Troubleshooting a Microbial CFU Assay

[kparduc]

Hello Microbiologists!

 

I am having some issues with majority of my experiments. I am performing microbial assays on Pseudomonas aeruginosa with the use of CFU assays (incubation, serial dilution, and spot plating). However the bacteria are not growing after I incubate them for three hours. They are growing fine when I do not incubate them.

 

Have any of you been experiencing similar problems with similar assays - perhaps with other microorganisms?

Here are some details:
- buffer pH is fine at 7.4

- we've made fresh stocks of our buffers (correct salt content and molarities) - we even tried it in PBS + TSB!

Perhaps there has been a mutation in the bacteria that causes them to become vulnerable to environmental changes? We place them on ice after incubating them, wash them with a centrifuge, and the incubated group we put back in the shaking incubator at 37C.

 

I hope someone can help me out! This has been bugging me for a while.

- Kevin

[CharonY]

The protocol is not quite clear to me. What precisely do you with "the bacteria are not growing after I incubate them for three hours."?

Do you mean after harvesting the bacteria from plate or liquid medium and incubating them in pure buffer? If so did you grow them again in media (full/minimal?). The source of the confusion is that incubation also refers to regular growth conditions. And thus

 

 

They are growing fine when I do not incubate them.

 

is kind of contradictory.

[iRNAblogger]

I am experiencing CharonY's confusion as well.

 

What exactly is the purpose of your initial 3-hr incubation step? Generally incubation is supposed to refer to growth conditions, but if you are incubating the bacteria with a ligand or something, then it MIGHT make more sense. I would suggest that for a CFU calculation, all you should be doing at the bench is diluting out your bacterial sample, spreading on plates, incubating (at optimum growth temperature), and then counting the colonies.

 

Are you testing the CFU after incubation with a ligand or substrate or chemical of some sort? That might be the source of the problem.