1.0 OBJECTIVE
To describe a procedure for Preparation and sterilization of the media used for microbial testing.
2.0 SCOPE
This procedure is applicable to preparation and sterilisation of media used in the microbiology lab.
3.0 RESPONSIBILITY
3.1 Microbiologist /Officer is responsible for the execution.
3.2 HOD or designee is responsible for review, effective implementation and compliance to the Standard Operating Procedures for execution.
4.0 PROCEDURE
4.1.1 Before preparing the media follow the instruction mentioned on the container for direction of media preparation, ingredients of the media, Purpose of the media, Storage Conditions, Expiry Date and Disposal of the used / unused media.
4.1.2 Ensure that the dehydrated media powder have the following characteristics.
4.1.2.1 Free flowing
4.1.2.2 Uniform color
4.1.2.3 Free from pungent smell
4.1.2.4 Absence of lumps
4.2 Preparation of media :
4.2.1 Preparation of media follows as per the label claim of the manufacturer direction, like as mentioned in the Attachment #1 for each specified media.
4.2.2 Weigh the required quantity of dehydrated media in a clean butter paper using top pan balance and Record the weigh details in Attachment #2.
4.2.3 If the container is having new / different batch check for growth promotion tests of media.
4.2.4 Suspend the media in a glass / Stainless Steel container containing desired volume of Purified water. Add a small volume of water and swirl to dissolve completely.
4.2.5 Make up the volume by adding water from the side walls of the container.
4.2.6 Boil this hydrated medium if required to dissolve completely using, hot plate/water bath.
4.2.7 Take care to avoid excessive heating or scorching of the medium.
4.2.8 Check the pH of the medium; if necessary adjust the pH by 0.1N NaOH or by 0.1N HCl.
4.2.9 Allot the Media Lot No to the each medium as ‘AA/000’.
• Where, Alphabets “AA”denotes the medium code in numeric as
01, 02, 03 and so on and Numeric “000”denotes the Lot number Refer Format Attachment #3
• For example, Nutrient agar, Lot No can be written as 28/001.
4.2.10 Mix well and boil it to dissolve the medium, using a glass rod. Distribute the boiled media into glass flasks/bottles as per the requirement.
4.2.11 Keep 1 bottle with around 50ml of medium of the same Lot for pH after sterilization.
4.2.12 Use plastic cap for closing the bottles. Dispense the completely dissolved media as desired i.e., in tubes and bottles.
4.2.13 Close the tubes or bottles with non-absorbent cotton plugs or screw caps according to the containers used.
4.2.14 Load the media and operate the double door autoclave to sterilize the media.
4.2.15 After sterilization, use the media accordingly.
4.2.16 Allow to cool down the medium around 40-50°C for agars and room temperature for liquid medium. Check the pH and record in Attachment #2
4.2.17 If the pH is in the range as mentioned in Attachment #1 format no.: QCP019/F01 then, use the medium for analysis.
4.2.18 If the pH is not in the range discard the complete medium.
4.3 Preparation of 0.1N NaOH and 0.1N HCl.:
• Dissolve 40 g of Sodium Hydroxide in 1000 ml and 18 ml of Hydrochloric acid in 1000 ml to prepare 1 N solution.
• Make a 1:10 dilution of the above solutions to obtain 0.1N strength.
4.4 Preparation of 7.2 buffer solution:
Dissolve 34 g of monobasic Potassium phosphate in about 500 ml of water contained in a 1Lt volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of 4 % w/v aqueous solution of Sodium hydroxide (about 175 ml), add water to volume and mix. Dispense, sterilize and store under refrigeration. For use, dilute the Stock solution with water in the ratio of 1 to 800, and sterilize in an autoclave.
ATTACHMENT # 1: PREPARATION OF MEDIA
Weigh the quantity of dehydrated media as per the manufacturer label claim and prepare accordingly as per the requirement of the medium required for the analysis. Here are some details of media for reference. (Make: Himedia)
Soyabean Casein Digest Agar Medium
1.Weigh 40.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.3 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 50-55°C and use the media for the proposed analysis or as follows.
8.After cooling mix well the media and pour in to the sterile petriplates/ in tubes to make slants.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation).
10.After preincubation use the plates/tubes for inoculation.
Soyabean Casein Digest Medium
1.Weigh 30.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purifed water and mix well.
4.Adjust the final pH (at25°C) 7.3 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Transfer the dissolved media 100 ml each into the sterilized glass bottles/tubes of capacity.
7.Place all the bottles in a container that is in turn kept in an autoclave.
8.Sterilize all the bottles contained media in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
9.After sterilization cool the media to 25°C and store in a cool dark place preferably below 25°C store in a cool dark place place in an incubator 24 hours (i.e. for preincubation).
10.After preincubation use the tubes/bottles for detection.
Baird - Parker Agar
1.Weigh 65.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle
3.Add exactly 950 ml of distilled /purifiedwater and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Avoid over heating.
7.After sterilization cool the media to 50°C
8.After cooling aseptically add 50 ml Egg Yolk emulsion (FD045) and 3 ml Sterile 3.5% Potassium Tellurite solution (FD047) or 50 ml Egg Yolk Tellurite emulsion (FD046) if desired and rehydratred contents of 1 vial BP Sulpha supplement (FD069)
9.Mix well the media and poured into the sterile petriplates.
10.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
11.After preincubation use the plates for inoculation.
Brilliant Green Agar Medium
1.Weigh 29.0 g of media in a butter paper in a clean weighing balance
2.Transfer the weighed media in to a clean sterilized beaker /bottle
3.Add exactly 500 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 6.9 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Avoid over heating.
7.After sterilization cool the media to 50°C
8.After cooling aseptically add rehydrated contents of 1 vial of Sulpha Supplement (FD068)
9.Mix well the media and poured into the sterile petriplates.
10.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation).
11.After preincubation use the plates for inoculation.
Bismuth Sulphite Agar Medium
1.Weigh 40g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle
3.Add exactly 1000 ml of distilled /PURIFIED water and mix well.
4.Adjust the final pH (at25°C) 7.6 ± 0.2.
5.Heat to boiling to dissolve the medium completely by keeping the beaker / bottle on the hot plate/water bath.
6.Do not autoclave / over heat.
7.After cooling mix well to disperse precipitated Bismuth Sulphite in suspension and poured into the sterile petriplates.
8.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
9.After preincubation use the plates for inoculation.
Cetrimide Agar Medium
1.Weigh 45.3 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.2 ±0.2.
5.Boil to dissolve the medium completely by keeping the beaker / bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Avoid over heating.
7.After sterilization cool the media to 40 - 50°C.
8.After cooling the media is poured into the sterile petriplates / in tubes to make slants.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
Deoxycholate Citrate Agar Medium
1.Weigh 69.2 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.3 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6. Do not Autoclave the medium.
7.After cooling mix well and pour into the sterile petriplates.
8.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
9.After preincubation use the plates for inoculation.
10.It should not be remelted and the surface of the plates should be dried before use.
Fluid Lactose Medium
1.Weigh 13.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 6.9 ± 0.2
5.Heat if necessary to dissolve the medium completely. Mix well and distribute into tubes / bottles and if required with inverted Durham’s tubes or as per requirement.
6.Sterilize by autoclaving at 15 lbs pressure (121°C) for 20 minutes.
7.The concentration of medium is adjusted in accordance with the sample.
8.After sterilization cool the media to 25°C and store in a cool dark place in an incubator for 24 hours (i.e. for preincubation)
9.After preincubation use the tubes for detection.
Levine Eosin –Methylene Blue Agar Medium
1.Weigh 36.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.2 ± 0.2.
5.Heat to dissolve the medium completely by keeping the beaker / bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.Avoid over heating of the media.
8.After autoclaving cool the medium to 50° C and shake in order to oxide the methylene blue (i.e to restore its blue Colour) and to suspend the flocculent precipitate.
9.If the medium is inoculated on the same day,it may be used with out autoclave sterilization.
10.After cooling the media is poured in to the sterile petriplates
11.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
12.After preincubation use the plates for inoculation.
Mannitol Salt Agar Medium
1.Weigh 111.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.4 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 50°C.
8.If desired add 5% v/v Egg Yolk Emulsion (FD045) and mix well.
9.After cooling the media is poured in to the sterile petriplates / in tubes to make slants.
10.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
11.After preincubation use the plates for inoculation.
MacConkey Agar Medium
1.Weigh 51.53 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.1 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Avoid over heating.
7.Cool the media to 40 - 50°C.
8.After cooling the media is poured into the sterile petriplates / in tubes to make slants.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
MacConkey Broth Medium
1.Weigh 40.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.4 ± 0.1.
5.Heat if necessary to dissolve the medium completely by keeping the beaker / bottle on the hot plate/water bath.
6.Distribute the dissolved media into the tubes with inverted Durham’s tubes or as per requirement.
7.Place all the test tubes in a container that is in turn kept in an autoclave.
8.Sterilize all the test tubes contained media in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
9.After sterilization cool the tubes before inoculation.
Nutrient Agar Medium:
1.Weigh 45.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /Purified water and mix well.
4.Adjust the final pH (at25°C) 7.2 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the media in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.Cool the media to 40 - 50°C and used the proposed analysis.
8.After cooling the media is poured into the sterile petriplates / in tubes to make slants.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
Nutrient Broth Medium
1.Weigh 13.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.4 ± 0.1.
5.Heat to dissolve the medium completely by keeping the beaker/bottle on the hot plate/water bath.
6.Transfer the dissolved media 100 ml each into the sterilized glass bottles of 125 ml capacity.
7.Place all the bottles in a container that is in turn kept in an autoclave.
8.Sterilize all the bottles contained media in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
9.After sterilization cool the tubes before inoculation.
10.After cooling use the media for the proposed.
Pseudomonas Agar Medium for Detection Of Flourescein
1.Weigh 38.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 50°C.
8.Mix well the media and pour in to the sterile petriplates.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
Pseudomonas Agar Medium for Detection of Pyocyanin
1.Weigh 46.4 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 50°C.
8.Mix well the media and pour in to the sterile petriplates.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
R-2A Agar
1.Weigh 18.12 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.2 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Do Not Over Heat
7.After autoclaving cool the medium to 50°C and use the media for the proposed.
8.After cooling mix well the media and pour in to the sterile petriplates.
Sabouraud Dextrose Agar Medium
1.Weigh 65.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 5.6 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 40-50°C and use the media for the proposed.
8.After cooling mix well the media and pour in to the sterile petriplates/ in tubes.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates/tubes for inoculation.
Selenite F Broth
1.Weigh 23.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.3 ± 0.2.
5.Warm to dissolve medium completely.
6.Transfer the dissolved media in sterile test tubes.
7.Sterilize in a boiling water bath or free flowing steam for 10 minutes.
8.Do Not Autoclave.
9.After sterilization cool the media to 25°C and store in a cool dark place preferably below 25°C.
10.After cooling use the media for the proposed.
Tetrathionate- Bile- Brilliant Green broth Medium
1.Weigh 63.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Heat just to boiling to dissolve the medium completely.
6.Do not Autoclave / Over heat.
7.Distribute the medium into sterile test tubes / bottles.
8.After cooling the medium to 25°C, store in a cool dark place preferably below 25°C.
9.After cooling use the medium for the proposed.
Triple Sugar-Iron Agar Medium
1.Weigh 65.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Mix well and distribute into test tubes.
7.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
8.After sterilization allow the medium to set in sloped form with a butt about 1 inch long.
9.After the medium in the test tubes is solidified, place the test tubes in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the test tubes for inoculation.
Vogel-Johnson Agar Medium
1.Weigh 61.0 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker / bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.2 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes. Avoid over heating
7.After sterilization cool the medium to 45°C and add aseptically 20 ml of sterile 1 %potassium tellurite solution (FD052).
8.After cooling mix gently and pour into sterile petriplates.
9.After the medium in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the petriplates / test tubes for inoculation.
Xylose-Lysine-Desoxycholate Agar Medium
1.Weigh 56.68 g of media in a butter paper in a clean weighing balance.
2.Suspend the weighed media in to a clean sterilized beaker /bottle
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.4 ± 0.2.
5.Boiling to dissolve the medium completely by keeping the beaker / bottle on the hot plate/water bath.
6.Do not autoclave / over heat.
7.After boiling cool the medium and distribute in into sterile petriplates.
8.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
9.After preincubation use the plates for inoculation.
Plate Count Agar
1.Weigh 23.5 g of media in a butter paper in a clean weighing balance.
2.Transfer the weighed media in to a clean sterilized beaker /bottle.
3.Add exactly 1000 ml of distilled /purified water and mix well.
4.Adjust the final pH (at25°C) 7.0 ± 0.2.
5.Boil to dissolve the medium completely by keeping the beaker /bottle on the hot plate/water bath.
6.Sterilize the medium in an autoclave at 15 lbs pressure (121°C) for 20 minutes.
7.After autoclaving cool the medium to 50°C and use the media for the proposed analysis.
8.After cooling mix well the media and pour in to the sterile petriplates.
9.After the media in the petriplates is solidified, place the petriplates in an incubator for 24 hours (i.e. for preincubation)
10.After preincubation use the plates for inoculation.
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