OBJECTIVE :-
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
IMPORTANT POINT/NOTE/PRECAUTIONS:-
SAFETY:
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
PROCEDURE :-
a) MAINTAINENCE OF CULTURE
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
contaminated
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
container
b) METHOD OF SUBCULTURING
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
c) DESTRUCTION:
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
PERSONNEL TO BE REPORTED IN CASE OF NON COMPLIANCE:- QA Executive
FREQUENCY OF OPERATION :- Fortnightly
NO OF ATTACHMENTS:-
RETENTION OF RECORDS :- 5 Years
Guide for Pharmaceutical Manufacturing pharmacy students pharmacy colleges and pharmacists pharmaceutical companies in health care
Standard operating procedure SOP for microbiology and aseptic techniques
Following are some of articles which will be useful for you in further understanding of aspects of sterile dosage form manufacturing
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