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Methods For Standardization of Antibiotics

The document describes several methods for standardizing antibiotics, including the cylinder-plate method, turbidimetric method, and assay of streptomycin. The cylinder-plate method involves diffusing an antibiotic from a cylinder through solid agar in a Petri dish, and measuring the zone of growth inhibition of microorganisms. The turbidimetric method similarly measures inhibition of microorganism growth but uses transmittance measurements rather than zone sizes. The streptomycin assay specifically tests samples against Bacillus subtilis to determine the potency of streptomycin, an aminoglycoside antibiotic derived from Streptomyces griseus that is active against gram-negative bacteria such as those causing tuberculosis.

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251 views4 pages

Methods For Standardization of Antibiotics

The document describes several methods for standardizing antibiotics, including the cylinder-plate method, turbidimetric method, and assay of streptomycin. The cylinder-plate method involves diffusing an antibiotic from a cylinder through solid agar in a Petri dish, and measuring the zone of growth inhibition of microorganisms. The turbidimetric method similarly measures inhibition of microorganism growth but uses transmittance measurements rather than zone sizes. The streptomycin assay specifically tests samples against Bacillus subtilis to determine the potency of streptomycin, an aminoglycoside antibiotic derived from Streptomyces griseus that is active against gram-negative bacteria such as those causing tuberculosis.

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rhittum1802
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METHODS FOR STANDARDIZATION OF ANTIBIOTICS

Antibiotic is a medical preparation, containing sufficient amount of chemical entity which is caused to
produce naturally (by a microorganism) or by artificially (semi- synthetic way) and that possesses the
inherent ability to either destroy (bactericidal) or inhibit (bacteriostatic) microorganisms in relatively
dilute solutions.

METHODS FOR STANDARDIZATION OF ANTIBIOTICS

➤ There are mainly three important points are for standardization of antibiotics:

• FDA (Food Drug Administration) regulations governing all aspects of antibiotics testing are completely
detailed and are subject to periodic amendment.

• FDA regulations need to be referred with regard to prescribed method for the assay of individual
antibiotics and their preparations.

• During the evaluation of potency of antibiotic substances, the actual and apparent measured effect is
the degree of inhibition of the growth of a suitable strain of microorganism i.e. the prevention of the
multiplication of the test organisms.

METHODS FOR STANDARDIZATION OF ANTIBIOTICS

Cylinder-plate (or cup-plate) Method

The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a
solidified agar layer in a Petri dish. The growth of the added micro- organism is prevented entirely in a
zone around the cylinder containing a solution of the antibiotic.
The cylinder plate method depends on the diffusion of an antibiotic from a vertical cylinder through a
solidified agar in a Petri dish. The growth of the added microorganism is prevented through the action of
antibiotics around the cylinder. After incubation, the diameter of the inhibition zone is measured. The
diameter depends on the concentration of the antibiotic used and its specific activity. This method has
accuracy and is so used in the commercial preparation of antibiotics.Antibiotic assay helps physicians for
proper selection of antibiotics which is more effective against the causative microorganism of a

particular disease.
Requirements:
Media: Muller Hintons media for bacteria, Potato Dextrose media for fungi.
Microbial culture: 24 hours fresh bacterial culture or fungal culture
Glasswares: Sterile Petri dishes, inoculating wire loop, forceps, sterile test tubes, Cork Borer, Paper disc,
Glass marking pencil.
Apparatus: Autoclave, Hot air oven, Incubator.

Method:
Prepare media as per standard procedure and subject it to sterilization in an autoclave.
Prepare the bacterial suspension which has to be examined for antibiotic assay
Immediately pour the sterile media into sterile Petri dishes.
Evenly spread the bacterial culture on the surface of media with the help of a sterile swab under aseptic
conditions.
Prepare a solution of antibiotic having a concentration equal to a reference standard.
Form the wells in the media with the help of cork-borer.
Apply the solution of the antibiotic in the cavity. Similarly, apply reference standard into another cavity
of the same Petri plate. (Note: in place of using the same plate for sample and reference material one
can use a separate plate for individual substance.)
Alternatively, instead of the formation of the cylinder, a paper disc is impregnated in the solution of
antibiotic and reference substance.
Using flame sterilized forceps, gently press each disc to the agar media.
In a single plate, a maximum of three dilutions of reference can be used at a time along with a sample
under testing.
Incubate the plate overnight in an incubator at 37º C.
Examine the zone of inhibition on the surface of media and around the cavity.
Zone size is measured from the edge of the disk to the end of the clear zone.
Observation: Observe the formation of a zone of inhibition around the well of antibiotics.

Result:
Positive result: Formation of clear zone around the well indicates positive test and show zone of
inhibition. It indicates that the microbes are sensitive to a given concentration of antibiotics.
Negative Result: If the zone of inhibition is not formed around the well indicates a negative result which
means that microbes are resistant to a given concentration of antibiotic.

Turbidimetric Method

The cylinder-plate method depends upon diffusion of the antibiotic from a vertical cylinder through a
solidified agar layer in a Petri dish. The growth of the added micro- organism is prevented entirely in a
zone around the cylinder containing a solution of the antibiotic.
The steps involved in this method are the same as that of the titrimetric method but the only difference
is that it involves the preparation of uninoculated blank tubes which contain only Basal medium-plus
sterile water and in place of titration with NaOH it involves measurement of transmittance by using a
colorimeter.
Adjust transmittance to 100% at 640 nm by using the uninoculated blank tube.
Record transmittance for all test tubes.
Plot the graph of % transmittance (Y-axis) vs. volume of cyanocobalamin (X-axis).
From graph compare the activity of the test sample by placing % transmittance of the test sample and
extrapolating on the x-axis.

Result: Compare activity of test sample by using the graph.

Media Preparations

All the required ingredients are dissolved in sufficient water to produce 1000 ml and added sufficient
amount of 1 M Sodium hydroxide or 1 M Hydrochloride acid, as required so that after sterilization the
pH is between 6.5 to 7.5.

METHODS FOR STANDARDIZATION OF ANTIBIOTICS

Buffer Solution Preparation:

Buffer solutions are prepared by dissolving the required quantities of dipotassium hydrogen phosphate
and potassium dihydrogen phosphate in sufficient water to produce 1000 ml and pH adjusted with 8 M
phosphoric acid or 10 M potassium hydroxide.

METHODS FOR STANDARDIZATION OF ANTIBIOTICS

Standard Preparation:

Standard preparation is an authentic sample of the appropriate antibiotic for which the potency has
been determined by reference to the appropriate international standard.

The potency of the standard preparation is expressed in µg per mg of an international unit of the pure
antibiotic.

■ A stock solution is prepared by dissolved a required amount of weighed quantity of the standard
preparation of a given antibiotic.
METHODS FOR STAR METHODS FOR STANDARDIZATION OF ANTIBIOTICS
Preparation of the Sample Solution:

Dilutions of sample or unknown samples are prepared at the range of dilutions of standard prepared
dilutions.

Assay Method of Streptomycin:

Streptomycin is bactericidal antibiotic belongs to class aminoglycosides. It is derived from


actinobacterium Streptomyces griseus.

It is used against gram negative bacteria, mainly against tuberculosis by inhibiting protein synthesis of
microorganisms. It is water soluble.

Bacillus subtilis is used for the microbiological assay of Streptomycin.

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