MacConkey Agar Techniques

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MacConkey Agar Techniques

 
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  Let's start off with a little about MacConkey agar (MA): Fermentation is metabolism in the absence of oxygen.Yeast is an alcoholic fermenter of sugars (it converts sugar to ethanol in the absence of oxygen). E. coli is a "mixed acid fermenter of sugars." That means that E. coli will convert sugars to a wide variety of organic acids in the absence of oxygen (anaerobic growth). Those acids are acetic, formic, succinic, etc., etc. Many other Gram(-) bacteria are mixed acid fermenters - BUT they do now make formic acid (HCOOH), which is a rather strong organic acid. So Dr. MacConkey, in the early 1900's, concocted his medium so that it contained the sugar lactose, which few other Gram(-) bacteria can use, a pH indicator that turned red under acidic conditions, a lot of phosphate buffer that had to be overcome by all the "mixed acids" including the formic acid in order for the pH to drop into the "red" zone of the indicator, and a lot of nitrogenous material (amino acids, for example) that could sustain the growth of non-E. coli, if they happened to be present. But those non-E. coli forms would ferment the amino acids into cell mass and NH3, which would make the pH go basic rather than acidic. Thus the color would not be red. One final thing the inventor thought of was that the dye, when acidic must be insoluble so that it stays right there wherever the bacteria happen to be growing.

Thus E. coli would make red colonies as shown on the left side of the plate, while other Gram(-) bacteria would not be red and actually be a very light pink as shown on the right side of the plate. (MA usually comes with lactose in it so that you can see that E. coli ferments lactose to make red. However, you can also buy "MA without lactose", and then add your own types of sugar to it to test whether they can use those sugars and thus detect what kinds of sugar mutations might be incorporated into those strains that you have.


MAKING "QUICKIE" MacCONKEY PLATES.

  1. The previous day you have cleaned and dried the required number of petri plates. Today, array them on your freshly wiped countertop. (A suggestion would be to set the plates on aluminum foil so that any spilled medium will not stain your countertop.) Remove the tops (without getting your fingertip inside either of the tops or the bottoms!) and set them elsewhere, but nearby In your kitchen. Take some clean paper towel from a roll (try touching only the ends), and lay a strip of it across all the plates to cover them.
  2. Measure out the required amount of MacConkey agar powder into a sauce pan, and then add the required amount of COLD water. Stir to totally suspend the powder. The agar will not dissolve and will remain particulate UNTIL the mix is heated to boiling. (You use cold water just as you would for making a smooth gravy. Both have long-chain polysaccharides (agar and starch in the flour or corn starch). Cold water aids in suspending the polysaccharides while hot water added to powdered polysaccharides makes a gloppy goo.) Once the mix is homogeneous, turn on the heat and stir constantly, just like you would in making gravy. When the mix comes to a boil, stir for another half-minute so that the agar granules will dissolve, and then pour into dry petri plates.
  3. Lift off the paper towel and set it down on some other clean, DRY surface. Again only handle it by its edges.
  4. Pour the molten MA into the plates making them 2/3 full.
  5. IMMEDIATELY, rinse the pan with HOT water to help in your clean-up procedures. Any excess agar should not be put down the drain (hardens in the pipes and you need to call the plumber - and you will have a lot of explaining to do!) Pour excess agar into a plastic bag lined garbage can. The agar will solidify in the garbage.
  6. Burst any bubbles or foam by wafting a flame rapidly across the surfaces of the molten agar.
  7. Replace the paper towel over the open plates. Steam will be able to escape the hot plates through the paper.
  8. When the agar has gelled, remove the paper towel a bit at a time by rolling it back as you replace the tops onto the plates.
  9. About 30 minutes before needing the plates, preheat your oven to about 150°F, and then place all your plates onto the racks with the tops separate from the bottoms, and ALL openings facing down. Close the door and turn off the oven. Allow the plates to dry of any residual condensation.
  10. Once all the droplets are gone, reach into the oven and one by one put tops and bottoms together stacking the plates so that the agar-filled bottoms are on top (the plates are upside down).
  11. The plates should then be used immediately. (Some fungal spores will probably have blown in, and these will grow slowly. But so slowly that any E.coli work you do will outrace the fungi for the first couple of days.) (Why won't any of the bacterial spores germinate and grow?)


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