11. Preparation of assay Test organism :- The test organism for each antibiotic is listed in table.

12. Procedure The nutrients agar is melted,cooled suitably, poured into petri dish. Required quantity of microbial test inoculated on the surface of the solidified agar by spred plate technique.

13. Procedure Cups and cavity are made by using a sterile boror.

14. Procedure Now different concentration such as 0.2,0.4,0.6 ml of antibiotic is poured into the cups of agar plate and then incubated on suitable temperature for 24 hours. If the antibiotic has any antibacterial effect it will show the zone of inhibition.

15. Procedure Zone of inhibition:- The clear region around the paper disc saturated with an antimicrobial agent on the Agar surface

16. Procedure The quantitative estimations of antibiotic is done by accurately measuring The diameter or areas of a circular inhibition zones

17. Dose responce curve The dose responce curve is sigmoidal for both test and standard, the distance between the two curves (x) measures the effectiveness of the test material relative to the standard. This is the log potency ratio The two lines Are coincide if standard And test are identical.

18. Turbidimetric method Turbidimetric method of assay of antibiotics are based on the turbidity produce by the test sample campare to standard. Principle :- The turbidimetric method is a method to determine the antimicrobial potency of an antibiotic. Mesurement of the inhibition of growth of a microbial culture in fluid medium. The inhibition of growth of a test organism is photometrically measured as changes in turbidity of microbial culture.

19. Preparation of assay Test organism :- We will select the organism for the assay from the following example listed below. Staphylococcus aurers ATCC 6538P For antibiotic assay Staphylococcus aurers ATCC 9144 For antibiotic assay Staphylococcus aurers ATCC 10537 For antibiotic assay Klebsiella pneumuniae ATCC 10031 For antibiotic assay

20. Preparation of assay Preparation of culture media :- For the growth of microbial organism we will prepare culture media. Following ingredients are used. Glucose 1gm/litre Peptone 6.0gm/litre Meat extract 1.5 gm/litre Yeast extract 3.0gm/litre Sodium chloride 2.5 gm/litre Agar 15gm/litre Water 1000 ml for make a volume. Mix all the ingredients and sterilize and adjust the pH of the solution so that it will be 6.5 to 6.6 after sterilization.

21. Preparation of assay Preparation of stock suspension of test organism :- Inoculate the test organism onto the slant of agar medium incubate the inoculated slant at 32°c to 37°c for 16 to 24 hours. Suspented the test organism in approximatly 10 ml of the liquid medium. Add approximately 150 ml of the same medium to give 85% Transmittance at a wavelenght of 650 nm.

22. Preparation of assay Standard solution of antibiotic :- Use the standard solution of the antibiotic specified in the indivisual monograph and make a 5 diffrent concentration of the standard solution of the antibiotic such as 0.1, 0.2 ,0.3 ,0.4, 0.5 Sample solution of antibiotic:- use the sample solution of unknown potency of antibiotic & take a diffrent concentration in each test tube such as 0.1, 0.2, 0.3, 0.4, 0.5

23. Preparation of assay Control tubes are also prepared :- For the test purpose we will also prepared 3 control tubes. One containing the inoculated culture (culture control) Second containing inoculated culture with 0.5 of dilute formaldehyde solution (As Blank ) Third one conatin uninoculated culture medium

24. Procedure Take a 5 test tubes in one set and 5 test tubes in another set 1 ml of standard solution of antibiotic are placed on the first set of antibiotic. 1 ml of the sample soltution of antibiotic are placed on the second set which is test set of antibiotic. In each test tube 9 ml of the nutrient medium previously seeded with the appropriate test organism is added.

25. Procedure Cover with a suitble lid or a cottan plug and then incubate in a water bath maintained at 35°c to 37°c for 3 to 4 hours. After incubation add 0.5 ml of formaldehyde sol. To each test tube. The growth of the test organism is measured by determining the extinction of each of the sol. In the test tube against a blank at 530 nm.