Subculturing (Cell Passaging) in Microbiology Lab

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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