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Standard operating Procedure for Subculturing (Cell Passaging) in microbiology and its maintenance. Subculturing also referred to as passaging cells, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.

Subculturing (Cell Passaging)

1.0   Objective :

• • To lay down the procedure to describe step wise procedure for procurement, maintenance and Subculturing (Cell Passaging) of pure cultures to maintain their purity.

2.0   Scope

• • This SOP is applicable for maintenance and preservation of microbial culture and procedure for Subculturing (Cell Passaging) in Microbiology laboratory.

3.0   Procedure for Subculturing (Cell Passaging) :

• • ATCC (American type culture collection) cultures / MTCC (Microbial type culture collection) equivalent Strains shall be procured from authenticated source.

• • Store the cultures at 2-8°C, incubation conditions and media used for Subculturing (Cell Passaging), as per the Table-I instructions.

• • All accessories & Glassware’s should be sterilized and LAF work bench should be neat and clean to prevent contamination.

• • Microbiologist should wear Sterile boiler suit and hand gloves washed with filtered 70% IPA.

• • Disinfect the outer surface of ampoule with 70% IPA and flame.

• • If the mother culture is lyophilized score the middle of the ampoule with an cutter.

• • Disinfectant the ampoule with 70 % v/v IPA solution which is moistened with lint free cloth.

• • Wrap the sterile cotton cloth around the ampoule and break it carefully under aseptic condition pull the cotton wool plug using sterile forceps.

• • Aseptically add about 0.3 ml of sterile saline solution with the help of micropipette and mix well.

• • Transfer a loop full suspension in a duplicate sterile media slant and incubate at a temperature as per table -1, and observe the growth after incubation.

• • Take a loop full new microbial culture in to 10 ml sterile of SCDM Incubate the culture of bacteria at 30-35°C for 24 to 48 hours, culture of mold at 20-25°C for 72-120 hours and culture of yeast at 20-25°C for 48 to 72 hours.

• • Check the purity of culture by staining.

• • If the cultures are not show their physical characteristics as per table -1, discard the cultures as per SOP.

• • After ensuring Purity of mother culture three master culture shall be prepare of each culture by transferring a loop full suspension from mother culture to fresh slant and mark as MC1, MC2, MC3.

• • Note:- Proceed Subculturing (Cell Passaging) of microbial cultures as per the Figure-1. (NMT 5 Subculturing (Cell Passaging) from the Seed Lot cultures).

Figure – 1 – Flow Chart of Subculturing (Cell Passaging)

• • Both the tube shall be incubate at specified temperature and period as per table -1 and observe the growth after Incubation.

• • The slants shall be incubate at specified temperature and period as per table -1.

• • Purity shall be check by staining of the culture.

• • If the cultures are not show their physical characteristics as per table -1, discard the cultures as per SOP.

• • If the cultures show their physical characteristics as per table -1, transfer a loopful growth  from MC1 to a fresh seven set of slant and mark  as M1, M2, M3 , M4, M5, M6, and

• • All slants shall be incubate at specified temperature and period.

• • After incubation growth shall be observe, label the culture and store the culture at 2-8⁰ C for specified period.

• • Cultures shall be used as per flow chart for seed  lot culture maintenance, (Fig- 1).

• • For the 1st month 2^nd month 3^rd month 4^th month 5^th month 6^th month use M1, M2, M3, M4, M5, M6 for preparation of weekly culture. If one of the above culture get contaminated use M7 culture.

• • Transfer a loopful growth from monthly culture i.e. of slant M1, M2, M3 , M4, M5, M6,after ensuring the purity of culture to a five fresh slant and mark as W1, W2, W3, W4, and W5 (control).

• • Record the details of Subculturing (Cell Passaging) and Subculturing (Cell Passaging) number.

• • After each Subculturing (Cell Passaging) of the organism, each tube shall be labeled with following information as per Annexure No VI.

• • For 1^st ,2^nd , 3^rd and 4^th week of every month  use W1,W2 W3 and  W4 cultures respectively.

• • If one of the above culture get contaminated W5 control culture shall be use.

• • Prepare daily working culture by transferring a loopful growth to a fresh four slants and mark  as D1 ,D2, D3 ,D4 ,(Daily culture).

• • All the slants shall be incubate at specified temperature and period.

• • Every year new lyophilized cultures shall be procured as given in the Table-1

• Usage and Destruction of Microbial culture:-

  • For daily microbiological analysis only daily working culture shall be used  .

• • Use one D1 culture for two days ,D2 for next two days ,D3 for next two days and respectively.

• • After completion of work ,the culture slants shall be discard.

• Important:

• • Before and after reviving culture burn the UV light for one hour for surface sterilization.

• • Only one culture should be revived under LAF at a time to avoid cross contamination.

• • All the material used during the revival of culture should be properly sterilized and decontaminate after the completion of the work in an autoclave.

• • Complete and sterile gowning should be taken while working with live cultures.

• • Common microbiological and safety procedures should be followed while handling cultures.

• Frequency of Subculturing (Cell Passaging).

• • First generation- Once in year ± 5 day.

• • Second generation- Once in six month ± 5 day.

• • Third generation- Monthly ± 5 day.

5.0   Reference Document:

• • IP / USP

4.0   Annexure – Subculturing (Cell Passaging):

Annexure I : Reconstitute freeze-dried culture record

Name of Microbial culture      :

Date of reconstituted              :

Streak on agar plate (Media __________________________ Lot No. ____________ ) for purity check and inoculate in 10 ml SCDM,

(Lot no.    __________ ). After streaking /inoculating on slants/broth. Incubate the culture of bacteria at 30-35°C for 24 to 48 hours,

Culture of mold at 20-25°C for 72-120 hours and culture of yeast at 20-25°C for 48 to 72 hours.

Gram staining                         :

Biochemical characterization :

Remark:

Check all the tubes for copious growth after completion of incubation.  Subculture on slants/broth which is previously prepared & pre-

Incubated. Store the slants/broth at 2-8°C temperature.

Quantity of prepared tubes ______________

Remark:

Annexure II : Identification of pure culture

Annexure III : Yearly Subculturing (Cell Passaging) from the Master culture

Annexure IV : Monthly Subculturing (Cell Passaging)

Annexure V : Weekly Subculturing (Cell Passaging)

Annexure VI : Subculturing (Cell Passaging) Label

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