Procedure for Media Preparation

1. OBJECTIVE
To provide a method for Preparation, Storage, Sterilization and Growth promotion test of microbiological Media

2. SCOPE
It is Applicable for preparation of solid and liquid media which are to be used for
Microbiological testing

3. RESPONSIBILITY
• Microbiologist

4. SAFETY INSTRUCTIONS
• Handle the Media very carefully

5. PROCEDURE
5.1 For liquid media preparation:
5.1.1 Weigh the required quantity of 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 the ready-made dehydrated media is not available, prepare the media as per pharmacopeia.
5.1.3 Dissolve the media by heating with continuous stirring. When a large quantity of media is continuous stirring and add remaining quantity of water to make up the volume. Mix it properly.
5.1.4 Adjust the pH by adding 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 and discard it. The pH should be within the 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 a 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.
5.1.12 After sterilization, cool down the media at room temperature& proceed for the pre-incubation and growth promotion test of the same.
5.2 For Solid Media Preparation:
5.2.1 As per the instruction, weigh the specify quantity of the media powder in a beaker whose capacity is double to 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 it by using 0.1N HCL/ 0.1 N NAOH if required.
5.2.6 While in the molten state, distribute the specific volume of the media in appropriate glass Containers.
5.2.7 Seal the mouth of glass container by using cotton plugs
5.2.8 Cover the cotton plugs with wrapping papers.
5.2.9 Unless specified, sterilize the media at 121 °C for 20 minutes
5.2.10 After completion of sterilization solid media are used in a different forms according to the requirements.
5.2.11 For Slant Preparation:
Place the molten media tubes slanted and let it solidify.
5.2.12 For Stab Preparation:
Keep the media tubes in upright position and let it solidify.
5.2.13 For the Preparation of Plate:
To prepare plates pour the media in sterile petri plates under LAF and let it solidify.
5.2.14 Solid media for another 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.
5.2.15 Sterile solid plates should be stored at 20° -25 °C while sterile stabs and slants should be stored at freezer 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 indicate growth (in case of liquid media solution become 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 the 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 solid media plates with control organism known to produce a positive reaction. In case 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 the indicator organismas 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 a 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 plates at 20°-25 °C for use.
5.4.5 Store all the slants and stabs at freeze temperature.
5.5 Storage:
5.5.1 Medium more than 3 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 should be stored at freeze temperature should be used within two months.

6. RELATED DOCUMENTS
N/A

7. DISTRIBUTION INDEX
Original Copy will be retained by Quality Assurance Department.

8. ABBREVIATIONS
HCl Hydrochloride
NaOH Sodium Hydroxide
LAF Laminar Air Flow
SOP Standard Operating Procedure

9. REVISION HISTORY