Prof.Mr.Kiran K. Shinde (M.Pharm), Assistant professor (VNIPRC) Pharmaceutical microbiology (Second year b.pharm) (3rd semester) Introduction Methods Of Different microbiological assays Principles of Assays with Procedure Methods For Standardization of 1. Antibiotics 2. Vitamins 3. Amino Acids Assessment of new Antibiotic

1. MICROBIOLOGY ASSAYS
• INTRODUCTION
• PRINCIPLES AND METHODS OF DIFFERENT MICROBIOLOGY ASSAY
• METHODS FOR STANDARDIZATION OF ANTIBIOTICS, VITAMINS AND AMINO
ACIDS
• ASSESSMENT OF NEW ANTIBIOTIC
PREPARED BY,
PROF. SHINDE KIRAN (M.PHARM)
ASSISTANT PROFESSOR (VNIPRC)
PHARMACEUTICAL MICROBIOLOGY-I
SECOND YEAR B.PHARM
IIIRD SEMESTER
Vidya Niketan Institute
Of Pharmacy &
Research Centre, Bota

2. INTRODUCTION
1. A microbiological assay may be defined as qualitative or quantitative
determination of any chemical compound from a single or even complex
material with the use of micro-organisms.
2. Many therapeutic agents, which either inhibit the growth of micro-organisms
[antibiotics] or essential for their growth [vitamins and amino acids] can Be
standardized by microbiological assays.
3. Microbiological assays are relatively as accurate as chemical methods.
4. It is a simple, specific, inexpensive and convenient method.
5. Microbial assays are more difficult to perform as compared to chemical and
physical assays and also require proper calibration.
6. They are less reproducible and has greater error as compared to other assays.
7. They are not used if a good alternative physical or chemical assay is available.

3. The microbiological assay of antibiotics may be carried by 2 methods:
ASSAY METHODS
Method A: Cup-plate or
cylinder-plate method
Method B: Turbidimetric
or tube assay method
The microbiological assay of vitamin B12 may be performed by 2 methods:
Method 1. Titrimetric
method
Method 2. Turbidimetric
method

4. MICROBIOLOGICAL ASSAY OF ANTIBIOTICS
The inhibition of microbial growth under standardised conditions may be
utilised for demonstrating the therapeutic efficacy of antibiotics.
Principle:
The microbiological assay is used upon a comparison of the inhibition of
growth of micro-organisms by measured concentrations of the antibiotics to
be examined with that produced by known concentrations of a standard
preparation of the antibiotic having a known activity.

5. Principle:
This method depends on the diffusion of an antibiotic from a vertical
cavity or a cylinder, through the solidified agar layer in a petri plate.
Procedure:
1. The nutrient agar is melted, cooled suitably, poured into petri dish.
2. Spread 0.2 ml of known concentration of inoculum on the surface of the
solidified agar [Spread Plate Technique].
3. Cups or cavities are made y using a sterile borer.
4. now 0.2, 0.4, 0.6, 0.8, 1.0 ml of antibiotic is poured into the cups of agar
plate and then incubated at 37C for 24 hrs.
5. If the antibiotic has any anti-bacterial effect it will show the zone of inhibition
Method A: Cup-Plate or Cylinder-plate method

7. Method B: Turbidimetric or tube assay method
Principle:
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. Not used for turbid or cloudy preparations.
Procedure:
1. Five different concentrations of the standard solution are prepared by diluting
the stock solution for making standard curve.
2. A median concentration is selected and the test sample of the antibiotic solution
is adjusted y dilution to obtain approximately this concentration.
3. 1 ml of each concentration of std. solution and of sample solution are placed in
each of the tubes in duplicates.

8. 4. To each tube, 9ml of nutrient medium previously seeded with appropriate test
micro-organism is added.
5. At the same time,3 control tubes, 1st containing the inoculated culture medium,
another identical with it but treated immediately with 0.5 ml of dilute
formaldehyde solution and a 3rd containing uninnoculated culture medium are
prepared.
6. All tubes are placed in incubator at specific temperature for 4-5 hrs. after
incubation add 0.5 ml formaldehyde solution to each tube.

9. MICROBIOLOGICAL ASSAY OF CYANOCOBALAMIN
[VITAMINS B12]
Vitamins are important growth factors needed for growth and multiplication
of micro-organisms. They are very sensitive to small amounts of growth
factors. Its ability of these micro-organism to synthesize the factor being
assayed that forms the basis of the microbiological assay to vitamins and
amino acids.

10. Procedure:
1. Clean 10 + 4 test tubes with 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0ml resp.
of standard cyanacobalamin solution.
2. To each test tube add 5ml basal medium stock solution.
3. Adjust final volume 10ml by using water.
4. To other 4 test tubes, add 1ml, 2ml, 3ml,4ml resp. the test solution to be assayed.
5. To each test tubes add 5 ml basal medium stock solution. Adjust final volume 10ml
by using water.
6. Sterilize all test tubes in autoclave at 120C for 5 min. afterwards cool at room
temperature & inoculate with 1 drop of inoculum [lactobacillus leichmonii ATCC
7830] for 64-72 hrs. at chosen temperature within 30-37 C
7. Titrate the content of each tube with 0.05 N NaOH, using 0.1 w/v bromothymol
blue as an indicator [coverts green].
1. Titrimetric method

11. 8. Determine the average of titration values for each level of std. and test sample.
9. Plot the graph of titration value versus std. cyanacobalamin solution
concentration.

13. 2. Turbidimetric method

14. ASSESSMENT OF NEW ANTIBIOTIC:
MINIMUM INHIBITORY CONCENTRATION
[MIC]
Minimum Inhibitory Concentration [MIC] is the lowest concentration of
antimicrobial compound found to inhibit the growth of a particular test
micro-organism. It may e applied to assess new disinfectants, antiseptics,
preservatives and antibiotics. MIC values are usually expressed in terms of
g/ml or units/ml. MIC od different antimicrobial compounds may be
determined by
THE LIQUID DILUTION METHOD or
THE SOLID DILUTION METHOD

15. 1. Liquid Dilution method or
test tube method
Procedure:
Use a series of test tubes which contain a double-strength medium
1. In 1st test-tube inoculum not added & used for checking the sterility of the
medium.
2. All other 11 test-tube inoculum is added to reach the final concentration.
3. In all test tubes, test chemical is added ranging from 0.5-5ml expect in the
uninnoculated and control tube.
4. The 2nd tube [control] is used to check the suitability of the medium for
growth of the test micro-organisms and the viability of the inoculum.
5. The final volume 10 ml is adjusted in all test-tubes by sterile water.
6. Properly mixed and incubated for 2-3 days at 37C.

16. 7. After incubation, all test tubes are examined for growth and minimum MIC
recorded. Also necessary to conduct preliminary experiment to determine
the approximate range

17. 2. Solid dilution method
Procedure:
1. In this method, test chemical is first mixed into molten agar and then poured
into petri plates.
2. After solidification, the inoculum is spread on the surface of agar medium.
3. All plates are incubated at 37C for 2-3 days.
4. After incubation, all plates are observed for growth of inoculum and the
minimum inhibitory concentration of the test chemical is calculated.
5. The advantages are-
• Several micro-organisms can be tested at the same time y use of multipoint
inoculator.
• Contaminants are easily detected, because colony features on solid media are
more distinctive than turbidity difference in fluid media.

18. Thank-you