Culture Maintenance:-

Culture maintenance refers to the practices and procedures used to sustain and preserve living microbial cultures in laboratory settings. Proper culture maintenance is essential for ensuring the viability, purity, and genetic stability of microbial cultures over time.

 

DEFINITION:

ASEPTIC TECHNIQUE:

Aseptic technique refers to the procedure used to avoid the introduction of microorganisms into the vulnerable site. The principle aim of an aseptic technique is to protect the patient/articles/materials from contamination by pathogenic organisms.

SEED LOT TECHNIQUE:

Seed Lot technique is used to provide a method for generation of a working culture no more than 5 passages removed from the original stock from the national repository. To provide a method for generation of a pure culture for laboratory work. To provide a method for generation of a traceable culture for laboratory investigations

REFERENCE STRAINS

Microorganisms defined at least to the genus and species level, catalog and described according to its characteristics and preferably stating its origin. Normally obtained from a recognized national or international collection center

WORKING CULTURE

A primary subculture from a reference stock used as a reference in Microbiological analysis.

BIOLOGICAL SAFETY CABINET (BSC):

A ventilated cabinet with unidirectional HEPA-filtered air flow and HEPA-filtered exhaust to protect the worker from hazardous drugs. A BSC used to perform an activity must be capable of providing an ISO Class 5 environment for the activity.

PASSAGE:

Transfer of organisms from a viable culture to a fresh medium with growth of the microorganisms. Any form of sub culturing is considered to be transfer/passage.

1.0 OBJECTIVE:

To lay down the procedure for Reviving & maintenance of microbial cultures.

2.0   SCOPE:

The scope of this document is to describe the procedure for maintenance and enumeration of standard Microbial cultures used for various microbiological purposes.

3.0   RESPONSIBILITY:

A.     Microbiologist

I.       To perform the activities as per procedure.

II.      To report any non compliance to the procedure

III.     To prepare and maintain a schedule of the activity as per procedure

B.     Head Microbiology/Designee

I.      To ensure the activities are performed as per procedure

II.     To ensure the activities are performed as per the laid down schedules.

III.    To investigate any non compliance to the procedure

4.0   ACCOUNTABILITY :

A. Head Quality Control & Head Quality Assurance

 I.      To ensure the procedure is implemented at their site

II.      To verify investigation of any non compliance to the procedure.

5.0  PROCEDURE:

A. Procedure for Reviving  Freeze Dried Cultures/lyophilized Ampoules

1. Remove freeze dried cultures/lyophilized ampoule from refrigerator and allow the temperature of cultures to reach at room temperature.

2. Mention  the details in the receipt and reconciliation record

3. Revive the freeze dried cultures as per manufacturer’s instructions. If instructions are not available, follow the below stated general procedure.

4. Prepare 100 mL tubes of Sterile Soybean Casein Digest Medium + 10% glycerol for revival of all cultures.

5. Disinfect the Bio-Safety Cabinet (Culture handling room) bench with filtered 70%v/v  Isopropyl alcohol.(IPA+ Water mixture)

6. Sanitize the ampoule and cutter by spraying filtered 70% v/v IPA and score the middle of the ampoule with  sterilized/sanitized ampoule cutter.

7. Wrap the ampoule with thick sterilized cotton to cover the base of the ampoule and break it carefully at the marked area.

8. Gently remove the pointed top of the ampoule. Snap opening will draw the cotton plug  to one end, hasty opening will release fine particles of dried organisms into the air of the laboratory.

9. Aseptically remove the cotton plug and transfer culture bead to sterile medium or add about 0.3 to 0.4 mL of the sterile SCDM+10% glycerol with sterile pipette/micro pipette. Avoid frothing or creating aerosols.

10. Mix well and transfer the cell suspension of each organism to 100 mL of sterile SCDM  in the test tube.

11. Ensure that the ampoule and the cotton wool is disposed off in an appropriate disinfectant and thereafter  follow the procedure for discard of cultures preferably by Autoclaving at 121ºC for NLT 30 min. or as per validated period of time..

B. Procedure for Reviving of cultures received in the form of slants

1. Remove the culture received in the form of slants from refrigerator and allow the temperature of cultures to reach at room temperature.

2. Enter the details in the receipt and reconciliation record

3. Revive the cultures as per manufacturer’s instructions. If instructions are not available, follow the below stated general procedure.

4. Prepare 100 mL tubes of Sterile Soybean Casein Digest Medium+ 10% glycerol for revival of all cultures.

5. Disinfect the LAF/UAF/BSC (Culture handling room) bench with filtered 70%v/v  Isopropyl alcohol.(IPA+ Water mixture)

6. With the help of sterilized inoculation loop remove one loop full of culture from the slant and inoculate in 100 mL of sterilized SCDM+ glycerol in the test tube.

7. Sterilize the inoculation loop properly or if pre sterilized inoculation loops are use dispose off in an appropriate disinfectant and thereafter  follow the procedure for discard of cultures preferably by Autoclaving at 121ºC for NLT 30 min. or as per validated period of time.

C. Procedure for Reviving of cultures received in the form of culture sticks

1. Remove the culture received in the form of culture sticks from refrigerator and allow the temperature of cultures to reach at room temperature.

2. Enter the details in the receipt and reconciliation record

3. Revive the cultures as per manufacturer’s instructions. If instructions are not available, follow the below stated general procedure.

4. Prepare 100 mL tubes of Sterile Soybean Casein Digest Medium+10% glycerol for revival of all cultures.

5. Disinfect the LAF/UAF/BSC (Culture handling room) bench with filtered 70%v/v  Isopropyl alcohol.(IPA+ Water mixture)

6. Remove the stick and inoculate the stick in 100 mL of sterilized SCDM +10% glycerol in the test tube. Rotate the stick manually in the medium.

7. Dispose off the stick  in an appropriate disinfectant and thereafter  follow the procedure for discard of cultures preferably by Autoclaving at 121ºC for NLT 30 min. or as per validated period of time.

D. Incubation and Observation:

1. 1. Incubate the inoculated SCDM+10% glycerol test tube as per the conditions mentioned in Table 1

   2. During the incubation period, check the enriched tubes for any growth in terms of turbidity for bacterial and yeast culture and growth of mycelium in case of fungal/mold culture at an interval minimum of 24 hours.

   3. If growth is observed during the 24 hours observation in the enriched tubes, proceed for the next stage of Purity check and culture transfer.

   4. If growth is not observed within the stipulated time period as mentioned in the Table 1 then proceed for revival of culture from the next  procured set.

A. Maintenance of Cultures:

1. Use 24-48 hours pre incubated media slants/ plates for sub-culturing of cultures.

2. Label all the culture containers (slants, Cryo vials etc.)  with details like the name of the organism, ATCC No etc., Passage No., date of preparation, Use before date and prepared by.

E. Preparation of master cultures:

1.   Two methods can be followed of preparing and maintaining master     cultures:

2.    Method A- Conventional Method

3.     Method B- Seed lot technique

Flow Diagram of Sub culture

F.    METHOD A (Conventional Method)

1. From each of the revived culture suspension (Mother culture), streak aseptically a loop full of microorganisms on to the respective sterile, pre incubated, pre labelled slants. These slants shall be named as Master cultures i.e. M1, M2, M3, M4, M5, M6, M7 and so on till M 12.

2. Prepare a few pre determined number of extra master cultures (preferably 3) for use in case of contingency (i.e. The extra master cultures shall be labelled as M 13, M14 and M 15) and shall be used only in case of urgencies.

3. Simultaneously streak one sterile, pre incubated, pre labelled SCDA media plate from each re-hydrated culture for purity test. Streak fungal culture on sterile, pre incubated, pre labelled SDA media plate

4. Label each container with details

5. Keep one blank slant for negative control and Incubate the slants at appropriate temperature as specified in Table

G. METHOD B (seed lot technique)

1. Revive the 6 cultures as mentioned in Pt. No. A 1 to 6
2. The remaining amount of revived culture suspension(Mother culture) shall be used for preparation of Cryo vials.
3. Prepare defined numbers of Cryo vials containing 4.0mL suspension from the revived culture suspension(Mother culture) each and store vials at below -30º C. These cryo vials shall be  treated as Master Cultures and shall be labeled as M1, M2, M3, M4, M5, M6, M7 and so on.
4. Prepare one master culture cryo vial for 1 month, depending on the total period as  defined in the site specific SOP.
5. Usually master cultures prepared by seed lot technique shall be used for 2 years (24 months), provided they are maintained below -30º C.
6. Label each cryo vial with details as mentioned
7. Follow the schedule of culture transfer and record the data of transfer and other details in Format
8.  Purity check of the revived culture suspension(Mother culture) shall be confirmed prior to dispensing into the cryo vials.
9. Till the confirmation of purity the Mother culture shall be stored at  2-8ºC.