1.0 OBJECTIVE:To lay down a procedure for media preparation.
2.0 SCOPE:This SOP shall be applicable for media preparation in microbiology section.
4.0 ACCOUNTABILITY:Head QA & QC
5.1 Method for preparation & use of microbiological media:5.1.1 Use dehydrated culture media from Hi Media. Check the in house use before date prior to use of media.
5.1.2 The method for preparation of media is provided on the pack. It gives the quantity of powder to be suspended per litre of Water for Injection.
5.1.3 Weigh required quantity of powder and add required quantity of freshly collected WFI. Note down the weights, mix well to dissolve and check the pH of the media before sterilization. Sterilize the media as per instructions given on the pack. After autoclaving; let the media cool to 55- 60°C, store at about 55 ± 5°C or pour into the plates as per requirements.
5.1.4 This stored media can be used for 7 days. Maintain the record for media preparation usage and destruction in format.
5.1.5 Broth media:
184.108.40.206 Weigh the quantity of dehydrated culture media and add WFI as per direction given on pack of medium. Note down the weight taken for each medium.
220.127.116.11 Dissolve the solids in WFI, warm gently (if necessary) to affect a uniform solution, Cool to room temperature & adjust the required pH (specified on the respective medium pack) by using either 0.1 M HCl or 0.1M NaOH solution.
18.104.22.168 Distribute the suitable quantity of the medium in appropriate flask or test tubes. Plug it with non-absorbent cotton, wrap with aluminium foil.
22.214.171.124 Sterilize the medium as per the instructions given on the pack. Autoclaving should be done at 121°C, 15 lbs/inch² for 20 minutes or time specified for respective media.
126.96.36.199 After sterilization, cool the medium to room temperature, check the pH again by using either pH indicator strip or pH meter and use for analysis.
188.8.131.52 Store the sterilized broth media below 25°C till its use.
5.1.6 Agar Media:
184.108.40.206 Weigh the quantity of dehydrated culture media in WFI as per direction given on pack of medium. Note down the weight taken for each medium.
220.127.116.11 Dissolve the solids in WFI, warm gently (if necessary) to effect a uniform solution, Cool to room temperature adjust the required pH (Specified on the respective medium pack) by using either 0.1 M HCl or 0.1 M NaOH solution.
18.104.22.168 Distribute the suitable quantity of the medium in appropriate flask. Plug it with non-absorbent cotton, wrap with aluminium foil.
22.214.171.124 Sterilize the medium as per the instructions given on the pack. Autoclaving should be done at 121°C, 15 lbs/inch² for 20 minutes or time specified for respective media.
126.96.36.199 After sterilization, allow the medium cool to 60ºC.
188.8.131.52 Store the molten media in oven maintained at 55-60ºC, till further use.
184.108.40.206 In certain cases, e.g. Selenite cysteine broth, do not autoclave the solution. After preparing solution, follow the procedure of sterilization as given on the pack of medium. Then cool to room temperature & use.
220.127.116.11 For agar media, after autoclaving, cool the medium to about 45-50°C then pour the media aseptically into sterile petridishes in 15 to 20 ml quantity in each plate. Allow these plates to solidify. Close the plates invert them and store in BOD incubator (Below 25°C).
18.104.22.168 All the media prepared in a day should be assigned a respective batch no. and these batch nos. should be entered in media preparation, utility & destruction record.
5.2 Assigning batch numbers:All the media prepared in one autoclaved cycle should have respective batch numbers & these batch numbers should be entered in media preparation & record. Batch No. of autoclaved media should be allotted in following manner:
Where XXX : Autoclave Load No. Starting from 001 for every year.
ZZZZZ: Batch No. of Media provided by manufacturer.
Sterilize the microbial culture media within 2 hrs. From their preparation.
5.3 Storage of microbiological media:Prepared media :
Broth: Stored below 25°C.
Molten agar media : Stored between 55-60°C
Prepared plate : Stored below 25°C in BOD
5.4 Testing of Media:5.4.1 pH:
From each quantity of nutrient media, which is prepared in one run, take a sample after autoclaving and let it cool down to room temperature. Measure the pH value of solid media (before solidify), and liquid media with electrode. Record the result. If the measured pH does not comply with the requirement, the pH value is determined for another two containers of the same batch. If the results from the two repetitions lie within the limits, the medium batch may be used for the test. If the results don’t comply, the batch must not be used and is to be discarded.
5.4.2 Pre incubation:
Pre incubate the media for 48 hrs. After incubation physically inspect the media for any contamination (Macroscopic growth of evidence), if there is contamination discard all the media as per SOP for disposal of media.
5.4.3 Growth Promoting Properties:
Growth promoting properties of selective and non-selective media are tested using indicator organisms as per SOP.
SOP : Standard Operating Procedure
QA : Quality Assurance
QC : Quality Control
BOD : Biological Oxygen Demand
M : Molarity
WFI : Water for injection
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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