Microbial Assay of Antibiotics STANDARD PREPARATION AND UNITS OF ACTIVITY Preparation of media Buffer solutions Standard solution Sample solution Test organisms Preparation of inoculum Method -1 Method 2 Method 3 Method 4 Determination of Inoculum Apparatus Assay design Assay method cylinder plate method One level assay with standard curve Estimation of potency Turbidimetric method

1. MICROBIAL ASSAY
OF
ANTIBIOTICS
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Prepared by:
ADITYA SHARMA
NIPER Guwahati

2. Microbial assay of antibiotics
◦ It is based upon the comparison of the inhibition of growth of microorganisms by
measured concentration of the antibiotics under examination with that produced by
known concentration of standard preparation of antibiotic having a known activity.
◦ Two general methods are usually employed
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Method A
Cylinder plate method or
Plate(USP)
Cup-plate (IP)
Diffusion method (BP)
Method B
Turbidimetric method or
Tube assay

3. 3
Cylinder plate
method
Depends on diffusion of the
antibiotic from a vertical cylinder
through a solidified agar layer in a
Petri dish or a plate
The growth of specific
microorganisms incoculated into the
medium is prevented in the circular
area or zone around the cylinder
containing the solution of antibiotic
Turbidimetric
method
Depends on the inhibition of growth of micro organism in a
uniform solution of the antibiotic in a fluid medium that is
favourable to the growth of microorganism in the absence
of antibiotic.

4. STANDARD PREPARATION AND
UNITS OF ACTIVITY
◦ A standard preparation is a authentic sample of the appropriate antibiotic, for which potency has
been precisely determined by the reference to an appropriate international standard.
◦ The potency of the standard preparation may be expressed in International Units or in µg per mg of
the pure antibiotic.
◦ µg of activity is based on single active ingredient.
◦ Unit is used when there are more than one active ingredient in the antibiotic.
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5. Preparation of media
◦ Preparation of test microorgnism inocula from the ingrdients listed
in the following table.
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• Dissolve the ingredients
in sufficient water to
produce 1000ml.
• Add sufficient 1M
sodium hydroxide or hcl,
as required, so after
sterilization the pH is as
given.

6. Buffer solutions
◦ Dissolve given quantities of dipotassium hydrogen
phosphate and potassium dihydrogen phosphate in
water to produce 1000ml.
◦ Adjust the pH with 8M phosporic acid or 10M
potassium hydroxide, after sterilization.
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7. Standard solution
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• Dissolve standard preparation of
antibiotic in the solvent specified.
• Dilute to required concentrations
• Store in a refregirator and use within the
period indicated.
• From th stock solutions prepare 5 or more
test dilutions
• the succesive solutions increasing in
stepwise in concentrations in the ratio of
1:2.5 for method A and smaller for
method B

8. Sample solution
◦ Assign assumed potency per unit
weight or volume.
◦ On the day of assay prepare a stock
solution and test dilution as
specified for each antibiotic.
◦ Dilute the sample stock solution in
the specified final diluent to obtain
a nominal concentration equal to
median concentration of the
standard
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9. Test organisms
◦ Maintain a culture on slants of
the medium and under the
incubation conditions specified in
Table, and transfer weekly to
fresh slants.
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10. Preparation of inoculum
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11. 11
Preparation of inoculum
Method -1
Maintain the test organism on
slants of Medium A and transfer
to a fresh slant once a week.
Incubate the slants at the
temperature indicated for 24
hours.
Using 3 ml of saline solution,
wash the organism from the agar
slant onto a large agar surface of
Medium A such as a Roux bottle
containing 250 ml of agar.
Incubate for 24 hours at the
appropriate temperature.
Wash the growth from the
nutrient surface using 50 ml of
saline solution.
Store the test organism under
refrigeration.
Determine the dilution factor
which will give 25 per cent light
transmission at about 530 nm.
Determine the amount of
suspensions to be added to each
100 ml of agar of nutrient broth
by use of test plates or test
broth.
Store the suspension under
refrigeration.

12. 12
Method 2
Proceed as described in
Method 1 but incubate
the Roux bottle for 5
days.
Centrifuge and decant
the supernatant liquid.
Resuspend the sediment
with 50 to 70 ml of
saline solution and heat
the suspension for 30
minutes at 70º.
Wash the spore
suspension three times
with 50 to 70 ml of
saline solution.
Resuspend in 50 to 70
ml of saline solution and
heat- shock again for 30
minutes.
Use test plates to
determine the amount
of the suspension
required for 100 ml of
agar.
Store the suspension
under refrigeration.

13. Method 3
◦ Maintain the test organism on 10 ml agar slants of Medium G.
◦ Incubate at 32º to 35º for 24 hours.
◦ Inoculate 100 ml of nutrient broth.
◦ Incubate or 16 to 18 hours at 37º and proceed as described in Method I.
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14. Method 4
◦ Proceed as described in Method 1 but wash the growth from the nutrient surface using 50 ml
of Medium 1 (prepared without agar) in place of saline solution.
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15. Determination of Inoculum
◦ The inoculum prepared above is inoculated into 100ml of each type of the medium.
◦ Assay is performed as indicated in the monograph.
◦ In cylinder-plate assays, double-layer plates may be prepared by pouring a seed layer
(inoculated with the desired microorganism) over a solidified uninoculated base layer.
◦ 21 ml of base layer and 4 ml of the seed layer may be generally suitable.
◦ Each cylinder was filled with the median concentration of the antibiotic and
◦ Then incubate the plates.
◦ After incubation, examine and measure the zones of inhibition.
◦ The volume of suspension that produces the optimum zones of inhibition with respect to both
clarity and diameter determines the inoculum to be used for the assay.
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For method A-

16. Determination of Inoculum
◦ Proceed as described for Method A .
◦ The specific antibiotic assay was performed by running only the high and low
concentrations of the standard response curve.
◦ Read the absorbance's of the appropriate tubes.
◦ Determine which inoculum produces the best response between the low and
high antibiotic concentrations
◦ Use that inoculum for the assay.
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For method B-

17. Apparatus
◦ All equipment is to be thoroughly cleaned before and after each use.
◦ Glassware for holding and transferring test organisms is sterilized by dry heat or by steam.
◦ Thermostatic control is required at several stages of a microbial assay, when culturing a
microorganism and preparing its inoculum and during incubation in a plate assay.
◦ Temperature control during the conduct of the experiment is done by maintaining a continuous
heat through air or water
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18. Apparatus
◦ A spectrophotometer in which a 580nm(for inoculum preparation) or a 530 nm(for tube assay)
filter for allowing only required wavelengths.
◦ For the instrument to carry over the assay a flow through cell and a drain are arranged to
communicate the exchange of medium components whenever necessary.
◦ Set the instrument to zero using uninoculated broth.
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19. Apparatus
◦ Rectangular trays or glass or plastic petri dishes of particular dimensions are used
◦ Agar is made as gel and into which bores are made cylindrically into which
antibiotic solution is dropped on or paper discs wetted with antibiotic was placed
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Cylinder-plate Assay Receptacles

20. Apparatus
◦ Test tubes of particular dimension with uniformity in length, width and thickness
were maintained along with blemish and scratch free properties.
◦ The receptacles are cleaned with 2M nitric acid or chromic acid to take off the left
residues.
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Turbidimetric Assay Receptacles.

21. Assay design
Normal assay design:
◦ For a cylinder plate assay, comparative study of inhibition zone diameters.
◦ For a turbidimetric assay, comparison of turbidities
Alternative assay designs:
◦ A two level(or 3 level) factorial assay.
◦ Single level assay with a standard curve.
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22. 22
◦ For factorial assay, prepare 2 or 3 test dilutions of both standard and test on the day of
assay.
◦ For single level assay, prepare 5 dilutions of standard and a single test solution such that it
falls in the median value of the corresponding standard preparations.
◦ If the computed potency is less than 60% or greater than 150% repeat the assay by adjusting
the assumed potencies.
◦ Smaller independent assays carried over large number of days is more reliable in potency
estimation than a single large assay carried using same plates and tubes.

23. 23
Assay method
cylinder plate method
Inoculate the specified organism
in a liquid medium at 40-50
degrees and pour into a petri dish
so that depth of 3-4 mm is
reached(1-2mm for Nystatin) .
Prepare standards and
test solutions as
mentioned in the
monographs.
Apply these solutions
into the cylinders or agar
cavities or two sided
sterilized (lamp)paper
discs
Leave the dishes for about
4 hrs at room
temperature to
equilibrate all of them as
the time of applying
solution differs between
the dishes.
Maintain the
incubation
temperature
and leave for
18hrs
Calculate the
inhibition
zone
diameters.
Select the assay
design as
specified in the
monograph.

24. One level assay with standard curve
◦ prepare from the stock solution, 5 dilution (solutions S1to S5) representing 5 test
levels of the standard and increasing stepwise in the ratio of 4:5.
◦ From the information available, assign to antibiotic an assumed potency per unit
weight or volume .
◦ a stock solution with same solvent as used for the standard.
◦ Prepare from this stock solution a dilution to a concentration equal to the median
level of the standard to give the sample solution.
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Standard solution
Sample solution

25. 25
A.One level assay with standard curve
(cont.)
For preparing the standard
curve, use a total of 12 petri
dishes or plates to
accommodate 72 cylinders or
cavities.
A set of 3 plates (18 cylinders
or cavities) is used for each
dilution.
On each of the three plates of a set
fill alternate cylinders or cavities
with solution S3 (representing the
median concentration of the
standard solution) and each of the
remaining 9 cylinders or cavities
with one of the other 4 dilutions of
the standard solution.
Repeat the process for the
other 3 dilutions of the
standard solution.
For each unknown
preparation use a set of 3
plates (18 cylinders or
cavities) and fill alternate
cylinders or cavities with the
sample solution and each of
the remaining 9 cylinders of
cavities with solution S3‘
Incubate the plates for about
18 hours at the specified
temperature and measure the
diameters or the zones of
inhibition.
METHOD

26. Estimation of potency
◦ Calculate average inhibition zones of each tested samples per plate and the inhibition zones of
S3 of 12 plates.
◦ The average value of S3 is considered as correction factor.
◦ From the averages of each sample’s concentration the correction factor is subtracted .
◦ Plot these values on a semi log paper using concentration in mcg/ml on ordinate and zone
diameter on abscissa.
◦ Highest and lowest zone diameters are calculated and the concentrations obtained after
averages are plotted through them.
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27. 27

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◦ If the averages of the zone sizes of the sample solutions is greater than that of the average of
the reference S3 then add the difference to the avg zone diameter of the reference.
◦ If the value is smaller then subtract the difference between them from the avg of the reference.
◦ Check for the concentrations corresponding to these corrected values from the response line.
◦ From the dilution factor potencies can be calculated.

29. Turbidimetric method
◦ The method has the advantage of a shorter incubation period for the growth of the test
organism (usually 3 to 4 hours)
◦ The presence of solvent residues or other inhibitory substances affects this assay more than the
cylinder plates assay
◦ Care should be taken to ensure freedom from such substances in the final test solutions.
◦ This method is not recommended for cloudy or turbid preparations.
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30. 30
To each tube add 9
ml of nutrient
medium previously
seeded with the
appropriate test
organism).
Place 1ml of each
concentration of the
standard solution
and of the sample
solution in each of
the tubes in
duplicate.
Select the median
concentration and dilute
the solution of the
substance being
examined (unknown) to
obtain approximately
this concentration.
Prepare five different
concentrations of the
standard solution for
preparing the standard
curve, increasing
stepwise in the ration
4:5.
Measure the growth of the test
organism by determining the
absorbance at about 530 nm of
each of the solutions in the
tubes against the blank.
After incubation add 0.5 ml of
dilute formaldehyde solution to
each tube.
Place all the tubes, randomly
distributed or in a randomized
block arrangement, in an
incubator or water-bath and
maintain them at the specified
temperature for 3 to 4 hours.
At the same time prepare three control tubes,
• one containing the inoculated culture medium (culture control),
• another identical with it but treated immediately with 0.5 ml of dilute formaldehyde
solution (blank) and
• a third containing uninoculated culture medium.

31. Estimation of potency
◦ Plot the average absorbance's for each
concentration of the standard on semi-
logarithmic paper with the absorbance's on
the arithmetic scale and concentrations on
the logarithmic scale.
◦ Construct the best straight response line
through the points either by inspection or
by means of the following expressions:
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32. THANKYOU...
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