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

1. TOPIC: Principle and method’s of different
microbiological assay
Subject: Pharmaceutical microbiology
SUBMITTED TO:
Dr. Dharemendra jain
( Asstt. Professor )
Department of
Pharmaceutical science’s
SUBMITTED BY:
Vaibhav Namdeo
(Y18150167)
B.PHARM, 3rd 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
1. Yeast
2. Moulds
3. 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
1. Cylinder plate method or cup plate method.
2. 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.
1. One containing the inoculated culture (culture
control)
2. Second containing inoculated culture with 0.5 of
dilute formaldehyde solution (As Blank )
3. Third one conatin uninoculated culture medium

24. Procedure
1. Take a 5 test tubes in one set and 5 test tubes in another
set
2. 1 ml of standard solution of antibiotic are placed on the
first set of antibiotic.
3. 1 ml of the sample soltution of antibiotic are placed on
the second set which is test set of antibiotic.
4. In each test tube 9 ml of the nutrient medium previously
seeded with the appropriate test organism is added.

26. 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.