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A 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

• • • • Biochemical characterization or

• • • • 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

• • • • Biochemical characterization or

• • • • 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)

Seed Lot Culture Label. (Annexure-6)

Working Culture Label. (Annexure-7)

 

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

Visiters also reading : 

A)       Isolation and Identification of Microorganisms

B)       Microbial Culture Management