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.

9. • 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

11. • 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.

12. Preparation of Test organism
• The test organism for each antibiotic is listed in Table, together
with its identification number in the American Type Culture Collection
(ATCC).

13. 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

14. Methods of Microbiological Assay
• A. Cylinder plate orCup plate method
• B. Turbidimetric orTube Assay method

15. A. Cylinder plate orCup 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.

16. Procedure:-
 A liquefied assay medium ( 43 -45 ˚ C) is inoculated by suspension of test
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
and assay of streptomycin is given as previous table.

17.  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.

20. 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

21.  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.

23. 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.

24. 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.
• Toeach test tube add 5ml basal medium stock solution.
• Adjust final volume 10ml by using water.
• Toother four test tubes, add 1ml, 2ml,3ml,4ml respectively of the test solution to be
assayed.
• Toeach test tube add 5ml basal medium stock solution.
• Adjust final volume 10ml by using water.

25. • Sterilize all test tubes in autoclave at 1210C 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- 37OC.
• 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.

27. 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.

28. 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 μg/ml or units/ml.

29. 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 105 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.

30.  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 370 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.

32. 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 370 C for 2- 3 days.
After incubation, all plates are observed for growth of
inoculum and calculate the MIC.

33. 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.