PREPARING YOUR MEDIUM
There are many ways in which media can be prepared. If you have a good method already you should continue to use it.
A. HOT PRE-DISSOLVED AGAR METHOD
- HEAT a litre of distilled water to boiling in a scrupulously clean container [ We use an electric kitchen kettle ]
- Immediately add about 250mI, of the just boiled water to a blender and immediately empty the contents of a 1L sachet of medium, replace the blender lid firmly, and flick the power on & off very quickly 2-3 times to start mixing the contents & heat the air above the water without blowing the lid off. NOTE THIS MUST BE DONE VERY CAREFULLY TO AVOID SCALDING YOURSELF. Now run the blender for 10 to 20 seconds for a thorough mix.
- Pour the mixture into your measuring vessel and rinse all the thick mixture from the blender into the measuring vessel with some more just boiled water.
- If banana pulp is required, weigh the banana, add it to the blender with some hot distilled water and blend to a fine pulp then add it to the measuring vessel and make to about 900mL &/or if required, measure & add coconut water &/or pineapple juice now also before making to 1000mL with more hot water and mix thoroughly before pouring into your flasks.
- If pH adjustment is required, allow to cool to about 50 degrees C, or use only 500mL of hot water to dissolve & rinse the agar into your make-up vessel then use cold distilled water to blend the bananas and make up to near final volume before checking, adjusting pH and adjusting to final volume. Mix thoroughly before pouring and pour quickly before it cools & sets.
- NOTE WHEN POURING, AVOID SPILLING MEDIUM ON TO UPPER NECK OR OUTER THREADED AREA AS THIS WILL ALLOW BACTERIA OR FUNGI TO GROW INTO YOUR FLASKS ON STORAGE VIA THE PATH OF SPILT MEDIUM.
- Make sure the flasks/containers you use & their lids can withstand the heat of pressure cooking or autoclaving to sterilise them & their contents.
B. COLD TECHNIQUE
- If required, weigh or measure the required amount of banana pulp &/or coconut water &/or pineapple juice.
- Add it to a blender with some of the distilled water and blend thoroughly.
- Add all of the contents of the sachet of medium plus some more water & blend very thoroughly.
- Rinse the contents of the blender into your measuring vessel with some more distilled water and make up to the final volume.
- Check pH & adjust as necessary.
- Mix thoroughly and pour only 1-2 flasks immediately then mix again and pour the next two flasks & so on until all are poured. [ This is necessary because the agar is heavier than water and not dissolved in cold water and so one must stir well and frequently between pourings or some will set like rocks whist others may not have enough agar in them set at all after sterilising. ]
- Transfer to pressure cooker or autoclave to sterilise. After sterilising, agar will be more concentrated at the bottom so it is necessary to gently swirl the flasks for a moment to suspend the agar evenly from top to bottom. [ DO NOT SHAKE FLASKS – JUST SWIRL GENTLY ]
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STERILISATION
To sterilize the medium, flasks and lids, the heat process must REACH AND MAINTAIN ALL THE MEDIUM AT 120 - 121 deg C FOR 15 MINUTES OR AT 115 - 116 deg C FOR 25 MINUTES - hence required autoclaving or pressure cooking time = time for all the contents to reach and be maintained at 120-121 or 115 - 116 deg C for 15 or 25 minutes respectively. NOTE :-
- After sterilization, de-pressurise slowly enough to avoid vigorous boiling within your culture vessels else the medium will boil and be forced out around the top, lid & thread area of the culture vessels. Later if a microbe were to land on the expelled medium it would be able to grow up along the residual medium, into the flask & set up contamination inside the flasks.
- If the depressurisation/cool -down process is overly slow, then the prolonged heat will cause the agar to lose some of its power to set properly. Cloning media containing plant growth regulators may not perform as expected if overheated. In general it should take about 10 minutes for your pressure cooker or autoclave to depressurise and allow you to remove the flasks of hot medium to a cool area to set. The ideal place for them to set is in your laminar airflow cabinet whilst it is running. NOTE also if the cooling area is too hot, the medium may cool too slowly & not set as firmly as it would if placed in a cooler area. Hot weather / hot environments may also be well avoided when preparing/ sterilising medium.
IMPORTANT NOTE :- FLASKS OF MEDIUM MUST NEVER BE TURNED UPSIDE DOWN ELSE THE FREE FLUID ON TOP OF THE AGAR MAY WEEP OUT TO THE THREADED AREA OF THE FLASK AND CAUSE CONTAMINATION AS MICROBES CAN THEN ENTER VIA THE NUTRIENT PATH SO MADE.
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