### Re: How do you calculate the number of bacteria colonies?

Date: Sun Sep 6 10:14:03 2009
Posted By: Billy Carver, Grad student, Biomedical Sciences, Vanderbilt University
Area of science: Microbiology
ID: 1252195062.Mi
Message:

This sounds like a really solid microbiology project. Here’s what I know: In the lab when I need to count the number of bacterial cells in a solution, I take a portion of that solution and plate it on an agar plate and then count the number of colonies. You said you didn’t have time to count all of them – and that’s obvious because you probably have tens of millions of bacteria per milliliter of medium. What you can do is take a tiny, tiny amount of the solution. So if you have 25 milliliters of your solution, you can take one milliliter of that solution and add 10mL of fresh medium to it. Then take a milliliter of THAT solution and add it to 10mL of fresh medium to it. Then plate 250 microliters of that second solution on an agar plate and watch for growth. Count the number of colonies you get and multiply that by 400 (10x for the first dilution, another 10x for the second dilution, and 4x because you only used 250 microliters…so 10x10x4, equals 400) and that will give you the number of bacteria per milliliter in your original culture. If you don’t have a device that will let you handle such small volumes of liquid you could also do this using teaspoons, with the knowledge that 1 teaspoon equals approx. 5 milliliters. I like to take a Sharpie ultra-fine tipped marker, turn the plate over and put a dot on each colony as I count…but I’m a dork like that.

There is another way to “count” bacteria, but it uses a sophisticated piece of equipment called a spectrophotometer. When you use a spectrophotometer to count cells you put some of your culture into a special tube called a cuvette. Light passes through the culture and depending on how many bacteria per volume are present (the turbidity), they absorb more or less light. The machine tells you how much light is passing uninterrupted through the cuvette and you can look at a graph to determine how many bacteria that represents. One drawback of this method is that dying or recently dead cells will also be counted, so it might be less useful for the viability assay you are proposing. My high school actually had a spectrophotometer, so you might check with your chemistry and/or biology teachers about that.

Finally, I want to make sure I understand your experimental procedure. You say that you want to put 25 milliliters of culture onto an agar plate. I highly suggest spinning down those cells in a centrifuge first, and then pouring off most of the medium before putting them on an agar plate. I have never used more than .5 milliliters to plate on agar before, and even that was a mess. Otherwise your experiment sounds really cool. Good luck! Billy Carver.

PS – I’ve included a single link to a laboratory protocol from a Polish university that contains a nice outline of how to count bacteria using both methods I mentioned about above. You can simplify them if you’d like, of course. It also has the graph that helps determine bacterial cells mass by spectrophotometry.

“Counting Bacteria” by Jackie Reynolds/Mark Farinha, Richland College http://www.biotech.univ.gda.pl/odl/doc/numbers.pdf

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