Determine how many plates you will need today. You will want to make 2 or three extra in case of accident.
Find a bottle of powdered MacConkey Agar Medium.
Read the instruction panel on the label.
- 300 ml of hydrated product will make about 9 or 10 plates.
- How many ml will you need to make?
- Weigh out the requisite amount of powder directly into an autoclavable bottle or flask using a powder funnel. (Never fill a container more than 2/3 full. It'll boil over in the autoclave.) Otherwise, use a weighing boat. (Do NOT make up a big batch thinking you will subdivide it into smaller flasks for autoclaving. The insoluble agar particles will quickly settle and won't distribute evenly. Eventually you will end up pouring some plates that will never solidify and those poured last will set like concrete!)
- Clean up, or be thrown to the Lions!
- Add the requisite amount of warm tap water (tap water contains minerals for growth).
- Swirl (do not shake) to disperse the powder components in the water. (Again: the agar will not dissolve until the water is nearly boiling.)
- Cap the bottle or flask with foil or a small beaker.
- Autoclave for 15 minutes on "liquid" cycle. (Now go to step 2 until autoclave is done.) (Here again MacConkey Agar has a hidden "plus" - autoclaving is not necessary! Merely boiling it to make the agar dissolve is all that is needed. - See the opening paragraph, above.)
- Remove bottle from autoclave. Screw the top on tightly. Swirl the bottle over a sink to disperse any agar gel on the bottom. (The sink is in case of superheated geysers. Bottles dropped in a panic!)
- Carefully wipe off your counter with a clean paper towel, which has been moistened with some dilute (1:30) household bleach. (Rids the area of spores.)
Array your plates about 1/2 inch apart on the counter top. Keep the covers on!
Pour molten agar into the plates filling them to about one third. Lift off only one cover at a time, replacing it after pouring in the requisite amount of molten agar. Do not talk during pouring (or whenever a plate is open) unless you want a lot of spurious colonies of oral bacteria to grow up!)
After a bottle's worth of molten agar has been poured, quickly waft a burner flame across the agar surfaces in the just-poured plates to burst any bubbles. (Don't do it so long that you melt plastic plates!) (You will need a lab burner that really shoots out a flame. If you don't have such a burner, you are fortunate because the substitute is much less expensive than a good quality lab burner - from your local hardware store purchase a plumber's propane torch set: canister of propane, nozzle, and sparker - all for less than $20.)
Just before use, the plates should be 'dried:' open each plate and place both the bottom and the top openside down on the 37° incubator's shelves for about 20 minutes. Excess moisture will evaporate and allow the additional moisture from serial dilutions to soak in quickly. (Here is a trick that can be done only with MacConkey or EMB agars - When you are about to pour the plates, remove all the covers and place them opening down on a clean piece of paper towell nearby. Pour the plates, burst the bubbles with the flame, and then carefully unroll a long piece of paper towel, and lay it across the open faces of the petri plates. The steamy humidity will be able to escape through the paper, and the plates solidify resulting in the surfaces' being ready to use, and, of course, the covers are dry!)
IT IS ABSOLUTELY NECESSARY
THAT THE AGAR SURFACES BE DRY AND FREE OF CONDENSATION.
If the agar surface is not dry, colonies will swarm and run together.
Once again, if further drying is needed, go to a 37° incubator, open the plates,
and place them on the shelves such that both the tops and the agar-filled bottoms
are facing downward so that dust won't fall in them.