Chapter 6 1

MICROBIOLOGICAL ASSAY OF
ANTIBIOTICS
Many therapeutic agents, which either inhibit the growth of micro-organisms(antibiotics) or
essential for the growth (vitamins and amino acids) can be standardized by microbiological 2
assays.
Any change in antibiotic molecule which may not be detected by chemical methods will be
revealed by a change in the anti-microbial activity and hence microbiological assays are very
useful for resolving doubts regarding possible change in potency of antibiotics.
Three methods used for the assay-
1- Cylinder method or cup plate method
Depends upon diffusion of the antibiotic from the vertical cylinder on the surface of the agar plate
to an extent such that growth of the added micro-organism is prevented entirely in a zone around
the cylinder containing a solution of antibiotic.
2- Disc method
Depends upon diffusion of the antibiotic from the discs placed on the surface of the agar plate to
an extent such that growth of the added micro-organism is prevented entirely in a zone around the
discs containing a solution of antibiotic.
3- Turbidometric method or tube assay method
Depends upon the inhibition of 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.
ASSAY OF BENZYL PENICILLIN BY CUP PLATE METHOD
3
Aim: To perform microbiological assay of crystalline, sodium salt of benzyl penicillin by cup plate method.
Requirements:
Cultures- Bacillus subtilis
Medium- Antibiotic assay medium
Apparatus- Petri plate, conical flask, beaker, test tube, nichrome wire loop, glass spreader.
Equipments- Autoclave, hot air, oven, balance, pH meter, incubator, antibiotic zone reader, laminar air flow.
 
Principle:
This method is based on the diffusion of an antibiotic from a cavity through the solidified agar layer of a
petriplate used for the study. Growth of inoculated Mos is inhibited entirely in a circular area ‘zone’ around a
cavity containing a soln of antibiotics.
Procedure:
Dissolve 35 mg of benzyl penicillin in 100 ml of water. From this, take 4
1 ml and dilute to 100 ml of sterile water (each ml of this contain 6 unit
activity)
By using this stock solution, prepare 1,2,3,4 and 5 units/ml of standard
stock solution. Prepare a solution of unknown or a given sample of
antibiotics in a same solvent as that of standard solution and dilute it till
the level of standard preparation of the antibiotics.
Sterilize the antibiotic assay medium by autoclave and prepare petri
plates in laminar air flow. Spread the test MO (Bacillus subtilis) on the
surface of the petri plate by spread plate technique.
By using flame sterilized cock borer, prepare 5-6 cavities (cups) in each
plate keeping adequate dist. from each other.
Standard and test diluted antibiotic solutions (0.1-1ml, depending on
size of cavity) are added in each labelled cavity of plates.
Transfer all the plates in refrigerator for proper diffusion of antibiotics
at 4C for 1-2 hrs. Incubate all the plates in incubator at 32-35C for 24 to
48 hrs.
Observation and Results:
Observe the zone of inhibition around 5
each cavity. Measure the diameter by
antibiotic zone reader and record in
observation table. Plot the graph of the
diam. of the zone of inhibition v/s log
conc. of antibiotic and determine the
concentration of unknown antibiotic
sample from the graph. Concentration
of unknown sample is 4 unit/ml.
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ASSAY OF STREPTOMYCIN BY CUP PLATE METHOD
6
Aim: To perform microbiological assay of streptomycin by cup plate method.

Requirements:
Culture- 24 hrs broth culture of bacillus subtilis/E.coli
Medium- Nutrient agar plates/ Antibiotic assay medium
Apparatus- Petri plate, glass spreader, conical flask, sterile cork borer etc.
Equipment- autoclave, incubator, antibiotic zone reader and laminar air flow

Principle:
The microbiological assay is based upon a comparison of inhibition of growth of microorganism by measured
concentration of antibiotics to be used, examined with that produced by known concentration of a standard
preparation of antibiotic having known activity.
Procedure:
Dissolve 256 mg of streptomycin in 100 ml of sterile water. From this, 7
take 1 ml and dilute to 100 ml of sterile water (each ml of this contain
20 unit activity)
By using this stock solution, prepare 10,12,14,16 and 18 units/ml of
standard stock solution. Prepare a solution of unknown or a given
sample of antibiotics in a same solvent as that of standard solution and
dilute it till the level of standard preparation of the antibiotics.
Sterilize the antibiotic assay medium by autoclave and prepare petri
plates in laminar air flow. Spread the test MO (Bacillus subtilis) on the
surface of the petri plate by spread plate technique.
By using flame sterilized cock borer, prepare 5-6 cavities (cups) in each
plate keeping adequate dist. from each other.
Standard and test diluted antibiotic solutions (0.1-1ml, depending on
size of cavity) are added in each labelled cavity of plates.
Transfer all the plates in refrigerator for proper diffusion of antibiotics
at 4C for 1-2 hrs. Incubate all the plates in incubator at 32-35C for 24 to
48 hrs.
Observation and Results:
Observe the zone of inhibition around 8
each cavity. Measure the diameter by
antibiotic zone reader and record in
observation table. Plot the graph of the
diam. of the zone of inhibition v/s log
conc. of antibiotic and determine the
concentration of unknown antibiotic
sample from the graph. Concentration
of unknown sample is 18 unit/ml.
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ASSAY OF ANTIBIOTICS BY TURBIDOMETRIC METHOD
9
Aim: To perform microbiological assay of antibiotics using turbidometric or tube method.

Requirements:
Test tubes, antibiotics, microbial culture, spectrophotometer, laminar air flow, inoculating loop, Bunsen
burner.

Principle:
The microbiological assay is based on the growth of a microbial culture in a homogenous solution of the
antibiotic in a fluid medium that is favorable to its rapid growth in the absence of antibiotics. It is
advantageous because of shorter incubation period for the growth of MOs, but it is affected by inhibiting
substances and solvents. This method is not suitable for turbid or cloudy preparations.

Procedure:
Prepare 5 diff. conc. of std. solution for preparing the standard curve by diluting the stock solution of the
standard preparation of antibiotic.
Select the media conc. and dilute the solution of antibiotics.
Place 1 ml of each conc. Of standard solution and 1 ml of sample solution in separate tubes.
In each tube add 9ml of nutrient media previously inoculated by test micro-organism.
At the
   same time prepare three control tubes
1- inoculated culture media (culture control) 10
2- inoculated culture media + 0.5ml dilute formaldehyde solution
3- uninoculated culture media
Place all the tubes at specified temp. for 3-4 hrs.
After incubation add 0.5ml of dil. formaldehyde solution to each tube.
Measure the growth of test organism by determining the extinction at 530nm of each of the solutions in the tubes
against the blank.
Estimation of potency:
Plot avg. extinction for each conc. of standard on semilogarithmic paper with the extinctions on the arithmetic scale.
Equation-
L= calculated extinction for the lowest conc. of standard response line.
H= calculated extinction for the highest conc. of standard response line.
a,b,c,d,e= average extinction values for each conc. of standard response line lowest to highest resp.
Observation:
Plot the values of L and H. Avg. the extinction for the sample and read the antibiotic 11
conc. From the standard response line multiply the conc. by appropriate dilution
factors to obtain the antibiotic content of the sample.
ASSAY OF ANTIBIOTICS BY DISC METHOD
12
Aim: To evaluate antibiotic sensitivity of microorganisms by multiple disc technique.
Requirements:
Culture- any culture of microorganism.
Media- Nutrient agar plates.
Antimicrobial Sensitivity Discs- Penicillin, Ampicilin& Streptomycin.
Principle:
Antibiotics are defined as natural antimicrobial agents produced by microorganisms which are capable of killing
or inhibiting the growth of other microorganisms in small concentration.
Antimicrobial Sensitivity test evaluates susceptibility of micro-organisms to antibiotics or other chemotherapeutic
agents by allowing antimicrobial contained in a reservoir to diffuse out into the medium and interact with the test
organisms. The susceptibility of a micro-organism to a drug is determined by the size of zone of inhibition which
is dependent on the ability and rates of diffusion of the antibiotics into the medium and its interaction with the
test microorganism. Antibiotic-impregnated filter paper discs are applied gently under sterile conditions with a
sterile forceps. Discs are placed on the flat surface & firmly apply pressure to ensure adhesion. The disc should
preferably be deposited with centers at least 25mm to 30mm apart. The results are interpreted and reported as
“sensitive”, “moderately sensitive” & “resistant” by referring the diameter of zone of inhibition.
Various Factors affecting disc diffusion test:
1. Disc concentration. 13
2. Diffusibility of the drug.
3. Nature composition and depth of the medium.
4. pH of the medium.
5. Size of inoculums.
6. Incubation temperature.
7. Incubation period or length of incubation.
Based on spectrum of activity antibiotics are classified as:
1. Narrow spectrum antibiotics: are those which can kill or inhibit the growth of only a few species of
microorganisms. E.g. Penicillin is active against only Gram positive bacteria; Streptomycin is active against Gram
negative bacteria.
2. Broad spectrum antibiotics: are those which can kill the growth of many different species of microorganisms,
including both Gram positive and Gram negative bacteria.
E.g. Ampicillin, Tetracycline, Cephalosporin, etc.
Procedure:
Weigh the required amount of nutrient agar in conical flask and add sufficient quantity of water to it. 14
Heat if necessary to dissolve it in water and sterilize it in autoclave.
Pour the media aseptically in previously sterilized petri plate and allow it to solidify.
Inoculate given cultures of microorganisms by spread plate method and apply various discs of
antibiotics firmly over nutrient agar plate.
Petri plate was incubated at 37C for 24 – 48 hrs.
After incubation sensitivity and resistance of microorganism was determined by reading the zone of
inhibition.
Observation and results:
Observe the zone of inhibition around each cavity. Measure the diameter by antibiotic zone reader and 15
record in observation table against each type of antibiotic. As per the data of zone of inhibition, the
antibiotic disc with maximum zone of inhibition is considered to be more sensitive against the micro-
organism strain used.
ANTI-MICROBIAL SENSITIVITY TEST
The susceptibility of Mos to antibiotics or any other chemotherapeutic agents Is determined 16
by various methods
Various Mos are tested against various different groups of antibiotics to check the
sensitivity of antibiotics to Mos.
Same conc of antibiotic against diff. type of MOs can be checked or
Activity of Same kind of MO can be checked against different concentrations of antibiotics
and also with different type of antibiotics.
Chemo agents S. aureus Bacillus Ecoli
Subtilis
Penicillin sensitiv resistant
e
Streptomycin
Tetracycline
Chloramphenicol
Gentamicin