Preservation and Maintenance of Microbial Strain

1.0 Objective
To lay down a procedure for the preservation and maintenance of microbial strains.
2.0 Scope
This Standard Operating Procedure is applicable for the preservation and maintenance of microbial

strains to be followed at formulation plants of abc company.
3.0 Responsibility
3.1 Officer / Executive Quality Control Department are responsible for preservation and maintenance of microbial strains.
3.2 Head Quality control is responsible for the compliance of this SOP.
4.0 Abbreviations and Definitions
SOP : Standard Operating Procedure
QA : Quality Assurance
QC : Quality Control
No. : Number
LAF : Laminar Air Flow
DM : Demineralized
NCIM : National Collection of Industrial Micro organisms
MTCC : Microbial Type Culture Collection
LAF : Laminar Air Flow
I.P.A : Isopropyl Alcohol
% : Percentage
0C : Degree Celsius
ml : Millilitre

5.0 Procedure
5.1 Source: Organisms used for Microbiological analysis should be from recognized institute.
5.2 Approved Vendor:
MTCC, Chandigarh, India.
NCIM, Pune, India.
5.3 Method of Sub culturing by slants
5.3.1 Operate LAF as per SOP for operating instruction for LAF.
5.3.2 Wipe out the outside surface of the culture slant tubes with 70% soaked cotton.
5.3.3 Get fresh slant tube and wipe out outside surface of the tube with cotton wool soaked in 70% I.P.A.
5.3.4 Hold on the extreme end and heat the nichrome wire loop on the reducing flame till red-hot.
5.3.5 Allow to cool the wire loop and slowly open the cotton plug of culture tube.
5.3.6 Pick up the smear/growth with the help of loop after removing cotton plug of the slant tube.
5.3.7 Hold the nichrome wire loop with smear/culture growth, plug the culture slant tube, and put aside

the old slant culture.
5.3.8 Open the fresh slant tube, warm the mouth of the tube holding in other hand and carefully transfer

the culture that is held on the loop.
5.3.9 Spread the culture spirally on the surface of the slant, warm the mouth of slant and plug it.
5.3.10 Label the culture tube, incubate at specified temperature and period, and then store it in refrigerator at 2 to 80C.
5.4 Maintenance of Microbial culture by ampules/lyophilized
5.4.1 If the Mother culture is lyophilized, score the middle of the ampoule with an ampoule cutter.
5.4.2 Disinfect the ampoule with sterile 70% I.P.A solution which is moistened with lint free cotton.
5.4.3 Wrap the sterile cloth around the ampoule and break it carefully.
5.4.4 Aseptically add about 0.2 ml of sterile saline solution with the help of micropipette and mix well.
5.4.5 Transfer the suspension in a sterile SCDM medium and incubate at a temperature and observe the growth

after incubation.
5.4.6 If growth is present than culture is use for culturing otherwise discard the culture.
5.4.7 Prepare three Master Culture of growth culture by transferring a loop full suspension from incubated

SCDM to fresh slant and treat as a MC1, MC2 and MC3.
5.4.8 Incubate the tubes at specified temperature, period and observe the growth after incubation.
5.4.9 If the cultures are not showing growth properly than discard the culture as per SOP.
5.4.10 For six month culture prepare from master culture and transfer a loop full growth from MC1 to a

fresh seven set of slant and treat as M1, M2, M3, M4, M5, M6 and M7 respectively.
5.4.11 Incubate the all the slant at specified temperature and period and assign

batch No. I.e. MC1/M1, MC1/M2, MC1/M3 up to MC1/M7.
5.4.12 After incubation observe the growth and label the culture, store the culture at 2 to 80C for their specific period.
5.4.13 Use the prepared culture as per Schematic diagram specified in SOP.
5.4.14 For the 1st month, 2nd month, 3rd month, 4th month, 5th month and 6th month use M1, M2, M3, M4, M5 and M6 respectively for preparation of weekly cultures. If one of the above culture get contaminated use M7 culture (Stock culture).
5.4.15 After incubation observe the growth and label the culture as per sample given in Annexure-1,

store the culture at 2 to 80C for their specific period.
5.4.16 For 1st, 2nd, 3rd and 4th Week of every month use working and standard cultures respectively.

If one of the above culture get contaminated use W5 culture.
5.4.17 Incubate all the slant at specified temperature & period , observe the growth and labeled properly.

Store the culture at 2 to 80C for their specific period.
5.4.18 After complication of six month, fallow the same procedure for reaming next sixth months.
5.5 Precaution
5.5.1 After transferring the culture, always return the cotton plug or caps to the correct tubes.
5.5.2 Never forget to sterilize the inoculating loop before returning it to the holder.
5.5.3 Always flame the tips of the tube before you insert the loop and after withdraw the tube.
5.5.4 Do not dip the needle into the agar while inoculating slant.
5.5.5 Never leave the culture tube open for longer time than the time needed to transfer the culture.

6.0 Forms and Records
6.1 Format for Culture Preparation and discard Record – Annexure-1
6.2 Label for cultures – Annexure-2
6.3 Culture maintenance – Annexure-3
7.0 Distribution
7.1 Master copy – Documentation Cell (Quality Assurance)
7.2 Control copy – Quality Control
8.0 History

Revision Number	Details For Change	Reason for Revision
00	New SOP	NA

Preservation and Maintenance of Microbial Strain

Reason for Revision

Leave a Comment Cancel reply