Serial Dilution Aggregatibacter actinomycetemcomitans - (Sep/15/2011 )

Hello everyone,

I can't figure out why my stock of bacteria are not growing. The bacterium, A. actinomycetemcomitans, was stored in a 1 mL 20% glycerol microcentrifuge tube and placed in the freezer. But before I put it into the freezer I forgot to mix the pellet of bacteria thoroughly with the glycerin. Could that be a problem?

In an attempt to determine the CFU's in the microcentrifuge tube, I performed serial dilutions twice up to 10^-6, 10^-7, 10^-8, 10^-9. Incubation time was 24-48 hrs @ 37 degrees C. The first go around, there were too many bacteria on all the plates (probably due to contamination) and this second time there were no bacteria at all. What could be going on?

The serial dilutions were performed with 10X TBS.

How do you know for sure you had contamination? Did you use antibiotics in the TBS? (not sure what you mean with TBS, is that TSB, a typo?)

And the second time no bacteria at all? This does sound weird to be honest... Because I can understand there is contamination.. but not on all plates and not like that.. esp if you dont have contamination the second time.. its weird.

But I dont understand what you mean by the "pellet"... Did you have a pellet? You could really see the pellet? Normally you dont store a "pellet"...

And how did you do the plating? you scabed a bit of the top of the frozen sample or?