Microbial Culture Management

Microbial CultureA microbial culture (microbiological culture) is a procedure of growing microbial organisms (reproduction) by allowing them to breed in programmed culture medium under controlled laboratory conditions.

Microbial cultures are initial and basic diagnostic methods used as a research tool in molecular biology.

Microbial Culture Management

Standard Operating Procedure (SOP)

1.0   PURPOSE:

    • To lay down the procedure for the management of microbial cultures.

2.0   SCOPE:

    • This Standard Operating Procedure is applicable at Microbiology Department.

3.0   REFERENCES:

    • Articles on Aseptic Technique for Microbiological Testing.
    • SOP for the procurement, transfer, preservation & disposal of microbiological cultures.
    • Disposal of used media and cultures SOP.
    • Isolation and identification of microorganisms.
    • USP/IP.

4.0   RESPONSIBILITY:

    • Officer or Executive of the Microbiology Department shall be responsible for the preparation of new or revision of existing SOPs.
    • Head of the department/designee of respective areas & QA shall be responsible for reviewing the SOPs.
    • Plant Head and Head-Quality shall be responsible for the approval of SOP.
    • QA shall be responsible for the distribution and control of SOPs to various departments.

5.0   ABBREVIATIONS:

    • ATCC     :  American Type Culture Collection
    • CC          :  Change Control
    • °C           :  Degree Celsius
    • IPA         :  Isopropyl alcohol
    • LAF        :  Laminar Air Flow
    • MTCC    :  Microbial Type Culture Collection
    • NA          :  Not Applicable
    • NCTC     :  National Collection of Type Cultures
    • NCYC     :   National Collection Of Yeast Culture
    • SCDA     :  Soya bean Casein Digest Agar
    • SDA        :   Sabouraud Dextrose Agar
    • SOP        :   Standard Operating Procedure
    • v/v          :   Volume by Volume

6.0   PROCEDURE FOR MICROBIAL CULTURING

    • Procurement of Cultures:

      • Prepare the list of ATCC / NCTC / NCYC / MTCC cultures required in the Microbiology section as per the details mentioned in Annexure-1.
      • Raise the purchase requisition for the cultures for procurement.
      • Procure the required cultures once in a year.
      • All cultures shall be procured from the authorized sources with certificate-based on permissible subculturing periods.
      • Ensure that the cultures shall not be more than 2 passages removed from the reference.
      • Upon receipt of the cultures, enter the details along with in house identification no.  in culture inward record as per Annexure-2.
      • Store these cultures in the refrigerator between 2º-8ºC or as per manufacturer recommendation.
      • For Example, E. coli received on 01/10/19 then given in house number should be E.coli 011019.

Also read: SOP for Isolation and Identification of Microorganisms

    • Reconstitution of Freeze-Dried Cultures:

      • Sanitized the surface of ampoule or vial or slant or loops using 70 % IPA.
      • Transfer the ampoule or vial or slant or loops under LAF/Biosafety cabinet and open the culture aseptically.
      • Add 0.5 ml to 1.0 ml of sterile water to the vial/ampoule/ slant to reconstituting the lyophilized / slant cultures or reconstitute the cultures as per the recommendation of the provider.
      • This culture will serve as mother Culture.
      • Record the details in Culture Maintenance Record as per Annexure-3.
    • Revival and Maintenance of Cultures:

      • Streak the mother culture on agar plates for confirmation of purity as per SOP for Isolation and identification of microorganisms (Annexure-4) or
      • Direct by automated identification system and simultaneously inoculate the total content of vial/ ampoule in 100 ml of sterilized Soya bean casein digest medium.
      • After transfer the mother culture in to the medium,
      • Dispose the remaining content and vial as per current version of SOP for Disposal of used media and cultures. and record the details in Annexure-8.
      • Use agar plate for purity check and
      • Use the liquid medium for preparation of Seed lot cultures.
      • Incubate the media containing
        • Bacteria (Cultures of Bacteria) at 32.5 ± 2.5º C for 24-48 hrs,
        • Molds (Cultures of Molds) at 22.5± 2.5º C for 72-120 hrs and
        • Yeasts (Cultures of Yeasts) at 22.5 ± 2.5º C for 24- 48 hrs.
      • Media and incubation conditions shall be followed for different cultures as recommended in Annexure-1.

      • After completion of incubation check the purity as per SOP on Isolation and identification of microorganisms of
        • Culture by colonial characteristics,
        • Microscopic examination,
        • Staining and
        • through automated identification System (BD Phoenix).
      • Add 10% v/v sterile glycerol in culture suspension in 1:1 ratio, mix well and dispense 2-3 ml into  the sterile cryo vial prepare 14 such vials which serves as Seed lot Culture (SLC).
      • Mark culture ID number as SLC-1, SLC-2, and SLC-3 and so on and store the cryo vials (Cryoprotective medium) at -30°C or below until use.
      • Label each cryo vial of SLC with the details like (as per the Annexure-6.)
        • Name of culture,
        • Strain no.,
        • Passage no.,
        • Culture ID.
        • Date of subculturing,
        • Sub cultured by and
        • Use before 
      • Use 12 cryovials of seed lot culture for subculturing up to 12 months (yearly) and Keep 2 cryovials as a stock which shall be used if any vial gets damaged or spillage.
      • Ensure that the cryovials shall not be used after one year.
      • Discard the remaining two cryovials after completion of yearly subculturing as per the current version of SOP on Disposal of used media and cultures.
    • Subculturing: (Microbial Culture)

      • Maintain the cultures as per the Schematic Flow for Subculturing as per Annexure-5.
      • For first-month subculture streak five slants of agar medium from the cryovial of SLC-1 and mark culture ID numbers as
        • 1.0            SLC-1WC-1,
        • 2.0            SLC-1WC-2,
        • 3.0            SLC-1 WC-3,
        • 4.0            SLC-1WC-4, and
        • 5.0            SLC-1WC-5.

Visiters also reading : 

A)       Isolation and Identification of Microorganisms

B)       Microbial Culture Management

      • Simultaneously streak on the plates of agar medium for purity check as per SOP for “Isolation and identification of microorganisms” of culture by
        • Colonial characteristics,
        • Microscopic examination,
        • Staining and
        • through an automated identification System (BD Phoenix).
      • Incubate the slants and plates containing cultures of
        • Bacteria at 32.5±2.5ºC for 24-48 hrs,
        • Molds at 22.5± 2.5ºC for 72-120 hrs. and
        • Yeast at 22.5± 2.5ºC for 24- 48 hrs.
      • When proper growth observed on the transferred slants discard SLC-1 following the current version of SOP on “Disposal of used media and cultures” and record the details in Annexure-8.
      • Label each slant of working culture with the details like
        • Name of culture,
        • Strain no.,
        • Passage no.,
        • Seed lot culture no.,
        • Culture ID.,
        • Date of sub culturing,
        • Sub cultured by and
        • Use before as per Annexure-7 and
        • Store the working cultures at 2 – 8 ºC.
        • Use one working culture for each week for routine lab work for up to one month.

                              Discard the working culture at the end of the week or before using a new working culture.

      • The fifth working culture shall also be discarded at the end of the month if it remains unused and details shall be recorded in Annexure-8.
      • Start the same procedure with SLC-2 and so on, well before completing the cycle of previous SLC to get the working cultures ready to use for next month.
      • Check the purity of seed lot culture and working culture as per SOP on Isolation and identification of microorganisms  at the time of use by
        • Colonial characteristics,
        • Microscopic examination,
        • Staining and
        • Biochemical examination and
        • Record the observations in Annexure-4 or
        • through an automated identification system (BD Phoenix).
      • Record the details of subculturing in Annexure-3 at every step of sub-culturing.
      • Tentative Schedule for Maintenance of Microbial Cultures shall be prepared for subculturing at the time of the first revival of new cultures as per Annexure-9.
      • Ensure that the inoculates used shall not be more than 5 passages removed from the certified reference cultures.
    • Process Description: Live Cultures

      • During working with live cultures always use Gloves.
      • Segregate all live cultures from areas used for sample testing and optimally, handled in a different area of the laboratory within a Biosafety Cabinet.
      • Perform positive control dilutions and inoculation in a biological safety hood/cabinet.
      • Seal Agar plates containing fungal cultures with para-film to prevent spread of spores.
      • Surfaces in areas where a live culture plate, tube, bottle, pellet, etc., was opened shall be sanitized immediately after use by using an approved sanitizer for the appropriate contact time.
    • ANNEXURES:

      • List of Microbial Cultures. (Annexure-1)
      • Microbial Culture Inward Record. (Annexure-2)
      • Culture Maintenance Record. (Annexure-3)
      • Purity Check of Microbial Culture. (Annexure-4)
      • Schematic flow for Sub-Culturing. (Annexure-5)
      • Seed Lot Culture Label. (Annexure-6)
      • Working Culture Label. (Annexure-7)
      • Culture Disposal Record. (Annexure-8)
      • Schedule for Maintenance of Microbial Cultures. (Annexure-9)

Click to read article : Laminar Air flow (LAF) – Operation, Cleaning and Qualification

 

Schematic flow for Sub-Culturing. (Annexure-5)

Schematic Flow for Sub Culturing

Seed Lot Culture Label. (Annexure-6)

Seed Lot Culture

Working Culture Label. (Annexure-7)

working culture

 

Schedule for Maintenance of Microbial Cultures. (Annexure-9)

Schedule

Visiters also reading : 

A)       Isolation and Identification of Microorganisms

B)       Microbial Culture Management

 

pharmabeginers

Niranjan Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an updated pharmaceutical blogging platform. Email: [email protected]

Leave a Reply

Close Menu