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counting bacterial colonies - (Apr/10/2009 )

Hi all, am fairly new to the microbiological world, so please forgive me my ignorance.

I am trying to count the number of CFUs present in a mixed culture of lactic acid bacteria and bifidobacteria.
Planning on growing/culturing them in brain heart infusion broth (is that a good idea) and then I don't know how to measure the number of CFUs present in my culture. Any ideas??

Thanks
mutiny

-mutiny-

mutiny on Apr 10 2009, 10:42 PM said:

Hi all, am fairly new to the microbiological world, so please forgive me my ignorance.

I am trying to count the number of CFUs present in a mixed culture of lactic acid bacteria and bifidobacteria.
Planning on growing/culturing them in brain heart infusion broth (is that a good idea) and then I don't know how to measure the number of CFUs present in my culture. Any ideas??

Thanks
mutiny

You could indeed grown them on that media.

And how do you mean, you do not know how to count the CFU's?

You simply count the colonies you see? Check this link,at the bottom, CFU
I do not see what your problem might be? You mean that you can not see the difference between the different types of bacteria? or?

-pito-

Erm the problem I am having is the method to growing the colonies, do i just put a 10uL droplet onto a Brain Heart Infusion agar? and count - will I need to consider the difference that the different type of bacteria will grow at a different rate?

My question is that I need some step by step protocol. I have read that some methods include growing these bacteria in anaerobic conditions (setting it in warm agar) or in partially anaerobic conditions. Or is it okay to just grow it on the surface of regular bacto-agar?

-mutiny-

if they're strictly anaerobic then do the anaerobic thing .

U probably want about 50-100 ul on because u won't even have a chance to spread 10ul because it got absorbed into agar quick. sometimes this might even apply to 50ul.

morphology should be different. u must be able to differentiate these bacteria by their morphology.

-hanming86-

anaerobic thing? Not a real helpful answer. Lactobacillus spp are facultative anaerobes (grow with or without oxygen) and Bifidobacterium spp are anaerobic. Suggest you try a pour plate.

-GeorgeWolff-

*edited

Like hanming86 says, you better use 100Ál and then spread that out with a spatula (see: spatula)

You should ask someone in the lab how to, its not that hard, just make sure the spatula is sterile: dip into ethanol, hold in in the bunsenburner , make sure its not hot anymore when spreading your sample (push the spatula in the plate of agar , at the side where no sample is, too cool it down then spread the sample) You should be able to find a protocol for this on the net I think.

Just putting a drop on your plate doenst work, then you will simply have a drop with colonies. The point of CFU's is that you spread it on the entire plate and then count how many colonies you see.

But as GeorgeWolff allready stated: you better use a pour plate for the Bifidobacterium spp , for the Lactobacillus spp you could do what you allready wanted to do and what I explained here.


I hope this helps.

-pito-