This document discusses methods for standardizing antibiotics. It notes that there are three important points for standardization: following FDA regulations, referring to FDA methods for individual antibiotics, and measuring inhibition of microbial growth. Two common assay methods are described: the cylinder-plate method which measures zones of inhibition, and the turbidimetric method which measures prevention of microbial growth. Procedures for preparing media, buffers, standards and samples are outlined. Streptomycin is used as an example antibiotic, noting it is bactericidal, derived from Streptomyces griseus, and tested using Bacillus subtilis.

1. MS.NISHA MANDLOI
METHODS FOR STANDARDIZATION
OF ANTIBIOTICS

2. 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.

3. 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.

4. METHODS FOR STANDARDIZATION OF
ANTIBIOTICS
Two methods are usually
Cylinder-plate (or cup-plate)
Method
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 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.

5. METHODS FOR STANDARDIZATION OF
ANTIBIOTICS
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.
Media Preparation:

6. 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

7. Standard Preparation:
METHODS FOR STANDARDIZATION OF
ANTIBIOTICS
 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

8. Preparation of the Sample Solution:
Dilutions of sample or unknown samples are prepared at the range of
dilutions of standard prepared dilutions.
Test Organisms: Some of the test organisms for each antibiotic are
listed in
METHODS FOR STANDARDIZATION OF
ANTIBIOTICS

9. 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.
METHODS FOR STANDARDIZATION OF
ANTIBIOTICS