STANDARD OPERATING PROCEDURE Procurement, Confirmation and Maintenance Procedure of Microbial Cultures

	STANDARD OPERATING PROCEDURE
	Department: Quality Control	SOP No.: QC/MB/xxxx
Procurement, Confirmation and Maintenance Procedure of Microbial Cultures		Effective Date:
		Review Date:
		Page No.:

1.0            OBJECTIVE:

1.1                 To lay down a procedure for Procurement, Confirmation and Maintenance of            Microbial Cultures.

2.0            SCOPE:

2.1                     This SOP is applicable to all microbial cultures used in Microbiology laboratory at  Pharma Private Limited.

3.0            RESPONSIBILITY:

3.1                   Microbiologist – Responsible for procurement, confirmation and maintenance of microbial cultures.

3.2                 Section In-charge – Responsible for review the procedure and relevant documents.

3.3                 QC Head- Responsible for implementing the procedure.

3.4                 Head QA-System Compliance

4.0            PROCEDURE:

4.1                 Procurement and Confirmation of Master Cultures:

4.1.1             All master cultures shall be procured from any one of the following authorized national culture collection institutes.

4.1.1.1       Institute of microbial technology (IMTECH), Chandigarh.

4.1.1.2       National Collection of Industrial Microorganisms (NCIM), Pune.

4.1.1.3       American Type Culture Collection (ATCC), USA.

4.1.2             The master cultures shall be received in the form of slants / lyophilized.

4.1.3             On receipt of the microbial cultures ensure that the Certificate of Analysis is received along with the each set of microbial culture and verify the culture details like ATCC/NCTC/NCIM numbers, Expiry date, storage condition  in COA with that the label information on cultures. If details are matching then receive the cultures, label the received cultures with label mentioned in annexure VI and enter the details in annexure I (Master Culture inward record).

4.1.4             Store all the received microbial cultures at 2ºC to 8ºC in refrigerator.

4.1.5             The Identification and Confirmation of master cultures shall be done by the following tests.

4.1.5.1       Lyophilized cultures are revived as per manufacturer’s instructions mentioned in                 Annexure VIII (Manufacturer Instructions for revival of Lyophilized Cultures).

4.1.5.2       For Bacterial cultures, streak reconstituted culture onto the surface of two sterile Soyabean casein digest agar plates and incubate bacterial culture at 30ºC to 35ºC for 24 hours. One plate used for Identification tests and another plate used for sub culturing purpose.

4.1.5.3       For fungal cultures, streak reconstituted culture onto the surface of two sterile           Sabouraud dextrose agar plates and incubate at 20ºC to 25ºC for 72 hours. One plate used for Identification tests and another plate used for sub culturing purpose.

4.1.5.4       After completion of incubation period perform the Identification and confirmation tests    that include Biochemical Tests and Staining Techniques from one plate and store another plate in a refrigerator at 2°C to 8°C.

4.1.5.5       Biochemical tests and Staining techniques are performed as per Annexure-II and a report is generated for each culture as per Annexure-III.

4.1.5.6       After confirmation of master culture’s identification tests proceed for Sub culturing.

4.2                 Preparation of Slants:

4.2.1             Prepare Nutrient Agar and Potato Dextrose Agar/Sabouraud Dextrose Agar Slants by     using test tubes of size 25 X 150mm.

4.2.2             Prepare the required quantity of media as per current version of Media Preparation SOP QC/MB/021.

4.2.3             Boil the media and distribute 20ml of media into each test tube. Sterilize all the test tubes at 121ºC for 15 minutes.

4.2.4             For preparation of slants, after sterilization keep all the tubes in slanting position for solidification on glass rod under Laminar Air Flow unit.

4.2.5             After solidification incubate the Nutrient agar slants at 30ºC to 35ºC for 24 hours and Sabouraud Dextrose Slants/Potato Dextrose Slants at 20ºC to 25ºC for 48 hours to check the sterility of the slants (pre-incubation).

4.2.6             After confirming the sterility of the slants use them for sub culturing.

4.3                 Sub culturing and Maintenance of Microbial Cultures:

4.3.1             Take the sub cultured plates of all cultures from refrigerator and allow them to attain room temperature under laminar air flow cabinet.

4.3.2             Label the freshly prepared sterile slants as Stock Culture, Working Cultures and Identification Culture.

4.3.3             Subculture by taking a loopful of isolated colony from revived petri plate and streak on seven slants and incubate all slants as per annexure V (Temperature and time of incubation for maintenance of cultures).

4.3.4             For sub culturing and routine analysis use dedicated inoculation loops for each organism.

4.3.5             Label first slant as Stock culture, and second slant as Identification culture and remaining five slants as Working Culture-I to Working Culture-V.

4.3.6             Use stock culture for sub culturing of weekly cultures for next month.

4.3.7             Use identification culture slant for biochemical tests and staining techniques,   identification should be performed for every passage before working cultures are used for routine analysis.

4.3.8             Working cultures shall be used for routine analysis and for culture suspension    preparation. Use working culture-I for first week , working culture –II for second week, working culture – III for third week , working culture – IV for fourth week and working culture – V for fifth week (if applicable).

4.3.9             Before expiration of validity of the working cultures, the next set of stock, identification    and working cultures shall be sub cultured from previous stock culture.

4.3.10         The validity of the stock cultures shall be one month from the date of release and each     working culture shall be 7 days from the day of usage and identification culture shall be valid until the completion of identification tests.

4.3.11         Once the next passage Stock, Identification and Working cultures are released the previous passage cultures shall be discarded as per the procedure described in current version of SOP QC/MB/xx (Disposal of used media and cultures).

4.3.12         The above procedure will be followed for remaining all micro-organisms.

4.3.13         Enter all the Subculturing details in Annexure IX (Subculturing Record).

4.3.14         The above procedure is considered as one passage and not more than four passages shall be performed.

4.3.15         Only five passages shall be made from the original strain.

4.3.16         The procured master culture is considered as first passage.

4.3.17         Before completion of fifth passage, new cultures are to be procured.

4.3.18         A flow chart for preparation of passages is given in Annexure-IV.

4.3.19         Assign the lot number for each culture as per following manner.

4.3.19.1   For Master Culture

                       LXX-01/M1/Pn

                                                              Passage Number

                                                 Mother culture Number

                                                 Serial number which starts from 01

                                                Last Two digits of current year

                                              Lyophilized culture

4.3.19.2   For Stock Culture:

                      LXX-01/SC1/Pn

                                                                Passage Number

                                                   Stock culture Number

                                                   Serial number which starts from 01

                                                                                                                        Last two digits of Current Year

                                                  Lyophilized culture

4.3.19.3   For Identification Culture:

                       LXX-01/IC1/Pn

                                                                 Passage Number

                                                   Identification culture Number

                                                   Serial number which starts from 01

                                                   Last two digits of current year

                                                    Lyophilized culture

4.3.19.4   For Working Culture:

                       LXX-01/WC1/Pn

                                                                    Passage Number

                                                       Working culture Number

                                                       Serial number which starts from 01

                                                      Last two digits of current year

                                                       Lyophilized culture

4.3.20         Each Sub-cultured slant shall be labelled with the label mentioned in Annexure VII.

4.3.21         Master culture shall be stored till the completion of fifth passage of working cultures.

4.3.22         Master cultures and all its slants shall be stored at 2ºC to 8ºC.

4.4                 Precautions:

4.4.1             Handle the microbial cultures carefully as they are bio hazardous.

4.4.2             Cultures are used in the form of slants, liquid cultures and viable spores.

4.4.3             Always use sterile gloves, nose masks and goggles while handling cultures.

4.4.4             If any accidental spillage occurs, disinfect the area and clean the surfaces with 70%       IPA.

5.0            ABBREVIATIONS:

IPA: Iso Propyl Alcohol	QA: Quality Assurance
SOP: Standard Operating Procedure	ml: Milli liter
mm: Milli meter	cm: Centi mete
QC: Quality Control	°C: Degree centigrade

7.0            REFERENCES:

7.1                 European Pharmacopeia chapter 2.6.12 & 2.6.13 (Microbiological Examination of non-sterile Products: Microbial Enumeration test and Test for specified microorganisms).

7.2                 United States Pharmacopeia Chapter <61> &<62> (Microbiological Examination of non-sterile Products: Microbial Enumeration test and Test for specified microorganisms).

7.3                 United States Pharmacopeia Chapter <1117> (Best Microbiology Lab Practices).