Microbial Culture Management8 Archives - Pharmaceutical Guidelines

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)

PURPOSE:

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

SCOPE:

This Standard Operating Procedure is applicable at Microbiology Department.

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 01/10/20 then given in house number should be E.coli 011020.

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.

- 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.