sop for microbiological assay of erythromycin antibiotic

0.0 OBJECTIVE
To describe the procedure for determine the assay of Erythromycin Antibiotic by biological method using Bacillus pumilus MTCC-1607 or NCIM-2327
1.0 PURPOSE
It is the policy of Analytical Division that a written procedure shall be followed for determine the assay of Erythromycin Antibiotic by biological method using Bacillus pumilus MTCC-1607 or NCIM-2327
2.0 SCOPE
This SOP shall be applicable for in Microbiology Laboratory
3.0 RESPONSIBILITY
Persons along with their responsibilities are given below:
S. No Designation Responsibility
01 Microbiologist Preparation & revision of SOP.
02 QA Officer Distribution and retrieval of SOP.
03 Tech. Manager (Biological) Training & Implementation of SOP.
04 Quality Manager Approval & Implementation of SOP.
4.0 PROCEDURE
4.1 METHOD: Cylinder Plate Method.
4.2 ASSAY MEDIUM: Antibiotic assay No.:11 Agar Media.
4.3 INOCULUMS MEDIUM : Phosphate Buffer
KH2PO4 – 0.523 g
K2HPO4 – 16.73g
Water – 1000 ml
pH – 8.4 ± 0.1
Dispense in Flask, Autoclave, Cool & store at 4°C
4.4 PREPARATION OF CULTURE SUSPENSION
Inoculate loop full culture in 10 ml of Soyabean casein digest medium and incubate at 30-35°C for 24 hours.
4.5 PREPARATION OF ASSAY PLATES
Antibiotic assay No.-11 is sterilized in Validated autoclave cycle and cooled to 40-45°C, Add 2-3 ml of inoculum of Bacillus pumilus MTCC-1607 in 100 ml Antibiotic assay No.-11 Agar media. Mix gently and thoroughly. Distribute 25 to 30 ml of the inoculated medium in sterile petri plates. Allow to solidify the medium.
4.6 PREPARATION OF WORKING STANDARD DILUTIONS
Weigh accurately 100 mg eq. of Antibiotic Erythromycin Working Standard. Transfer it to 10 ml volumetric flask and make up the volume to 10 ml with methanol. Take 2.5 ml and dilute it to 100 ml (A) with phosphate buffer. Take 5 ml (A) and dilute it to 20 ml (B) with phosphate buffer.
4.7 PREPARATION OF SAMPLE DILUTIONS
Take Sample quantity which shall be eq. to 100 mg of Erythromycin. Transfer it to 10 ml volumetric flask and make up the volume to 10 ml with methanol. Take 2.5 ml and dilute it to 100 ml (A) with phosphate buffer. Take 5 ml (A) and dilute it to 20 ml (B) with phosphate buffer.
4.8 PREPARATION & INCUBATION OF PLATE
After set the medium make 4 wells in the four agar plate by 8.0 mm diameter borer at proper distance to avoid overlapping of the zones. Add 0.1 ml of the standard and test dilutions into opposite wells. Allow pre- diffusion for 30 min and incubate the plates at 30-37°C for 18-24 hours.

4.9 MEASUREMENT OF DIAMETER
Measure the diameter of every zone of growth. Note down every reading.

4.10 CALCULATIONS
Sum up readings of individual dilution.
Formula – Percent potency (%P) = Antilog (2 + a log I)
Where,
(TH + TL) – (SH + SL)
a = ———————————-
(TH – TL) + (SH – SL)
Where,
TH – Sum of Test Higher Dilution
TL – Sum of Test Lower Dilution
SH – Sum of Standard Higher Dilution
SL – Sum of Standard Lower Dilution
Where, log I = ratio of dilution i.e. 1:4 therefore log 4 = 0.6020

Assay = % P x Assumed potency of the sample
100

5.0 ACCEPTANCE LIMIT: Not less than 90 percent or accordingly to individual monograph.

6.0 ABBREVIATIONS & DEFINITION
MPL Multani Pharmaceuticals Limited (Analytical Division)
QA Quality Assurance
SOP Standard Operating Procedure
QM Quality Microbiology
S. No. Serial Number
ml Milliliter
°C Degree centigrade
min Minutes
MTCC Microbial culture collection bank
NCIM National Collection of Industrial Microorganisms

Plate No.	Zone of inhibition in mm
Plate No.	TH	TL	SH	SL
1
2
3
4
SUM

sop for microbiological assay of erythromycin antibiotic

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