SOP for Standard Culture transfer and its maintenance in microbiology lab.
1.0 PURPOSE:
To lay down a procedure for Culture transfer and its Maintenance in Microbiological Laboratory.
This procedure is applicable for Culture transfer and its Maintenance in Microbiology Laboratory.
3.0 RESPONSIBILITIES:
Microbiologist : To perform the procedure of Culture transfer as per SOP.
Head-QC : To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0 PROCEDURE:
4.1 Procurement of Culture:
4.1.1 Generally microbial cultures must be procured from Institute of National chemical Laboratories, Pune (NCL) or other authorized agencies.
4.1.2 After receipt, record all the details like ATCC No., Receiving date, Received by and Expiry date and labeled as Master Culture and store at 2–8°C.
4.1.3 Before sub culturing perform the purity and viability checking of microbial culture.
4.1.4 In every stage of subculturing, check the purity of culture by gram’s staining and by streaking on selective media. Record their physical characteristic and colony characteristics.
4.1.5 If the cultures are not showing their physical characteristic and fail in viability checking of culture, discard the culture.
4.1.6 Proceeded sub culturing at every stage, after qualifying the purity and viability test (six monthly, monthly and weekly sub cultures).
4.2 Procedure for Subculture:
4.2.1 Perform subculturing under LAF.
4.2.2 Wipe out the outside surface of the culture slant tubes with cotton soaked or tissue paper with freshly prepared 70% IPA.
4.2.3 Hold on the extreme end and heat the nichrome wire loop on the reducing flame till red-hot.
4.2.4 Allow to cool the wire loop and slowly open the cotton plug of old culture tube and gentle warm the mouth of the tube.
4.2.5 Pick up the smear/growth with the help of loop.
4.2.6 Hold the nichrome wire loop with smear/culture growth, plug the culture slant tube, and put inside the old slant culture.
4.2.7 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.
4.2.8 Spread the culture spirally on the surface of the slant, warms the mouth of slant and plug it.
4.2.9 For anaerobic bacteria, insert the loop into a deep stab of slant, warm the mouth of slant and plug it.
4.2.10 Label the culture tube, and incubate it at specified temperature and period, and then store it in refrigerator at 2 – 8°C.
4.2.11 If the Master Culture is lyophilized, score the middle of the ampoule with an ampoule cutter or Glass cutter.
4.2.12 Disinfect the ampoule with sterile 70% IPA solution which is moistened with lint free cotton or tissue paper.
4.2.13 Wrap the sterile cotton cloth around the ampoule and break it carefully.
4.2.14 Aseptically add about 0.3-0.4 ml of sterile saline solution with the help of micropipette and mix well and pour into 5 ml saline test tube to make suspension.
4.2.15 Transfer a loop full suspension in a duplicate sterile media slant and simultaneously inoculate on nutrient agar media plates for checking of viability of cultureand incubate at specified temperature. Observe the growth after incubation. These Culture suspensions are stored at 2 – 8°C for their specific period.
4.3 Maintenance of Culture:
4.3.1 After ensuring the purity of master culture, prepare three mother cultures (six monthly) of each culture by transferring a loop full suspension from master culture to fresh slant, and give identification number as a MC1, MC2 and MC3.
4.3.2 Incubate all the tubes at specified temperature and period, as indicated in Annexure-I and observe the growth after incubation.
4.3.3 Check the purity of culture by staining and record their physical characteristic.
4.3.4 If the culture showing their physical characteristics as per Annexure-I, transfer a loop full growth from MC1 to a fresh seven set (monthly culture) of slant and treat as M1, M2, M3, M4, M5, M6 and M7 respectively.
4.3.5 Incubate the all the slant at specified temperature and period, and assign identification number i.e. MC1/M1, MC1/M2, MC1/M3 up to MC1/M7.
4.3.6 After incubation observes the growth and store the culture at 2 – 8°C for their specific period.
4.3.7 Use the prepared culture as per Schematic diagram.
4.3.8 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.
4.3.9 Transfer a loop full growth from Monthly Cultures i.e. M1, M2, M3, M4, M5, & M6 after ensuring the purity of cultures to a five fresh slant (Weekly culture) and treat as a W1, W2, W3 W4 and W5. Assign the batch number i.e. MC1/M1/W1, MC1/M1/W2, MC1/M1/W3, MC1/M1/W4 and MC1/M1/W5.
4.3.10 After incubation observes the growth and label the culture. store the culture at 2 – 8°C for their specific period.
4.3.11 For 1st, 2nd, 3rd and 4th Week of every month use W1, W2, W3 and W4 cultures respectively. If one of the above cultures gets contaminated, use W5 culture.
4.3.12 The sub culturing of pure microbial culture should not be more than 5 passages (Generation).
4.4 Precaution:
4.4.1 After transferring the culture, always return the cotton plug or caps to the correct tubes.
4.4.2 Never forget to sterilize the inoculating loop before returning it to the holder.
4.4.3 Always sterilize the tips of the culture tube in the flame before you insert the loop and after withdraw the tube.
4.4.4 The plug after removal must be kept in hand. Never place the plug on the work surface or touch it to anything except the flamed tip of culture tube.
4.4.5 Do not dip the inoculation loop into the agar while inoculating slant, only for anaerobic microbes makes the stab culture.
4.4.6 Never leave the culture tube open for longer time than the time needed to transfer the culture.
4.5 Frequency:
· First Generation (Master culture) – Yearly.
· Second Generation (Mother culture) – Six monthly.
· Third Generation (Working culture) – Monthly.
· Fourth Generation (Routine culture) – Weekly.
5.0 SCHEMATIC DIAGRAM:
Where:
MC1, MC2 & MC3 = Mother Culture (Six monthly)
MC1/M1 to MC1/M7 = Working Culture (Monthly)
MC1/M1/W1 to MC1/M1/W5 = Routine Culture (Weekly)
6.0 Annexure-I:
Sr. No.
|
Microorganism
|
Growth Medium
|
Physical characteristics
|
Incubation Time/ Temp.
|
1
|
Escherichia coli
|
Nutrient Agar
|
Gram –ve , short rods
|
30-35°C for 24-48 hrs.
|
2
|
Bacillus subtilis
|
Nutrient Agar
|
Gram +ve, rods
|
30-35°C for 24-48 hrs.
|
3
|
Candida albicans
|
Sabouraud dextrose agar
|
Oval shaped
|
20-25°C for 72-120 hrs.
|
4
|
Pseudomonas aeruginosa
|
Nutrient Agar
|
Gram –ve rod shaped
|
30-35°C for 24-48 hrs.
|
5
|
Aspergillus brasiliensis
|
Sabouraud dextrose agar
|
Mycelial spores
|
20-25°C for 72-120 hrs.
|
6
|
Salmonella
|
Nutrient Agar
|
Gram –ve rod shaped
|
30-35°C for 24-48 hrs.
|
7
|
Staphylococcus aureus
|
Nutrient Agar
|
Gram +ve, cocci in clusters
|
30-35°C for 24-48 hrs.
|
8
|
Clostridium sporogenes
|
Nutrient Agar
|
Gram +ve bacilli
|
30-35°C for 24-72 hrs.
|
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