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Tuesday, August 11, 2009


SOP for Handeling microbial culturs and method of subculturing 


Cultures of microorganisms are frequently used in microbiological laboratory for

routine microbiological work It is important that microorganisms in frequent use

do not become genetically altered as a result of sequential transfer and do not get

contaminated due to improper aseptic techniques A safe handling of all microorganisms

including non pathogens is of prime importance as all microorganisms may cause

certain amount of health and environment hazards

UNIT OF RESPONSIBILITY:- QA Executive and trained QA Assistants



The type of culture handled in the microbiology lab are in the form of slants, stabs,

liquid culture and viable spores

1 The personnel handling the culture must protect themselves with suitable

means such as use of gloves and mask and any spillage of accidental

contamination that may occur during the handling must be taken care of, properly

2 The gloves and the mask should be removed and disinfected using a

disinfectant and or by sterilizing by autoclaving

3 The hand or any other portion of the body exposed to contamination must

be washed thoroughly with soap water and disinfected immediately


1 The culture recieved from the source like FDA, or any other authentic source

should be labelled as mother culture and suitably batch numbered and stored

in a cool condition at a temperature of 8-15 deg C for not more than six months

2 Mother culture should be checked for purity and for identity as per SOP for indentification

of micro organisms and stored for not more than six months It should be subcultured

in duplicate

3 One of the two mother cultures should be used for preparation of working culture and

stock culture The other is intended as reserve in the event of first mother culture getting


4 Inocula from mother culture must not be used directly in microbiolgoical testing

Prepare a stock culture and a working culture and label that as generation 1

5 After 15 days subcultrue from the generation 1, prepare stock culture and working

culture label them as generation 2 (Detailed outline for maintenance of culture - Refer

6 Prepare a routine culture from the working cultures for the microbiological purpose

7 If all the cultures become contaminated or fail to grow it is necessary to revert to the

mother culture for preparation of new working culture

8 The culture slants should be labelled as follows:

Name of the Organisms with classified codes:

Batch No / Generation no of culture

Date of Subculturing

Use before date

9 All the working cultures must be stored at a temperature of 8-15 deg C in a covered



1 Clean the area where subculturing is to be done with the suitable disinfectant

(70 % isopropyl alcohol)

2 Wear previously sterilised gloves and nose mask

3 Wipe out the outside of the old culture tube with cotton wool soaked in 70 % IPA soln

4 Get fresh slants/stab culture of maintainence medium and wipe out outside of

tube with cottom wool soaked in 70 % IPA solution

5 Hold on the extreme end and heat the nichrome wire loop on the reducing flame till

red hot

6 Allow it to cool to 40-45 deg C and slowly open the cottom plug of old culture tube

and warm the mouth of the tube

7 Pick up the smear with the help of the loop after having removed cottom plug of the

test tube

8 Plug tube again still holding nichrome wire between two fingers and thumb

9 Open plug of the slant/stab tube Warm the mouth of the tube holding in other hand

and carefully transfer the culture that is held on the loop

10 Spread the culture spirally on the surface of the slant if the organism is aerobic and

stab the medium if the organism is anaerobic in nature

11 Warm mouth of the tube and immediately plug the cotton into the mouth

12 Label tubes with the name of the organism Classified code,Generation number, Batch No

and date of subculturing

13 Incubate fresh subcultured organism tube at 30-35 deg C for 18-24 hrs and then

store it in refrigerator

14 Each generation of subcultured organism should be manitained for months


1 Follow the SOP Disposal of Microbial Waste Material

Destruction pattern for stock culture generation

The subculturing data and the destruction should be recorded as per attachment No 1





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