This document describes the microbiological assay method for quantifying antibiotics. It involves preparing standard solutions of known antibiotic concentrations and measuring their zones of inhibition against a test microbe. An unknown sample is also tested and its zone of inhibition is compared to the standard curve to determine its concentration. The key steps are: 1) Preparing standard solutions in increasing concentrations and an unknown at a median level. 2) Inoculating agar plates with a test microbe and creating wells for the solutions. 3) Measuring the zones of inhibition and calculating averages for the standards. 4) Comparing the unknown's zone of inhibition to the standard curve to determine its concentration.

1. MICROBIOLOGICALASSAY
OF ANTIBIOTICS
PROF.DR.S.SAISIVAM
PRINCIPAL
N.R.VEKARIA INSTITUTE OF
PHARMACY, JUNAGADH

2. DEFINITION
• Therapeutic efficacy of Antibiotics can be
exhibited by inhibiting the microbial growth.
• Any small change in the structure of antibiotic
can be easily detected by this method. Even
chemical methods may not reveal this.

3. PRINCIPLE
• Standard graph: known concentration Vs. ZOI.
• Comparing the zone of inhibition produced by
standard with that of unknown

4. METHODS AVAILABLE
• Method A: Cylinder Plate (or cup plate):
Diffusion of antibiotic from a vertical cylinder
or a cup through a solidified agar layer in a
petridish. It prevents the growth of organism
around the cylinder.
• Cylinder size of outer dia with 8mm, inner dia
of 6mm and length of 10mm.
• Instead of cylinder, cup of 6mm diameter can
be made with sterile borer.

5. METHOD B: TURBIDIMETRIC
(TUBE ASSAY)
• Inhibition of test organism growth in presence
of varying concentration of antibiotic solution.
• Assay tube size is 16 X 125mm or 18 X
150mm
• Tubes used should be same size and not some
with 16 X 125mm and some with 18 X
150mm.

7. STANDARD PREPARATION AND
ITS UNITS OF ACTIVITY
• Standard preparation is an authentic sample.
• The potency expressed in International Units
or in µg or mg of the pure antibiotic.
• Maintained at the Central Drug Laboratory,
Calcutta.
• A standard preparation may be replaced by a
working standard prepared by any laboratory
which should be compared at definite intervals
under varying conditions with the standard.

8. MEDIA
• The media required for the preparation of test
organism inocula is to be referred from Indian
Pharmacopoeia. They are termed as Media A
to J. The pH of the medium after sterilization
can be adjusted using sterile 1M NaOH or 1M
HCl.

9. Ingredient A B C D E F G H I J
Peptone 6 6 5 6 6 6 9.4 - 10 -
Pancreatic
casein
4 - - 4 - - - 17 - 15
Yeast ext 3 3 1.5 3 3 3 4.7 - - -
Beef ext 1.5 1.5 1.5 1.5 1.5 1.5 2.4 - 10 -
Dextrose 1 - 1 1 - - 10 2.5 - -
Papaic
casein
- - - - - - - 3 - 5
Agar 15 15 - 15 15 15 23.5 12 17 15
Glycerin - - - - - - - - 10 -
Polysorbate
80
- - - - - - - 10 - -
Nacl - - 3.5 - - - 10 5 3 5
K2HP04 - - 3.68 - - - - 2.5 - -
KH2P04 - - 1.32 - - - - - - -
pH 6.5 6.5 6.95 7.8 7.8 5.8 6 7.1 6.9 7.2

10. PREPARATION OF STANDARD
• Dissolve the weighed quantity of the standard
(if stated, previously dried) in the specified
solvent to get the required concentration.
• Store in a refrigerator and use within the
specified period.
• On the day of assay from the stock solution,
test solutions of 5 or more are prepared in
increasing concentration at the ratio of 1:1.25
using final diluents specified.

11. PREPARATION OF SAMPLE
• By assuming the potency labeled as correct,
the sample can also be similarly prepared as
like standard preparation both in case of stock
and in case of test solution.
• The assay with 5 levels of the standard
requires only one level of the sample or
unknown at a concentration assumed equal to
the median level of the standard.

12. FINAL DILUENT FOR THE TEST
SOLUTION
• It can be simply water or various buffer
solutions coded as B1 to B6.

13. BUFFER SOLUTION
Buffer No. K2HPO4(g) KH2PO4(g) pH adjusted after
sterilization to
1 2.0 8.0 6.0± 0.1
2 16.73 0.523 8.0± 0.1
3 ---- 13.61 4.5± 0.1
4 20.0 80,0 6.0± 0.1
5 35.0 --- 10.5± 0.1*
6 13.6 4.0 7.0± 0.2

14. Table 3 details of the Indian
Pharmacopoeia (2010)
• It furnishes following details:
• Name of the antibiotic to be assayed
• Assay method to be followed (method A or B)
• Whether to be dried before making the
solution or not. For example,
Bleomycin = dry at 25°C for 4h
Novobiocin = dry at 100 °C for 4h
Gentamycin = Dry at 110°C for 3h

15. Table 3 details (contd.)
• What is the initial solvent for preparing the standard
stock solution?
• Solvents = Water, DMF, 0.01M HCl, B1, B6,
Methanol, B2, Ethanol
• What is the final stock concentration per ml?
• Within how many days you have to use the stock?
• What is the final diluent for making the test
solutions?
• What is the median dose in µg or units per ml to be
selected?
• What is the incubation temperature?

16. TABLE 4: Test organisms for different
antibiotics
Antibiotic Test organism ATCC NO.
Amikacin,Tetracycline,
Tobramycin
Staphylococcus aureus 29737
Chlortetracycline Bacillus pumilus 14884
Erythromycin Micrococcus luteus 9341
Framycetin Bacillus pumilus
Bacillus subtilis
14884
6633
Gentamycin , Neomycin,
Novobiocin
Staphylococcus epidermidis 12228
Kanamycin sulphate Bacillus pumilus
Staphylococcus aureus
29737
Streptomycin, Vancomycin,
Rifampicin
Bacillus subtilis 6633
Nystatin Saccharomyces cerevisiae 2601

17. METHODS OF PREPARING
INOCULUM
• Method 1: Maintain the test organism on slants of
Medium A and transfer to a fresh slant once a
week. Incubate for 24h at specific temp.
• Using 3 ml saline, wash the agar surface and
transfer this into Roux bottle containing 250 ml of
the same Medium A. Incubate for 24h at specific
temp.
• Wash the growth from the agar surface using 50
ml saline and store under refrigeration.
• Determine the dilution factor which will give 25
% light transmittance at about 530 nm.

18. Method 2
• Proceed as per method 1 but incubate the Roux bottle
for 5 days.
• Centrifuge and decant the supernatant.
• Resuspend the sediment with 50 to 70 ml saline and
heat the suspension for 30 minutes at 70°C.
• Wash the spore suspension 3 times with 50 to 70 ml
saline. Resuspend in 50 to 70 ml saline and heat shock
again for 30 minutes.
• Determine the dilution factor to get 25% light
transmittance and dilute using sterile saline
• Store the spore suspension under refrigeration.

19. Method 3
• Maintain on 10 ml agar slants of Medium G.
• Incubate at 32°C to 35 °C for 24h. Rest is
similar to Method 1.

20. Method to determine the volume of
inoculum to be added
• Prepare Assay medium of 4 X 100 ml
• To each 100 ml add, varying volume of inoculum like 50 µl,
100 µl, 150 µl and 200 µl.
• Seeded medium of 27 ml is transferred from each culture
bottle into a sterile petriplate and allowed to solidify.
• Cups of 6 mm dia were made in each petriplate and placed
the median concentration of test antibiotic solution.
• After 1h of refrigeration, plates can be incubated in an
inverted condition at the specified temp.
• Based on clarity of zone of inhibition, the volume of
inoculum can be decided.

21. TABLE 5 DETAILS OF INDIAN
PHARMACOPOEIA
• Which method for preparing the inoculum?
• Which medium for inoculum prepartion?
• The incubation temperature and time
• Suggested dilution factor for the inoculum
• Assay medium to be used
• Amount of inoculum to be added
• What are the antibiotics can be assayed using
the specific organism?

22. ASSAY DESIGNS
• One level factorial assay: Prepare 5 test
dilutions of the standard and a solution of a
single median test level of the unknown.
• Two or three level factorial assay: Prepare
solutions of 3 or 2 corresponding test dilutions
for both the standard and the unknown.

23. ONE LEVEL FACTORIAL ASSAY
• Means 5 test dilutions of the standard and one
unknown concentration of the sample.
• Labelled as S1,S2,S3, S4 and S5 for test
dilutions of the standard and UK for the
sample.

24. TO ASSAY THE STREPTOMYCIN
• Pharmacopoeial suggested method: Both A and B.
• Test organism suggested: Bacillus subtilis
ATCC6633(MTCC 619).
• Dry the standard and sample of 100mg each at
60ºC for 3h.
• Initial solvent : Water sterile
• Stock concentration: 1 mg/ml
• Use the stock within 30 days
• Final diluent for test dilution : Sterile water

25. PROCEDURE
• To a 420 ml of Streptomycin assay medium,
add determined amount of volume of Bacillus
subtilis spore suspension(for example 200µl).
• Transfer 27 ml each into the 15 sterile
petriplates of 9 cm dia which will produce 3to
4 mm thickness.
• Allow to solidify by refrigerating.
• Make six cups using borer of 6mm diameter
in each petriplate.

26. PROCEDURE(Contd.)
• Each standard test dilution is to be tested in
triplicate except S3.
• Similarly prepare 3 plates for Unknown.
• Alternative cups are to be filled with 50 µl of S3
solutions in all the petriplates of standard test
dilution and unknown.
• Refrigerate for half an hour to allow the diffusion
of antibiotic solution and also to minimize the
growth of test organism during diffusion.

27. S1
S1S1
S3S3
S3
S1
S1S1
S3S3
S3
S1
S1S1
S3S3
S3
S2
S2S2
S3S3
S3
S2
S2S2
S3S3
S3
S2
S2S2
S3S3
S3
S4
S4S4
S3S3
S3
S4
S4S4
S3S3
S3
S4
S4S4
S3S3
S3
Avg. S1: 9S1/9
Avg. S2: 9S2/9
Avg. S4: 9S4/9

28. S5
S5S5
S3S3
S3
S5
S5S5
S3S3
S3
S5
S5S5
S3S3
S3
U
UU
S3S3
S3
U
UU
S3S3
S3
U
UU
S3S3
S3
Avg. S5: 9S5/9
Avg. U: 9U/9
Avg. S3: 36S3/36

29. Diameter of zone of inhibition
(cm)
Diameter of zone of
inhibition(cm)
1 3 5 Avg. 2 4 6 Avg.
S1:S3
S1:S3
S1:S3
S2:S3
S2:S3
S2:S3
S4:S3
S4:S3
S4:S3
S5:S3
S5:S3
S5:S3
UK:S3
UK:S3
UK:S3
OBSERVATIONS

30. Concn. of
std.antibiotic
solutions
Log
Concentration
Actual zone of
inhibition
Corrected Zone
of inhibition(cm)
S1: 20 units/ml 1.3010 Average of 9
readings =
S2: 25 units/ml
1.3979 Average of 9
readings =
S3: 30 units/ml 1.477 Average of 36
readings=
S4: 35 units/ml 1.544 Average of 9
readings =
S5: 40 units/ml 1.6020 Average of 9
readings =
Unknown(UK) Average of 9
readings =

31. Diameter of zone of inhibition
(cm)
Diameter of zone of
inhibition(cm)
1 3 5 Avg. 2 4 6 Avg.
S1:S3 1.2 1.2 1.2
1.25
1.4 1.4 1.4
1.36S1:S3 1.3 1.3 1.25 1.3 1.3 1.4
S1:S3 1.3 1.3 1.2 1.4 1.4 1.3
S2:S3 1.4 1.4 1.4
1.36
1.35 1.4 1.4
1.38S2:S3 1.3 1.3 1.4 1.3 1.4 1.4
S2:S3 1.3 1.3 1.35 1.4 1.4 1.4
S4:S3 1.5 1.5 1.55
1.51
1.4 1.45 1.4
1.416S4:S3 1.5 1.5 1.5 1.4 1.4 1.45
S4:S3 1.5 1.5 1.55 1.4 1.4 1.45
S5:S3 1.5 1.6 1.55
1.51
1.4 1.4 1.4
1.38S5:S3 1.5 1.5 1.5 1.4 1.4 1.45
S5:S3 1.5 1.5 1.55 1.3 1.3 1.4
UK:S3 1.5 1.5 1.6
1.5
1.35 1.4 1.4
1.38UK:S3 1.5 1.4 1.5 1.3 1.4 1.4
UK:S3 1.5 1.5 1.5 1.4 1.4 1.4
OBSERVATIONS
1.38 (Avg. of 36 readings of S3)

32. Concn. of
std.antibiotic
solutions
Log
Concentration
Actual zone of
inhibition
Corrected Zone of
inhibition(cm)
S S3
S1: 20 units/ml 1.3010 1.25 1.36 1.38 -1.36 = 0.02 is less in
S3 avg and hence, S1 will be
corrected as 1.25 + 0.02 =
1.27
S2: 25 units/ml 1.3979 1.36 1.38 1.36
S3: 30 units/ml 1.477 1.38(Avg. of
36 readings)
1.38
S4: 35 units/ml 1.544 1.51 1.42 1.42-1.38 =0.04 in excess
and hence S4 will be
corrected as 1.51 – 0.04
=1.47
S5: 40 units/ml 1.6020 1.51 1.38 Average of 9 readings =1.51
Unknown(UK) May be 1.58
equals to 37.5
units
1.5 1.38 Average of 9 readings =1.5

33. Meandiameter(mm)ofZOI
Conc. of bacteriostatic agent (µg/mL)
S1
S2
S3
S4
S5

34. FORMULA FOR ESTIMATION OF
POTENCY
• If we are not able to plot a graph as explained
before, then following formula can be used.
3a+ 2b+c-e 3e+ 2d+c –a
L = ------------- H = --------------
5 5
Where L= calculated zone diameter for the lowest
concentration of standard response line
H= Calculated zone diameter for the highest
concentration of standard response line

35. Contd.
• C= average zone dia of 36 readings of S3
• a,b,d,e = corrected average values for each
concentration of standard response line
• A plot can be drawn only by using L and H
value on Y axis and concentration on X axis.

36. TURBIDIMETRIC ASSAY OF
STREPTOMYCIN
• Test organism : 18 to 24 h culture
• Medium: 150 ml assay broth
• 15 sterile cotton plugged tubes of 16 X 125mm
or 18 X 150 mm, out of which 12 tubes for
assay and 3 for control
• Dilute Formaldehyde solution( 17 ml of
formalin + 33 ml of sterile water)
• Sterile pipettes of 10 ml, 5 ml capacity

37. PROCEDURE
• Transfer 10 ml of assay broth using sterile pipette
into the assay tube and label as negative control.
• Add 1 ml of 18 to 24h culture into the remaining
140 ml broth.
• Transfer 10 ml into the assay tube and label as
positive control.
• Transfer 10 ml into another assay tube and add
0.5ml of dilute formaldehyde solution and label as
blank.

38. Procedure ( contd.)
• Transfer 9.5 ml of inoculated broth into each of
remaining 12 assay tubes. Make 6 sets each
containing duplicate labeled as S1, S2,S3,S4, S5
and Unknown.
• Add respective concentration of standard or
unknown of 0.5 ml to each set and incubate at
37°C for 4 to 5 hours or till you can find the
turbidity difference in the tube of S1 and S5.
• Add 0.5 ml of dil.HCHO solution to each of the
12 tubes for arresting the growth.

39. Procedure ( contd.)
• Set the turbidometer at the λmax of 550 nm.
• Set zero with blank prepared previously.
• Measure the turbidities by starting from low to
high (means from S5 to S1) and then measure the
turbidity of Unknown.
• Plot a graph and find out the concentration of
unknown. If graphical points are not connectable,
then use the formula of L and H for drawing the
graph to find out the unknown concentration.

40. -ve control +ve control Blank
150 mL
10 mL 10 mL
10 mL
0.5 mL

41. 9.5 mL inoculated medium + 0.5 mL different conc. of Antibiotic + 0.5 mL Formaldehyde
S1
S1
S2
S2
S3
S3
S4
S4
S5
S5
U
U

42. Remember that Research is a
matter of honesty. Even result
opposite to that of your
expectation is also a research.