To provide a method of preparation, sterilization, storage and growth promotion test of microbiological media.
This procedure is applicable for preparation of solid and liquid media which are to be used for microbiological testing.
Doing : Technical Assistant
Checking : Executive /Manager
Head of the Department
5.1 For liquid media preparation :
5.1.1 Weigh the required quantity of the media powder for specific volume in a beaker as per the instruction given on the dehydrated media bottle or media preparation sheet.
5.1.2 In case if ready made dehydrated media is not available, prepare the media as per pharmacopoeia.
5.1.3 Dissolve the media by heating with continuous stirring. When large quantity of the media is to be continuous stirring and add remaining quantity of water to make up the volume. Mix it properly.
5.1.4 Adjust the pH with 0.1N HCl/0.1N NaOH if necessary .
5.1.5 Allot the Lot No. to prepared media as per SOP.
5.1.6 After sterilization check the pH from one container & discard it. The pH should be within limit.
5.1.7 Distribute the specific volume of the media in appropriate glass container intended for use.
5.1.8 Plug it either with cotton plug or metal caps.
5.1.9 Affix the label with media detail on each tube.
5.1.10 If cotton plugs are used, cover it with wrapping paper.
5.1.11 Unless specified, sterilize the media at 121°C for 20 minutes as per SOP.
5.1.12 After sterilization, cool down the media at room temperature & proceed for the preincubation & Growth promotion test of the same.
5.2 For solid media preparation:
5.2.1 As per the instruction, weigh specified quantity of media powder in a beaker whose capacity is double the final volume of the media to be prepared.
5.2.2 Add appropriate quantity of distilled water and mix it properly.
5.2.3 Allot the Lot No. to media as per SOP
5.2.4 Heat and dissolve the medium constituents with constant stirring to avoid charring.
5.2.5 Check the pH of the medium and set the pH using 0.1N HCl/0.1N NaOH, if required
5.2.6 While in the molten state, distribute the specific volume of the media into the appropriate glass containers.
5.2.7 Seal the mouth of the glass container using cotton plugs.
5.2.9 Cover the cotton plugs with wrapping paper.
5.2.10 Unless specified, sterilize the media at 121°C for 20 minutes SOP.
5.2.11 After completion of sterilization, solid media are used in different form according to the requirements.
[A] For slant preparation :
Place the molten media tubes slanted and let it solidify.
[B] For stab preparation :Keep the media tubes in the upright position and allow it to solidify.
[C] For preparation of plate :
To prepare the plates, pour the media into the sterile petri dishes under LAF and let it be solidify.
5.2.12 Solid media for other purpose should be kept in oven or water bath whose temperature should be between 45°C-50°C until use so that it should remain in molten state.
5.2.13 Sterile solid plates should be stored at 20°-25°C while sterile slants and stabs should be stored at freeze temperature.
5.3 Pre incubation of the media:
5.3.1 All the sterile solid as well as liquid media should be kept at 30°-35°C for 48 hrs. for pre-incubation
5.3.2 After 48 hrs.,if media indicates growth, (In case of liquid media solution becomes hazy & in case of solid media there may be some colonies) discard the contaminated media as per SOP.
5.3.3 Allot the lot No. to each container of medium and transfer in 20-25°C walk in-incubator.
5.4 Growth Promotion Test:
5.4.1 Check the growth promoting properties of the medium, either by inoculating <100 cells/ml of control organism in the liquid media or by streaking the surface of the solid media plates with the control organisms known to produce positive reaction. Incase of sterility test medium check the growth promoting quality by separately inoculating duplicate test container for each
5.4.2 During testing of total viable count keep the positive control with any one of indicator organism as for SCD agar B.subtilis SD agar C.albicans and Endo agar E.coli.
5.4.3 The medium is said to be satisfactory, if turbidity in liquid medium is observed within 48 hrs for bacteria and 5 days for fungi growth on solid agar is seen within 24-48 hrs at specific incubation condition.
5.4.4 After growth promotion test transfer all the pre-incubated sterile liquid medium tubes and solid medium plate at 20°-25°C for use.
5.4.5 Store all the slants & stabs at freeze temperature.
5.5.1 Medium more than three weeks old should not be used for sterility purpose.
5.5.2 Medium other than sterility test kept at 20°-25°C should be used within one month.
5.5.3 Slants and stabs stored at freeze temperature should be used within two months.
5.5.4 Maintain the media preparation record.
6.1 °C= Degree centigrade
6.2 ml= millilitre
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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