Problem with MPN

Daniel Sagan Daniel.Sagan at
Mon Aug 25 10:44:56 EST 1997


I want to count the number of the bacterium Agrobacterium tumefaciens in
100 ml of a nutrient solution. 
As I didn´t want to count hundreds of colonies on petri dishes I use the
MPN method. At this method you transfer a small quantity (normally 1, 0.1
and 0.01 ml) of the test water in a test tube with a fresh nutrient
solution. Normally you use 3 tubes for each volume of test water. After a
certain time (depends on the species) you count the number of postive tubes
of each volume. Then you look up in a table what this means for the
concentration of the bacteria in your test water.
My problem is, that I want to count the bacteria in a nutrient solution and
so I have to use transferring quantities of less than 1 µl (Huge amount of
A. tumefaciens!). That means that I can´t  use pipettes. So I first diluted
the solution in up to 5 steps up to a rate of 1/10exp5. Then I transferred
1 ml of the last step into an tube with nutrient solution. That is the same
as I would take 0.01 µl of the original solution. My question is if it is
allowed to transfer 3 times 1 ml of the "last step tube" into 3 parallel
tubes with nutrient solution and count them as if i would have inoculated
them 3 times with the original nutrient solution.

Thanks a lot for your answers,
to this newsgroup or email me: Daniel.Sagan at
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