1.0 Objective:
The objective of this sop is to lay down the procedure for procurement,
preservation and maintenance of microbial cultures
2.0 Scope:
This SOP covers all microbial
cultures used in the microbiological analysis
3.0 Responsibility:
Microbiologist:
Responsible for preparation the media, handling the organism and maintain the
necessary record.
QA Officer/QA Manager: Review the records and governing the document.
4.0
Procedure:
4.1 Procurement / Receiving of
Microbial Cultures:
1.0
2.0
3.0
4.0
4.1
4.1.1 Procure the equivalent
American Type Culture Collection (ATCC) lyophilized vials of microbial cultures
with certificate of authenticity as specified in culture certificate from
·
National
Culture of Industrial Microbiology, Pune or
·
Microbial Type Culture Collection, Chandigarh
4.1.2 After receiving the lyophilized vials from the culture collection
center, check the lyophilized vials for any breakage and match the ATCC/ MTCC /
NCIM number on the vial and certificate
4.1.3 The lyophilized culture shall be stored at 2 - 8ºC for not more than
one year.
4.1.4 Record the details of the lyophilized culture received in the format
given in Annexure – I.
4.1.5 Use
the media specified in certificate for the maintenance
of microbial cultures.
4.1.6
Prepare the media and subsequent version titled
“Preparation of Microbiological Media”.
4.1.7
Prepare media slants in clean 18 mm diameter rimless test
tubes / screw capped culture tubes and pre-incubate the slants for 48 hours at
specified temperatures as give in Annexure No. III to check any contamination.
Discard the contaminated slants. Prepared slants can be stored up to 7 days at
temperature not exceeding 25°C.
4.2
Sub-culturing of
micro-organisms from lyophilized cultures:
4.2.1 Check the cleaning and
sanitization status of the area and LAF bench before proceeding for
sub-culturing.
4.2.2 Sub-culturing shall be done in a laminar airflow clean station to
avoid the chances of contamination. Clean the LAF working bench before
proceeding for sub-culturing.
4.2.3 Open ampoule of lyophilized culture and aseptically transfer
contents equally into two different sterile vials under LAF and label vials as
X and Y respectively.
4.2.4 With the help of sterile inoculation loop, withdraw small quantity
of lyophilized culture from vial X and transfer the content in 10 ml test tube
containing sterile Soyabean casein digest medium (SCDM) for aerobic bacteria
and yeast and molds and in 10 ml sterile Fluid thioglycollate media (FTM) for
anaerobic bacteria. Label the tubes as mother culture tubes.
4.2.5 Incubate mother culture tubes at the temperatures mentioned in culture certificate. After completion of incubation period observe the tubes for
turbidity.
4.2.6 Using sterile inoculating loop, withdraw small quantity of culture
from mother culture tubes of SCDM / FTM and streak on 4 pre-incubated slants of
appropriate culture media as given in culture certificate.
4.2.7 Incubate inoculated slants / stabs at the temperatures mentioned in culture certificate.
4.2.8 After completion of incubation period, examine growth of
microorganisms visually, also carry out gram staining test Confirmation of the
purity of the cultures after sub culturing and subsequent versions titled “Confirmation of the purity of the
cultures” and observe microscopically for the purity of the culture in the
slant. Report the observations of gram staining and as per culture certificate. In
case of any contamination the slants shall be discarded and new set of slant
shall be prepared.
4.2.9 If any culture found contaminated discard it and get a new culture
from authentic source.
4.2.10 After observing the growth, label the Mother
culture tube as mentioned below
MOTHER
CULTURE
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Name of
Microorganism
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Strain No.
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Generation
No.
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Medium used
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Date of
Sub-culturing
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Due On
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Sub-cultured
By
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WORKING CULTURE
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Name of
Microorganism
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Strain No.
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Generation
No.
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Medium used
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Date of
Sub-culturing
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Due On
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Sub-cultured
By
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4.2.11 After proper labeling of the slants, store both mother and working
cultures into refrigerator at 2 – 8 °C.
4.2.12 Withdraw maximum of 5 times [not more than 5 passages] for
sub-culturing from the lyophilized cultures or use up to one year from the date
of receipt whichever is less.
Note. If biochemical test of the culture shows positive
(viable) then it will be used for one more year.
4.3
Sub-culturing of
Microorganisms from Mother Culture:
4.3.1 Remove Mother culture from refrigerator and allow the slants to
reach at room temperature before proceeding for sub culturing. Streak / stab 2 new slants of appropriate
medium as given in culture certificate. Out of 4 subculture slants label 1 slant as mother
culture and 1 slant as working (monthly) culture.
4.3.2 Use mother culture for further sub-culturing and working (monthly)
culture for lab use.
4.3.3 Write complete information on label as given above and affix that
label on to prepared slants/stabs.
4.3.4 After proper labeling of the slants/stabs, store both stock and
working cultures into refrigerator at 2 – 8 °C.
4.3.5 After completion of the passage / series and after observation of
growth in new slants discard the used cultures as per current version of titled “ Disposal of Microbiological Used Media” for Disposal Used Media,
Culture and dehydrated media
4.4
Assign Generation Number
to Sub-cultures:
4.4.1 For first subculture starting from reference culture assign the
generation No. as AX/S1/G1.
Where, A – Stands for Lot No.
X –
Vial No.
S1 –
Stands for Series 1.
G1 –
Stands for Generation 1.
4.4.2 This sub-culture may be further sub-cultured up to 3 times [Total
not more than 5 times starting from lyophilized culture]. To this subculture
assign generation No. as AX/S1/G1 this will go up to AX/S1/G3.
4.4.3 After completion of series S1 up to generation AX/S1/G3 discard the
used cultures and again start sub-culture from reference culture vial X and assign generation No. AX/S2/G1
and this will again go up to AX/S2/G3.
4.4.4 After completion of series S2 up to generation AX/S2/G3 discard the
used cultures and again start sub-culture from reference culture vial X and assign generation No. AX/S3/G1
and this will again go up to AX/S3/G3.
4.4.5 After completion of series S3 up to generation AX/S3/G3 discard the
used cultures and again start sub-culture from reference culture vial X and assign generation No. AX/S4/G1
and this will again go up to AX/S4/G3.
4.4.6 After completion of series S4 up to generation AX/S4/G3 discard the
used cultures and again start sub-culture from reference culture vial Y and assign generation No. AY/S1/G1
and this will again go up to AY/S4/G3.
4.4.7 If subculture is being started from reference culture received 2nd
time [i.e. lot No. B] the generation No. will be BX/S1/G1 and will continue so
on up to BY/S4/G3.
4.4.8 Maintain a record for receipt of reference culture [lyophilized
vial] and for periodic sub-culturing
4.4.9 The sub-culturing shall be carried out
monthly as per the plan
4.4.10 The sub-culturing shall not be carried
out for more than five passages from the mother culture.
4.4.11 Preserve the newly prepared
culture tubes / screw capped tubes in the refrigerator at 2 – 8 °C.
4.4.12 The 1 stock cultures slants / stabs
shall be used for preparing culture suspensions. The other 1 working culture
slants / stabs shall be used for positive control, Growth Promotion test and
other laboratory testing requirements.
5.0 Precautions:
5.1 Handle the culture tubes carefully to
avoid breakage and contamination.
5.2 In case of breakage of the culture
tubes, disinfect the area immediately with 5 % v/v Dettol solution and discard
the tube.
5.3 In case spillage occurs on the
apparel, clean the area with 5 % v/v Dettol solution two to three times and keep the clothes
for washing.
