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SOP for Procurement, Confirmation and Maintenance Procedure of Microbial Cultures

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

SCOPE
This SOP is applicable to all microbial cultures used in the Microbiology laboratory at PharmaSky Limited.

RESPONSIBILITY
  • Microbiologist – Responsible for procurement, confirmation and maintenance of microbial cultures.
  • Section In-charge – Responsible for review the procedure and relevant documents.
  • QC Head- Responsible for implementing the procedure.
  • Head QA-System Compliance

PROCEDURE
Procurement and Confirmation of Master Cultures:
  • All master cultures shall be procured from any one of the following authorized national culture collection institutes.
  1. Institute of microbial technology (IMTECH), Chandigarh.
  2. National Collection of Industrial Microorganisms (NCIM), Pune.
  3. American Type Culture Collection (ATCC), USA.
  • The master cultures shall be received in the form of slants / lyophilized.
  • On receipt of the microbial cultures ensure that the Certificate of Analysis is received along with each set of microbial cultures and verify the culture details like ATCC/NCTC/NCIM numbers, Expiry date, and storage condition in COA with that the label information on cultures. If the details are matching then receive the cultures, label the received cultures with the label mentioned in annexure VI, and enter the details in annexure I (Master Culture inward record).
  • Store all the received microbial cultures at 2ºC to 8ºC in the refrigerator.
  • The Identification and Confirmation of master cultures shall be done by the following tests.
  • Lyophilized cultures are revived as per the manufacturer’s instructions mentioned in Annexure VIII (Manufacturer Instructions for a revival of Lyophilized Cultures).
  • 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 is used for Identification tests and another plate is used for sub-culturing purposes.


  • 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 is used for Identification tests and another plate is used for sub-culturing purposes.
  • After completion of the 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.
  • Biochemical tests and Staining techniques are performed as per Annexure-II and a report is generated for each culture as per Annexure-III.
  • After confirmation of master culture’s identification tests proceeds for Subculturing.

Preparation of Slants:
  • Prepare Nutrient Agar and Potato Dextrose Agar/Sabouraud Dextrose Agar Slants by using test tubes of size 25 X 150mm.
  • Prepare the required quantity of media as per the current version of SOP for Culture Media Preparation.
  • Boil the media and distribute 20 ml of media into each test tube. Sterilize all the test tubes at 121ºC for 15 minutes.
  • For the preparation of slants, after sterilization keeps all the tubes in a slanting position for solidification on the glass rod under the Laminar Air Flow unit.
  • 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).
  • After confirming the sterility of the slants use them for sub culturing.

Sub-culturing and Maintenance of Microbial Cultures:
  • Take the sub-cultured plates of all cultures from the refrigerator and allow them to attain room temperature under a laminar airflow cabinet.
  • Label the freshly prepared sterile slants as Stock Culture, Working Culture and Identification Culture.
  • Subculture by taking a loopful of an 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).


  • For sub-culturing and routine analysis use dedicated inoculation loops for each organism.
  • Label the first slant as Stock culture, and second slant as Identification culture, and the remaining five slants as Working Culture-I to Working Culture-V.
  • Use stock culture for sub-culturing of weekly cultures for next month.
  • 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.
  • Working cultures shall be used for routine analysis and for culture suspension preparation. Use working culture-I for the first week, working culture –II for the second week, working culture – III for the third week, working culture – IV for the fourth week and working culture – V for the fifth week (if applicable).
  • Before the expiration of the validity of the working cultures, the next set of stock, identification,    and working cultures shall be subcultured from the previous stock cultures.
  • 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 the identification culture shall be valid until the completion of identification tests.
  • Once the next passage Stock, Identification, and Working cultures are released the previous passage cultures shall be discarded as per the procedure described in the current version of SOP for Destruction of Culture Media.
  • The above procedure will be followed for remaining all micro-organisms.
  • Enter all the Subculturing details in Annexure IX (Subculturing Record).
  • The above procedure is considered as one passage and not more than four passages shall be performed.
  • Only five passages shall be made from the original strain.
  • The procured master culture is considered as first passage.
  • Before completion of fifth passage, new cultures are to be procured.
  • A flow chart for preparation of passages is given in Annexure-IV.
  • Assign the lot number for each culture as per following manner.


For Master Culture
LXX-01/YY/Pn

L: Passage Number
XX: Mother culture Number
01: Serial number which starts from 01
YY: Last Two digits of the current year
Pn: Lyophilized culture

For Stock Culture
LXX-01/YY/Pn

L: Passage Number
XX: Stock culture Number
01: Serial number which starts from 01
YY: Last Two digits of the current year
Pn: Lyophilized culture

For Identification Culture
LXX-01/YY/Pn

L: Passage Number
XX: Stock culture Number
01: Serial number which starts from 01
YY: Last Two digits of the current year
Pn: Lyophilized culture

For Working Culture
LXX-01/YY/Pn

L: Passage Number
XX: Stock culture Number
01: Serial number which starts from 01
YY: Last Two digits of the current year
Pn: Lyophilized culture

  • Each Subcultured slant shall be labeled with the label mentioned in Annexure VII.
  • Master culture shall be stored till the completion of the fifth passage of working cultures.
  • Master cultures and all its slants shall be stored at 2ºC to 8ºC.


Precautions
  • Handle the microbial cultures carefully as they are biohazardous.
  • Cultures are used in the form of slants, liquid cultures and viable spores.
  • Always use sterile gloves, nose masks and goggles while handling cultures.
  • If any accidental spillage occurs, disinfect the area and clean the surfaces with 70% IPA.

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

ANNEXURE
Annexure I - Master Culture inward record
Annexure II - Biochemical tests and Staining techniques
Annexure III - Biochemical tests and Staining techniques Report
Annexure IV - A flow chart for the preparation of passages
Annexure V - Temperature and time of incubation for maintenance of cultures
Annexure VI - Label for the received cultures
Annexure VII - Label for the Subcultured slant
Annexure VIII - Manufacturer Instructions for a revival of Lyophilized Cultures
Annexure IX - Subculturing Record


REVISION HISTORY
Nil

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