SOP For Maintenance and subculturing of reference culture is described in this post which you can follow in the section of the microbiological lab.
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Objective:
This procedure has been established for the maintenance and subculturing of reference culture.
Scope:
This procedure is applicable to the microbiology lab.
Responsibility:
Microbiologist.
Accountability:
Quality Control
Manager.
Procedure:
For preparing master/reference cultures specified media must be used for optimum growth.
Transfer all the lyophilized cultures and other items aseptically to microbiology testing area under the laminar flow cabinet.
Before starting the procedure switch "ON" the laminar flow cabinet disinfect it with 70% IPA properly.
Now light-up the flame and sterilize the loop on the flame till red-hot and then wait for a few seconds to let it cool.
Open the lid of the plate near the flame containing reference culture and pick the a colony of the culture carefully by the help of sterilized SS loop in 10ml of 0.9% normal saline Solution which is previously sterilized by autoclavation.
After sampling a colony of culture close the lid of the plate as soon as possible to avoid any cross contamination or spillage.
Gently shake or swirl the plate to mix the culture in the solvent and pour 20-25ml of the specific media over the plate and allow solidifying and set for incubation at a temperature of 32oC±2.5oC for a period of 18-24hrs in case of bacterial culture and 22oC±2.5oC for a period of 3-5days in case of fungal culture
Subculturing Procedure:
First of all prepare the specific media for each microorganism individually.
Transfer pre-incubated microbial-culture to the microbiology testing area
Form freshly prepared master or reference culture streak a loopful of culture onto the media plate and incubate at a specified temperature.
1st Dilution from reference culture =master culture
2nd Dilution from master culture =stock culture
3rd Dilution from stock culture= working culture
Label all the cultures as master, stock, working culture and preserve the culture at a temperature of 2 to 6°C.
Prepare atleast four plates for master culture, one plate for stock culture and one plate for working culture from previous culture plate.
Subculturing Frequency: atleast once in a month.
sequential subculturing will depict the purity of mother culture, stock culture by gram staining.
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