I am doing an experiment in which I am growing S. mutans in agar dishes, and I am not sure how I would measure the growth of the S. mutans. I am also not sure if I would do this by measuring cell mass or by cell count. Any ideas?
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$\begingroup$ please specify what exactly you starting with and what exactly you want to achieve $\endgroup$– aaaaa says reinstate MonicaDec 27, 2015 at 14:17
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1$\begingroup$ @aaaaaa he hasn't been here for over 2 years. $\endgroup$– Eli KorvigoDec 27, 2015 at 18:13
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2 Answers
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The most simple way is seeding the plates with a suspension of bacteria ensuring that you spread the solution properly. Then you can count the number of colonies, wich would be equal to the number of single cells.
If you want to mesure the growth speed, usually it's simpler to just measure the diameter of the colonies, always ensuring you inoculate the plates with the same amount of inoculum.
Lastly, it's even easier to estimate the growth if your culture is un liquid media and you measure the optic density with an spectophotometer.
answered Sep 25, 2013 at 13:28
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$\begingroup$ I don't understand the effect of not "always ensuring you inoculate the plates with the same amount of inoculum." $\endgroup$– jan-glxSep 25, 2013 at 16:33
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$\begingroup$ If you inoculate a plate with 100bacteria/ml and a second plate with 1000bacteria/ml, the second one will have more colonies, even if the effect of the experiment would have the opposite effect. $\endgroup$ Sep 25, 2013 at 19:39
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$\begingroup$ Thank you so much. And thank you for clarifying that statement. I was about to ask the same question. Also, I understand the second part, but I am still confused on what you mean by "seeding the plates with a suspension of bacteria ensuring that you spread the solution properly. Then you can count the number of colonies, which would be equal to the number of single cells." I'm sorry if this is a silly question, it's just that I'm in 8th grade and pretty new to this. I am doing this for science fair, actually. $\endgroup$– SohumSep 26, 2013 at 1:23
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$\begingroup$ Imagine you have a liquid media with a concentration of 100 bacteria/ml and you inoculate a plate with 1 ml. In theory, every single cell will grow to form a colony, so you must have around 100 colonies. If you don't spread it properly, all 100 colonies will appear clustered, and you won't be able to identify single colonies. That's why you must spread the inoculum yo form an homogenous layer over the agar, normally using an inoculation loop. $\endgroup$ Sep 26, 2013 at 6:28
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$\begingroup$ "Then you can count the number of colonies, wich would be equal to the number of single cells." - it won't. It will be equal to the number of CFUs, not cells. $\endgroup$ Dec 27, 2015 at 12:34
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you will almost certainly want to perform serial dilutions of your suspension, and plate the serial dilutions using the spread plate method (or pour plate method). The dilutions will make it much easier to count the number of bacteria.
Generally, it is not recommended to count more than 250 colonies on a plate for reasons of accuracy (but I never count more than like 50, because that is the whole reason for doing serial dilutions). Here is a nice picture of what I am talking about. It includes a description of how to calculate the number of organisms in the original solution. They have chosen to count the number of bacteria on the fourth plate (1:10,000 dilution) in order to estimate the number of bacteria in the original inoculum. Hope this helps
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1$\begingroup$ By the way, the prototypical selective media for S.mutans is mitis-salivarius agar. You should check out this paper about growing S. mutans jcm.asm.org/content/4/1/95.full.pdf $\endgroup$ Dec 27, 2015 at 12:28
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$\begingroup$ Your answer makes me happy, because it provides a real microbiological technique used in labs. You would make it even better by clarifying the difference between CFU counts and cell counts. And it would be nice to add that the process must be replicated to estimate error. $\endgroup$ Dec 27, 2015 at 12:36
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1$\begingroup$ Ehhh alright, well CFU stands for colony forming unit. Each one of those dots on the plate is a colony, and we are assuming that each colony is the result of a single cell from the original solution. So by counting the number of colonies on the plate we are also counting the number of single cells in the original solution that started the colonies on the plate, aka colony forming units(CFU). And sure, you could do this in dup or triplicate if you want and report the average of the counts, but this method is understood to be just an estimation, so choose the methodology that fits best for you. $\endgroup$ Dec 27, 2015 at 12:52