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.3.0 RESPONSIBILITY
Microbiologist4.0 ACCOUNTABILITY
Head QA & QC5.0 PROCEDURE
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 liter of Water for Injection.
5.1.3 Weigh the 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 are 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
5.1.5.1 Weigh the quantity of dehydrated culture media and add WFI as per direction is given on pack of the medium. Note down the weight taken for each medium.5.1.5.2 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.
5.1.5.3 Distribute the suitable quantity of the medium in an appropriate flask or test tubes. Plug it with non-absorbent cotton, wrap with aluminum foil.
5.1.5.4 Sterilize the medium as per the instructions are given on the pack. Autoclaving should be done at 121°C, 15 lbs/inch² for 20 minutes or time specified for respective media.
5.1.5.5 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.
5.1.5.6 Store the sterilized broth media below 25°C till its use.
5.1.6 Agar Media
5.1.6.1 Weigh the quantity of dehydrated culture media in WFI as per direction given on pack of the medium. Note down the weight taken for each medium.5.1.6.2 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.
5.1.6.3 Distribute the suitable quantity of the medium in the appropriate flask. Plug it with non-absorbent cotton, wrap with aluminum foil.
5.1.6.4 Sterilize the medium as per the instructions are given on the pack. Autoclaving should be done at 121°C, 15 lbs/inch² for 20 minutes or time specified for respective media.
5.1.6.5 After sterilization, allow the medium cool to 60ºC.
5.1.6.6 Store the molten media in an oven maintained at 55-60ºC, until further use.
5.1.6.7 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 the medium. Then cool to room temperature & use.
5.1.6.7 For agar media, after autoclaving, cool the medium to about 45-50°C then pour the media aseptically into sterile Petri dishes 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).
5.1.6.8 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 the following manner:XXX/ZZZZZ
Where XXX: Autoclave Load No. Starting from 001 for every year.
ZZZZZ: Batch No. of Media provided by the 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 the 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 Preincubation
Preincubate 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.6.0 ABBREVIATIONS
SOP: Standard Operating ProcedureQA: Quality Assurance
QC: Quality Control
BOD: Biological Oxygen Demand
M: Molarity
WFI: Water for injection
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After preparing media for how much time we can store it at room temperature before sterilization?
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