STANDARD OPERATING PROCEDURE

Topic Preparation Of Culture Suspension SOP No. EQC029

Revision
Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

Effective Date

Name Title Date

Prepared By

Reviewed By

Approved By

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 STANDARD OPERATING PROCEDURE
Topic Preparation Of Culture Suspension SOP No. EQC029
Revision
Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

REVISION SUMMARY

Revision
Effective Date Reason for Revision
Number
1. First issue (Supersedes SOPMB044-07).
2. Periodic Review.
00 3. Note is included that the volume of culture suspension
preparation shall be changed based on the
requirement .

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 STANDARD OPERATING PROCEDURE
Topic Preparation Of Culture Suspension SOP No. EQC029
Revision
Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

1.0 PURPOSE
To describe a Standard Operating Procedure for the preparation of culture suspension.
2.0 SCOPE
This procedure is limited to the preparation of culture suspension from weekly cultures
In Microbiology, APL, UNIT-V.
3.0 RESPONSIBILITY
Assistants and Executives shall be responsible for culture suspension preparation.
4.0 ACCOUNTABILITY
Head-Microbiology is accountable for compliance of the SOP.
5.0 PROCEDURE
5.1 Requirements
5.1.1 Pure culture slants
5.1.2 Micropipettes
5.1.3 Sterile pipette tips
5.1.4 Sterile Petri plates
5.1.5 Inoculation loop
5.1.6 Sterile 0.9% Saline
5.1.7 Vortex mixer
5.2 Take out the culture suspension bottle from the refrigerator stored at 2-8°C.
5.3 Using a sterile cotton swab / sterilized loop gently scrap the slant to suspend the
culture in 10mL sterile 0.9% saline and vortex properly (Stock Solution).
5.4 Keep back the culture slants in refrigerator at 2-8°C immediately.
5.5 Label the sterile 0.9% saline bottles with name of organism and dilution number and
perform serial dilution as below.
Vortex the suspension at each dilution steps.
1 ml from stock + 99 mL of saline 100 mL (10-2 dilution)
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 STANDARD OPERATING PROCEDURE
Topic Preparation Of Culture Suspension SOP No. EQC029
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Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

10 mL from 10 -2 dilution + 90 mL of saline 100 mL (10 -3 dilution)

10 mL from 10 -3 dilution + 90 mL of saline 100 mL (10 -4 dilution)

Perform the dilutions in the

Same manner up to required dilution
Note : The volume of culture suspension required shall be changed based on the
requirement .
5.6 After dilutions are prepared, take 1 mL from each dilution into sterile Petri plates in
duplicate and pour the agar medium as per Format No.EQC029-F02 Swirl the plates
properly.
Note : Ensure that the temperature of the agar media is between 40-45°C.
5.7 Store all the bottles containing culture suspension at 2-8°C immediately.
5.8 After solidification, incubate the plates in inverted position for specific period of
time at specified temperatures as per Format No.EQC029-F02.
5.9 On completion of incubation period, count the number of colonies as per
SOP EQC072 “Operation of colony counter & Reading of Petri plates” and record the
results in Format No. EQC029-F01.
5.10 Identify the dilution which gives 10-100 CFU/mL.
5.11 In case the desired number of CFU is not achieved, dilute further to get the 10-100
Cfu/mL
5.12 Store two dilutions preceding to 10-100 CFU/mL dilution which gives approximately
1000-10000CFU/mL and 10000-100000 CFU/mL for regular testing.
5.13 Rational for selecting the In house Isolates for routine testing :
5.13.1 Selection of in-house isolates for routine usage shall be done based on the
In-house isolates list. List shall be prepared once in a quarter.
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 STANDARD OPERATING PROCEDURE
Topic Preparation Of Culture Suspension SOP No. EQC029
Revision
Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

5.13.2 Most frequently found isolates shall be selected for the first quarter for
routine testing.
5.13.3 If the isolate is already selected and used in the first quarter and the same
isolate again repeating as the most frequently occurring in next quarter, the
second most frequently occurred isolate shall be selected next for routine
usage.
5.13.4 For third and fourth quarter isolate shall be selected as first & Second quarter.
5.13.5 Frequency: In-house isolates shall be rotated on Quarterly basis.
5.13.6 Record the culture details in culture suspension preparation record with Name
of the isolate and ATCC number.
5.14 Label the selected dilution with the following details:
Name of the Organism :
ATCC No. :
Valid Up to :
Approx.No of CFU/mL :
Prepared on / by :
5.15 The selected dilutions shall be stored at 2-80C and use for three days from the date of
release.
5.16 Enter the details in format EQC029-F01.

6.0 LIST OF DOCUMENTS & RECORDS:

EQC029-F01- Record for Culture Suspension Preparation.
EQC029-F02- Preparation of Culture Suspension.

7.0 REFERENCES
USP & EP Pharmacopoeias.

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 STANDARD OPERATING PROCEDURE
Topic Preparation Of Culture Suspension SOP No. EQC029
Revision
Department Quality Control 00
Number
Area Microbiology Regulatory Area APL UNIT-V Review Date

8.0 CROSS REFERENCES

ZQA001- SOP on SOP/Guideline
EQC072:Operation of colony counter and reading of petriplates.
9.0 ABBREVIATION

Abbreviation Full form

SOP Standard Operating Procedure
MB Microbiology
ATCC American type culture collection
CFU Colony forming unit

END OF DOCUMENT

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 AUROBINDO PHARMA LIMITED
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MICROBIOLOGY
RECORD FOR CULTURE SUSPENSION PREPARATION

AR No: Name of the Diluent: Diluent Wt. No :

Media Weight No. (SCDA) Media Weight No. (SDA) Tested By (Sign/Date)

Dilution → Dilution
S. Used s To be
Name of the organism
No Culture T D Avg T D Avg T D Avg T D Avg T D Avg T D Avg T D Avg stored
Slant ↓
Staphylococcus aureus
1.
ATCC 6538
Pseudomonas
2. aeruginosa
ATCC 9027
Escherichia coli
3. ATCC 8739
Salmonella Sps.
4.
ATCC 14028
Clostridium sporogenes
5.
ATCC 11437
Bacillus subtilis
6.
ATCC 6633
Aspergillus brasiliensis
7.
ATCC 16404
Candida albicans
8. ATCC 10231
EM Isolate
9.
Water Isolate
10

Legends:SCDA – Soyabean Casein Digest Agar, SDA – Sabouraud Dextrose Agar,T - Test, D - Duplicate, Avg – Average, TNTC: Too numerous To Count, EM – Environmental Isolate
Remarks: Valid up to______________________

Reported by Checked by
Sign
Date
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MICROBIOLOGY
PREPARATION OF CULTURE SUSPENSION

Incubation conditions
S. No Name of the organism Diluent Name Name of the media
Temp.ºC Time

Staphylococcus aureus
1 0.9% Saline Soybean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 6538
Pseudomonas aeruginosa
2 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 9027
Clostridium sporogenes
3 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 11437
Escherichia coli
4 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 8739

Salmonella sps.
5 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 14028

Bacillus subtilis
6 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days
ATCC 6633

Aspergillus brasiliensis
7 0.9% Saline Sabourouds dextrose agar 22.5  2.5ºC For ≤ 5 days
ATCC 16404

Candida albicans
8 0.9% Saline Sabourouds dextrose agar 22.5  2.5ºC For ≤ 5 days
ATCC 10231

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MICROBIOLOGY
PREPARATION OF CULTURE SUSPENSION

S. No Name of the organism Diluent Name Name of the media Incubation conditions
Temp.ºC Time

9 EM-Isolates 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days

10 Water Isolate 0.9% Saline Soyabean casein digest agar 32.5  2.5ºC For ≤ 3days

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Format No.: EQC029-F02-00-010116